Artificial expression constructs for selectively modulating gene expression in neocortical layer 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 TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 013,342, filed April 21, 2020, the entire contents of which are incorporated herein by reference as if fully set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States government support under Grant Nos. MH114126 and MH121274 from the National Institutes of Health. The United States government has certain rights in this invention.

[0003] Sequence Listing References The sequence listing associated with this application has been provided in text form in lieu of a paper copy and is hereby incorporated by reference. The text file containing the sequence listing is named A166-0024PCT_ST25.txt. This text file is 211 KB, was created on April 21, 2021, and has been submitted electronically via EFS-Web.

[0004] Field of Disclosure The present disclosure provides artificial expression constructs that selectively regulate gene expression in select central nervous system cell types. These artificial expression constructs can be used to selectively express synthetic genes or modify gene expression in neocortical layer 5 glutamatergic neurons, such as layer 5 glutamatergic extratelencephalic projection (L5 ET) neurons. [Background technology]

[0005] Background to the disclosure To fully understand brain biology, it is necessary to distinguish and define distinct cell types, and then to identify artificial expression constructs that can selectively label and perturb them for further study. In mice, recombinase driver strains have been used to great effect to label cell populations that share marker gene expression. However, the creation, maintenance, and use of such strains to label cell types with high specificity is expensive and often requires triple transgenic crosses, which results in low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans. Summary of the Invention

[0006] Disclosure Overview The present disclosure provides artificial expression constructs that selectively drive gene expression in target central nervous system cell populations, including neocortical glutamatergic layer 5 neurons, such as neocortical glutamatergic layer 5 extratelencephalic projection (L5 ET) neurons. In particular applications, embodiments of the present disclosure can be used to deliver therapeutic payloads to upper motor neurons, for example, those involved in amyotrophic lateral sclerosis (ALS) or other neurodegenerative diseases involving L5 ET neurons.

[0007] Certain embodiments of the artificial expression construct utilize the following enhancers to selectively drive protein expression in L5 glutamatergic neurons: 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 comprises a concatenated core of enhancers, such as the concatenated 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 a more rapid onset of expression compared to a single full-length original (native) enhancer.

[0009] In certain embodiments, the enhancer core comprises a sequence set forth in any one of SEQ ID NOs: 45-52. In certain embodiments, these cores are linked and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. SEQ ID NOs: 53-60 provide concatemers of 3 copies of a selected enhancer core.

[0010] Certain embodiments of 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] Certain embodiments are directed to the 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, 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.

[0012] Many of the drawings submitted herein are best understood in color, and applicants consider the colored drawings to be a part of the application documents as originally filed and reserve the right to submit images of the colored drawings by following the procedure described below. [Brief explanation of the drawings]

[0013] [Figure 1]An exemplary enhancer discovery schematic for viral tools is shown. To construct a cell-type-specific labeling tool, cells from adult mouse cortex can be isolated and sequencing (scATAC-seq) can be used to perform a single-cell assay for transposase-accessible chromatin. Samples can be clustered and compared with single-cell RNA sequencing (scRNA-seq) datasets to identify clusters. Single cells matching the same transcriptome type can then be pooled, and the genome can be searched for type-specific putative enhancers. Alternatively, a human motor cortex SNARE-seq dataset (Bakken et al., Nature. 2021 doi: https: / / doi.org / 10.1101 / 2020.03.31.016972) was used to select some enhancers. Selected putative enhancer regions can be cloned upstream of a minimal promoter in the AAV genome backbone, which can be used to generate self-complementary adeno-associated viral vectors (scAAV) or recombinant adeno-associated viral vectors (rAAV). These viral tools were delivered retroorbitally to label specific neocortical populations. In cells with matching cell types, enhancers recruit their cognate transcription factors to drive cell-type-specific expression. In other cells, the viral genome is present but no transcripts are expressed. However, not all enhancers behave as expected, so enhancer constructs must be tested for specificity. [Figure 2A] Mouse genomic coordinates for enhancer eHGT_451m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 2B]Epifluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2249 6.00E11GC. The genomic coordinates are for build Hg38 and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 3A] Mouse genomic coordinates for enhancer eHGT_452m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 3B] Epifluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2250, 1.45E12GC. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 4A] Mouse genomic coordinates for enhancer eHGT_453m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 4B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2251 7.00E11GC. Scale bar: 100 microns. [Figure 4C]Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex region of the mouse brain after retroorbital injection of CN2251 virus packaged in PHP.eB capsids. The number of cells mapped to the most distal end is shown in the bar graph below the dendrogram. The cell types of the transcriptome are indicated at the bottom. The data show that eHGT_453m enhancer-driven reporter expression is selective 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] Epifluorescence microscopy image of native SYFP2 fluorescence in a fixed brain slice from macaque primary motor cortex 64 days after stereotaxic injection of enhancer AAV vector CN2251 serotype PHP.eB. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 500 μm. [Figure 5A] Mouse genomic coordinates for enhancer eHGT_459m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 5B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2254 1.00E12GC. 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 retroorbital injection of CN2254 virus packaged in PHP.eB capsids. The number of cells mapped to the most distal end is shown in the bar graph below the dendrogram. The cell types in the transcriptome are indicated at the bottom. The data show that eHGT_459m enhancer-driven reporter expression occurs primarily in only one of four types of L5 ET neurons in the visual cortex region. The letters at the bottom, from left to right, are the same as those in Figure 4C. [Figure 5D] Epifluorescence microscopy image of native SYFP2 fluorescence in a fixed brain slice from macaque primary motor cortex 113 days after stereotaxic injection of enhancer AAV vector CN2254 serotype PHP.eB. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 500 μm. [Figure 6A] Mouse genomic coordinates for enhancer eHGT_460m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 6B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2255 6.50E11GC. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 7A] Mouse genomic coordinates for enhancer eHGT_462m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 7B] Epifluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2256, 6.50E11GC. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 8A] Mouse genomic coordinates for enhancer eHGT_464m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 8B]Epifluorescence micrograph showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2315 1.00E12GC. Scale bar: 100 microns. [Figure 8C] Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex of the mouse brain after retroorbital injection of CN2315 virus packaged in PHP.eB capsids. The number of cells mapped to the most distal end is shown in the bar graph below the dendrogram. The cell types in the transcriptome are indicated at the bottom. The data demonstrate that eHGT_464m enhancer-driven reporter expression selectively occurs in multiple types of L5 ET neurons within the visual cortex. The letters at the bottom, from left to right, are the same as those in Figure 4C. [Figure 8D] Epifluorescence microscopy image of native SYFP2 fluorescence in a fixed brain slice from macaque primary motor cortex 64 days after stereotaxic injection of enhancer AAV vector CN2315 serotype PHP.eB. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 500 μm. [Figure 9A] Mouse genomic coordinates for enhancer eHGT_461m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 9B] Epifluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2423 5.00E11GC. The genomic coordinates are for build Hg38 and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 10A] Mouse genomic coordinates for enhancer eHGT_463m (shaded region) and corresponding single cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 10B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex 2 months after retroorbital delivery of AAV vector #CN2424 from 7.30E11GC. Scale bar: 100 microns. [Figure 10C] Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex region of the mouse brain after retroorbital injection of CN2424 virus packaged in PHP.eB capsids. The number of cells mapped to the most distal end is shown in the bar graph below the dendrogram. The cell types in the transcriptome are indicated at the bottom. The data show 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 in Figure 4C. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. [Figure 11A] Mouse genomic coordinates for enhancer eHGT_647m (shaded region) and corresponding single-cell ATAC-seq peaks for each cell class and subclass are shown. Arrows indicate open chromatin peaks for the L5 ET neuron subclass. [Figure 11B] Epifluorescence micrograph image showing native SYFP2 expression in the visual cortex 1.5 months after retroorbital delivery of AAV vector #CN2847, 7.00E11GC. The genomic coordinates are for build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 12A] Sequences Supporting the Present Disclosure Enhancer Sequences Disclosed Herein Each of the artificial expression constructs described herein contains at least one of the enhancer sequences provided in Figure 12A. [Figure 12B] Sequences supporting the present disclosure. Exemplary components that can be included in (or encoded by) artificial expression constructs generated using the enhancer of (12A). [Figure 12C]Sequences supporting the present disclosure. Representative artificial expression constructs generated using the enhancer in (12A) and selected components in (12B). DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description To fully understand brain biology, we need to distinguish and define distinct cell types, and then identify artificial expression constructs that can selectively label and perturb them for further study. (Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018); Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017)) In mice, recombinase driver lines have been used to great effect to label cell populations that share marker gene expression. (Daigle et al., Cell 174, 465-480.e22 (2018); Taniguchi et al., Neuron 71, 995-1013 (2011); Gong et al., J. Neurosci. 27, 9817-9823 (2007)) However, the generation, maintenance, and use of such lines to label cell types with high specificity is expensive and often requires triple transgenic breeding, which results in low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans.

[0015] The present disclosure provides artificial expression constructs that selectively drive gene expression in target central nervous system cell populations, including neocortical glutamatergic layer 5 neurons, such as neocortical glutamatergic extratelencephalic projection (L5 ET) neurons. In particular applications, embodiments of the present disclosure can be used to deliver therapeutic payloads to upper motor neurons involved in, for example, amyotrophic lateral sclerosis (ALS) or other neurodegenerative diseases involving L5 ET neurons.

[0016] Particular embodiments of the artificial expression constructs 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 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 are utilized to selectively drive gene expression in L5 ET neurons.

[0017] In certain embodiments, the artificial enhancer element comprises the concatenated cores of the enhancers disclosed herein, such as the concatenated cores 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 more rapid onset of expression compared to a single full-length original (native) enhancer.

[0018] In certain embodiments, the enhancer core can comprise a core sequence set forth in any one of SEQ ID NOS: 45-52. In certain embodiments, these cores are linked and comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core. SEQ ID NOS: 53-60 provide concatemers of 3 copies of a selected enhancer core.

[0019] Certain embodiments of 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.

[0020] Certain embodiments are directed to the 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, 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.

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

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

[0023] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the level of transcription associated with a promoter, can function in either orientation relative to the promoter and coding sequence to be transcribed, and can be located upstream or downstream relative to the promoter or coding sequence to be transcribed. Methods and techniques for measuring the function(s) of an enhancer element sequence exist and are recognized in the art. Specific examples of enhancer sequences for use in the artificial expression constructs disclosed herein include: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_462m, eHGT_463m, eHGT_464m, eHGT_465m, eHGT_466m, eHGT_467m, eHGT_468m, eHGT_469m, eHGT_470m, eHGT_471m, eHGT_472m, eHGT_473m, eHGT_474m, eHGT_475m, eHGT_476m, eHGT_477m, eHGT_478m, eHGT_479m, eHGT_480m, eHGT_481m, eHGT_482m, eHGT_483m, eHGT_484m, eHGT_485m, eHGT_486m, eHGT_487m, eHGT_488m, eHGT_489m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT_49 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 For example, 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, a target central nervous system cell type enhancer is an enhancer that is uniquely or primarily utilized by the target central nervous system cell type. The target central nervous system cell type enhancer enhances expression of a gene in the target central nervous system cell type but does not substantially induce expression of the gene in other, non-target cell types, and thus has cell-type-specific transcriptional activity.

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

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

[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 characteristics is provided as follows:

[0028] Neocortical GABAergic subclasses: ●All: express the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2. • Lamp5, Sncg, Serpinf1, and Vip: developmentally derived from neuronal precursors from the caudal ganglion luminal (CGE) or preoptic area (POA). • Sst and Pvalb: developmentally derived from neuronal precursors in the medial ganglionic emittance (MGE). ●Lamp5: Found in many cortical layers, especially the upper layers (L1-L2 / 3), and mainly has glial and single plexus morphology. ● Sncg: Found in many cortical layers and has molecular overlap with Lamp5 and Vip cells, but expression of Lamp5 or Vip is inconsistent and expression of Sncg is more consistent. ●Serpinf1: Found in many cortical layers and has molecular overlap with Sncg and Vip cells, but expression of Sncg or Vip is inconsistent and expression of Serpinf1 is more consistent. ●Vip: Found in many cortical layers, but particularly abundant in the upper layers (L1-L4), where it highly expresses the neurotransmitter vasoactive intestinal peptide (Vip). Sst neurons are found in many cortical layers, but are particularly frequent in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic input to postsynaptic neurons. This subclass includes sleep-active Sst Chodl neurons (which also express Nos1 and Tacr1), which are very distinct from other Sst neurons but express several shared marker genes, including Sst. In humans, Sst gene expression is often detected in layer 1 LAMP5+ cells. Pvalb: Found in many cortical layers, but particularly frequent in the lower layers (L5-L6). They highly express the calcium-binding protein parvalbumin (Pvalb), express the neuropeptide Tac1, and often attenuate the output of postsynaptic neurons. Most GABAergic cells strongly express Pvalb. This subclass includes chandelier cells, which have a distinct chandelier-like morphology and express the markers Cpne5 and Vipr2 in mice and NOG and UNC5B in humans. Meis2: A distinct subclass defined as only the neocortical GABAergic type expressing the Meis2 gene, but not expressing some other genes expressed by all other neocortical GABAergic types (e.g., Thy1 and Scn2b). This type is found in L6b and subcortical white matter.

[0029] Neocortical glutamatergic subclasses: All: express the glutamate transmitters Slc17a6 and / or Slc17a7. They all express Snap25 and lack Gad1 / Gad2 expression. • L2 / 3 IT: Present mainly in layer 2 / 3 and has mainly intratelencephalic (cortico-cortical) projections. • L4 IT: Present mainly in layer 4 and have either primarily regional or intratelencephalic (cortico-cortical) projections. L5 IT: Predominantly in layer 5 and with primarily intratelencephalic (cortico-cortical) projections. Also called L5a. L5 ET: Predominantly located in layer 5 and primarily with cortico-subcortical projections. Also referred to as L5b or L5 CF (corticofugal), or L5 PT (pyramidal projection). This subclass includes cells that are corticospinal projection neurons located in the primary motor cortex and adjacent areas and associated with motor neuron / movement disorders such as ALS. This subclass includes thick-tufted pyramidal neurons, including distinctive subtypes found only in specialized areas, e.g., Betz cells, Meynert cells, and von Economo cells. ●L5 NP: Mainly resides in layer 5, with protrusions mainly in the vicinity. • L6 CT: Presents mainly in layer 6 and has mainly corticothalamic projections. • L6 IT: Present mainly in layer 6 and have mainly intracranial (cortico-cortical) projections. This subclass includes L6 IT Car3 cells, which are very similar to the intracranial cortical projection cells. • L6b: Present mainly in the neocortical subplate (L6b), with local (near the cell body) processes and some cortico-cortical projections from VISp to the anterior cyst, and cortico-subcortical projections to the thalamus. ●Cajal-Retzius cells, a distinct subclass defined as a single type in CR:L1, express distinct molecular markers Lhx5 and Trp73.

[0030] Cerebellar Purkinje cells: large GABAergic neurons, the only projection neurons and sole product of the cerebellum. Their cell bodies form a single layer called the "Purkinje cell layer" and express parvalbumin.

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

[0032] Non-neuronal subclasses: Astrocytes: Neuroectodermal-derived glial cells that express the marker Aqp4 and often GFAP, but not the neuronal marker SNAP25. They can have a distinct star-shaped morphology and are involved in the metabolic support of other cells in the brain. Multiple astrocyte morphologies are found in mice and humans. Oligodendrocytes: Glial cells derived from the neuroectoderm that express the marker Sox10. This category includes oligodendrocyte precursor cells (OPCs). Oligodendrocytes are a subclass of cells primarily responsible for myelination of neurons. • VLMC: Vascular leptomeningeal cells (VLMC) are part of the meninges that surround the outer layer of the cortex and express the marker genes Lum and Col1a1. Pericytes: Blood vessel-associated cells that express the marker genes Kcnj8 and Abcc9. Pericytes surround endothelial cells and are important in regulating capillary blood flow and are involved in blood-brain barrier permeability. SMCs: Specialized smooth muscle cells that are associated with blood vessels and express the marker gene Acta2. SMCs line the arteries of the brain and are involved in blood-brain barrier permeability. Endothelial cells: Cells that line the blood vessels in the brain. Endothelial cells express the markers Tek and PDGF-B. Microglia: Brain-resident macrophages and perivascular macrophages (PVMs) are hematopoietic-derived immune cells that may be transiently associated with brain tissue or included as a by-product of brain dissection. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

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

[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 those encoding expressible fluorescent proteins or expressible biotin, among others: blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, Amcyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™ (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0035] GFP, composed of 238 amino acids (26.9 kDa), was originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresces green when exposed to blue light. GFP from A. victoria has a major excitation peak at 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, in the lower green portion of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to its widespread potential and evolving research needs, many different variants of GFP have been genetically engineered. The first major improvement was a single point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the spectral properties of GFP, resulting in increased fluorescence, photostability, and a shift in the main excitation peak to 488 nm, with peak emission maintained at 509 nm. Adding the 37°C folding efficiency (F64L) point mutation to this backbone resulted in enhanced GFP (EGFP). EGFP has a 9.13 × 10-21 m, also referred to as 55,000 L / (mol cm). 2 The extinction coefficient (ε), also known as the optical cross section of the molecule, is ε. In 2006, we reported superfolder GFP, a series of mutations that allowed GFP to rapidly fold and mature even when fused to poorly folding peptides.

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

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

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

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

[0040] In certain embodiments, functional molecules include reporters of cell function and status, such as calcium reporters. Intracellular calcium concentration is an important predictor of numerous cellular activities, including neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels is via genetically encoded calcium indicators (GECIs). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor, designated GCaMP, is an efficient and widely used tool. GCaMP is formed by fusing M13 and calmodulin proteins to the N- and C-termini of a circular mutant GFP. Some GCaMPs exhibit distinct fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs with green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Additionally, GECIs with red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV8-CAG-GCaMP3 (catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog number: BS12-NXSAAV9), and AAV9-CAG-FLEX We provide AAV products including AAV9-Syn-FLEX-jGCaMP7f-WPRE (Cat. No.: BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (Cat. No.: BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (Cat. No.: BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (Cat. No.: BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (Cat. No.: BS7-NXBAAV8).

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

[0042] In certain embodiments, functional molecules include modulators of neuronal activity, such as channelrhodopsins (e.g., channelrhodopsin-1, channelrhodopsin-2, and their variants). Channelrhodopsins are a subfamily of retinylidene ion-gated proteins (rhodopsins) that function as light-gated ion channels. In addition to channelrhodopsin 1 (ChR1) and channelrhodopsin 2 (ChR2), several channelrhodopsin variants have been developed. For example, Lin et al. (Biophys J, 2009, 96(5):1803-14) described the creation of chimeras of the transmembrane domains of ChR1 and ChR2 combined with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6):631-3) described a red-shifted channelrhodopsin variant, VChR1. VChR1 has low light sensitivity and poor membrane trafficking and expression. Other known channelrhodopsin mutants include the ChR2 mutants described in Nagel et al., Proc Natl Acad Sci USA, 2003, 100(24):13940-5), ChR2 / H134R (Nagel, G. et al., Curr Biol, 2005, 15(24):2279-84), and ChD / ChEF / ChIEF (Lin, JY et al., Biophys J, 2009, 96(5):1803-14), which are activated by blue light (470 nm) but are insensitive to orange / red light. Further mutants are described in Lin, Experimental Physiology, 2010, 96.1:19-25, and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.

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

[0044] Sequences are publicly available, for example, lactase (e.g., GenBank: EAX11622.1), lipase (e.g., GenBank: AAA60129.1), helicase (e.g., GenBank: AMD82207.1), amylase (e.g., GenBank: AAA51724.1), alpha-glucosidase (e.g., GenBank: ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot: P08047.3), transcription factor AP-1 (e.g., NP_002219.1), heat shock factor proteins. 1 (e.g., UniProtKB / Swiss-Prot: Q00613.1), CCAAT / enhancer-binding protein (C / EBP) beta isoform A (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot: P14859.2), TGFβ (e.g., GenBank: CAF02096.2), platelet-derived growth factor receptor (e.g., GenBank: AAA60049.1), epidermal growth factor receptor (e.g., GenBank: CAA25240.1), vascular endothelial growth factor receptor (VEGF) (e.g., GenBank: AAC16449.2), interleukin-8 receptor alpha (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B isoform Ras-related protein Rab-4A isoform 1 (e.g., NP_001825.1), ras-related protein Rab-4A isoform 1 (e.g., NP_004569.2), ras-related protein Rab-11A (e.g., UniProtKB / Swiss-Prot: P62491.3), platelet-derived growth factor (e.g., GenBank: AAA60552.1), transforming growth factor-beta 3 (e.g., GenBank: AAA61161.1), nerve growth factor (e.g., GenBank: CAA37703.1), and EGF (e.g., GenBank: CAA34902).2), cocaine- and amphetamine-regulated transcript (chain A) (e.g., PDB:1HY9_A), protachykinin-1 (e.g., UniProtKB-P20366), oxytocin-neurophysin 1 (e.g., UniProtKB-P01178), somatostatin (e.g., GenBank:AAH32625.1), genetically encoded green calcium indicator NTnC (chain A) [synthetic construct] (e.g., PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g., GenBank:ACF93133.1), BRET-based self-luminescent calcium indicator [synthetic construct] (e.g., GenBank ADF42668.1), calcium indicator protein OeNL(Ca2+)-18u [synthetic construct] (e.g., GenBank BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (chain A) [synthetic construct] (e.g., PDB: 3EKJ_A), channellopsin 1 (e.g., UniProtKB-F8UVI5), channelopsin 1 (e.g., GenBank: AER58217.1), channelrhodopsin-2 (e.g., UniProtKB-B4Y105), channelrhodopsin 2 [synthetic construct] (e.g., GenBank: ABO64386.1), CRISPR-associated protein (Cas) (e.g., GenBank: AKG27598.1), Cas9 [synthetic construct] (e.g., GenBank: AST09977.1), CRISPR-associated endonucleases (ECs) Examples include nuclease Cpf1 (e.g., UniProtKB / Swiss-Prot: U2UMQ6.1), ribonuclease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot: Q05823.2), deoxyribonuclease II beta (e.g., GenBank: AAF76893.1), sodium channel protein type 1 subunit alpha (e.g., UniProtKB-P35498), potassium voltage-gated channel subfamily KQT member 2 (e.g., UniProtKB-O43526), ​​and voltage-gated L-type calcium channel subunit alpha-1C (e.g., UniProtKB-Q13936).

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

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

[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 more use in size-constrained constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct comprises an internal ribosome entry site (IRES) sequence. The IRES allows ribosomes to initiate translation at a second internal site on the mRNA molecule, leading to the production of two proteins from a single mRNA.

[0048] Coding sequences encoding the molecules (e.g., RNA, proteins) described herein can be obtained from publicly available databases and publications. Coding sequences can further contain various sequence polymorphisms, mutations, and / or sequence variants; such modifications do not affect the function of the encoded molecule. The terms "encode" or "encoding" refer to the property of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, mRNA, etc., that serves as a template for the synthesis of another molecule, such as a protein.

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

[0050] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can be strong promoters, weak promoters, constitutive promoters, and / or inducible promoters. Inducible promoters induce expression in response to specific conditions, signals, or cellular events. For example, a promoter can be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to effect transcription from the promoter. Specific examples of promoters include minBglobin, CMV, minCMV, and minCMV. * (minCMV * is minCMV with the SacI restriction site removed), minRho, minRho * (minRho *These include the SV40 immediate-early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous sarcoma virus (RSV) long repeat (LTR) promoter. Minimal promoters do not have the activity to drive gene expression by themselves, but can be activated to drive gene expression when linked to proximal enhancer elements.

[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 linked, e.g., inserted, into a vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication within the cell, or may contain a sequence that allows integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

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

[0053] An adenoviral vector is a construct containing sufficient adenoviral sequences to (a) support the packaging of an artificial expression construct and (b) express a coding sequence cloned therein in either the sense or antisense orientation. Recombinant adenoviral vectors contain genetically engineered forms of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb linear, double-stranded DNA virus, allows for the replacement of large elements of adenoviral DNA with up to 7 kb of foreign sequence. In contrast to retroviruses, adenoviral DNA can replicate episomalally without potential genotoxicity, so adenoviral infection of host cells does not result in chromosomal integration. Adenoviruses are also structurally stable, and no genome rearrangements have been detected after extensive amplification.

[0054] Adenoviruses are particularly suitable for use as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis-receptors necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain distinct transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in regulating the transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. Late gene products, including the majority of viral capsid proteins, are expressed only after significant processing of a single primary transcript derived from the major late promoter (MLP). MLP is particularly efficient during the late stages of infection, and all mRNAs derived from this promoter contain a 5'-triple leader (TPL) sequence, making them preferred mRNAs for translation.

[0055] Other than the requirement that the adenoviral vector be replication-defective, or at least conditionally defective, the nature of the adenoviral vector is not believed to be critical to the successful practice of certain embodiments disclosed herein. The adenovirus can be any of the 42 different known serotypes or subgroups A-F. In certain embodiments, adenovirus type 5 of subgroup C is the preferred starting material for obtaining the conditionally replication-defective adenoviral vectors used in certain embodiments, since adenovirus type 5 is a human adenovirus for which a great deal of biochemical and genetic information is known and has historically been used for most constructs using adenoviruses as vectors.

[0056] As indicated, typical vectors are replication-defective and lack the adenovirus E1 region. Therefore, it will be most convenient to introduce a polynucleotide encoding a gene of interest into the location where the E1 coding sequence has been removed. However, the insertion location of the construct within the adenovirus sequence is not critical. A polynucleotide encoding a gene of interest can also be inserted into an E3 replacement vector or helper cell line, or into the E4 region where the helper virus complements the E4 deficiency, replacing the deleted E3 region.

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

[0058] AAV DNA is 4.7 kilobases long. It contains two open reading frames flanked by two ITRs. The AAV genome contains two major genes, rep and cap. The rep gene encodes proteins involved in viral replication, while cap encodes the capsid proteins VP1-VP3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis-terminal components of AAV for chromosomal integration. Therefore, AAV can be used as a vector for delivery by removing all viral coding sequences and replacing them with gene cassettes. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their map locations. Transcription from p5 and p19 leads to the production of rep proteins, while transcription from p40 produces capsid proteins.

[0059] AAVs are prominent for use within the present disclosure due to their excellent safety profile and the fact that their capsids and genomes can be tailored to allow expression in selected cell populations. scAAV refers to self-complementary AAV. pAAV refers to plasmid adeno-associated virus. rAAV refers to recombinant adeno-associated virus.

[0060] Other viral vectors may be employed, such as those derived from viruses such as vaccinia virus, poliovirus, and herpesvirus, which offer several attractive features for a variety of mammalian cells.

[0061] Retroviruses are common tools for gene delivery. The term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear, double-stranded DNA copy and then covalently integrates the genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II, which induces the expression of RNA molecules encoding the structural proteins and enzymes required to produce new viral particles.

[0062] Exemplary retroviruses suitable for use in certain embodiments include: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0063] "Lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include: HIV (including human immunodeficiency virus, HIV types 1 and 2); visnamesi virus (VMV); capric arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) can be used.

[0064] Improved safety for the use of some vectors can be achieved by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used for this purpose include the simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate-early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to generate replication-competent virus due to the absence of the complete U3 sequence in the viral production system. In certain embodiments, heterologous promoters have the added advantage of controlling how the viral genome is transcribed. For example, the heterologous promoter can be inducible, so that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers can include one or more chemical compounds or physiological conditions under which the host cells are cultured, such as temperature or pH.

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

[0066] The "R region" refers to the region within a retroviral LTR that begins at the beginning of the capping sequence (i.e., at the beginning of transcription) and ends just before the beginning of the poly(A) tract. The R region is also defined as being adjacent to the U3 and U5 regions. The R region serves to allow the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

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

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

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

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

[0071] Particular 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 larger vector sequence, and can readily identify them based on the teachings of this disclosure (see Figure 12C). Nucleotides between the identifiable and listed subcomponents reflect restriction enzyme recognition sites used in construct assembly (cloning), and in some cases, additional nucleotides convey no discernible function. These segments of the complete vector sequence can be adjusted based on different cloning strategies and / or vector uses. Generally, short six-nucleotide palindromic sequences reflect vector construction artifacts that are not critical to vector function.

[0073] In certain embodiments, a vector (e.g., AAV) is selected that has a capsid that crosses the blood-brain barrier (BBB). In certain embodiments, the vector is modified to include a capsid that crosses the BBB. Examples of AAVs with viral capsids that can cross the blood-brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014;7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014;22(7):1299-1309), AAV1R6, AAV1R7 (Albright et al., Mol Ther. 2018;26(2):510), rAAVrh.8 (Yang, et al., supra), AAV-BR1 (Marchio et al., EMBO Mol Med. 2016;8(6):592), AAV-PHP.S (Chan et al., Nat Neurosci. 2017;20(8):1172), and AAV-PHP.B (Deverman et al., Nat Biotechnol. 2016;34(2):204), AAV-PPS (Chen et al., Nat Med. 2009;15:1215), and PHP.eB. In certain embodiments, the PHP.eB capsid differs from AAV9 and references AAV9, with the amino acids beginning at residue 586: S-AQ-A (SEQ ID NO:144) 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 BBB after intravenous injection. It transforms large portions of the central nervous system (CNS), thus enabling minimally invasive treatments, as described, for example, in connection with clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and CLN3 gene-associated neuronal ceroid-lipofusion syndrome (NCT03770572) (Naso et al., BioDrugs. 2017;31(4):317).

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

[0076] Two variants isolated from a library of chimeric AAV vectors, AAV1R6 and AAV1R7 (in which the AAV1 capsid domain was swapped with AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing significantly reduced liver and vascular endothelial transduction.

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

[0078] AAV-BR1 is an AAV2 variant displaying 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 the liver) (Korbelin et al., EMBO Mol Med. 2016;8(6):609).

[0079] AAV-PHP.S (Addgene, Watertown, MA) is a CREATE-generated variant of AAV9 that encodes the 7-mer sequence QAVRTSL (sequence number 147) and potently transduces neurons within the enteric nervous system and peripheral sensory afferents entering the spinal cord and brainstem.

[0080] AAV-PHP.B (Addgene, Watertown, MA) is a CREATE-generated variant of AAV9 that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 148). It transfers genes throughout the CNS more efficiently than AAV9, transducing a large proportion of astrocytes and neurons across multiple CNS regions.

[0081] AAV-PPS was created by inserting the DSPAHPS (SEQ ID NO: 149) epitope into the capsid of AAV2, and exhibits dramatically improved brain tropism compared to AAV2.

[0082] For more information regarding capsids crossing 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 (herein referred to as physiologically active components) of the present disclosure can be formulated with a carrier suitable for administration to cells, tissue slices, animals (e.g., mice, non-human primates), or humans. The physiologically active components in the compositions described herein can be prepared in neutral form, as a free base, or as a pharmacologically acceptable salt.

[0084] Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like, and salts formed with the free carboxyl groups are derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0085] Carriers for physiologically active ingredients can include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active ingredients is well known in the art. Except insofar as any conventional media or agent is incompatible with the physiologically active ingredient, it can be used with the compositions described herein.

[0086] The phrase "pharmaceutically acceptable carrier" refers to a carrier that does not produce an allergic or similar untoward reaction when administered to a human, and in certain embodiments, when administered intravenously (e.g., into the retroorbital plexus).

[0087] In certain embodiments, the compositions can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or intranasal inhalation, or for direct injection or application to one or more cells, tissues, or organs.

[0088] The composition may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles.

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

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

[0091] Injectable compositions may include sterile aqueous solutions, or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468). For injectable delivery, the form is sterile and fluid to the extent that it can be delivered by syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage and, optionally, contains one or more preservatives to protect against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and / or by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation includes isotonic agent(s), such as sugar(s) or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption into the composition, for example, aluminum monostearate and gelatin. The injectable composition can be suitably buffered, if necessary, and the liquid diluent can be first rendered isotonic with sufficient saline or glucose, for example.

[0092] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. As indicated, under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0093] Sterile compositions can be prepared by incorporating the physiologically active ingredient in an appropriate amount of a solvent containing other optional ingredients (e.g., as described above), followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized physiologically active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients (e.g., as described above). In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method can be vacuum drying and freeze-drying technology, which yields a powder of the physiologically active ingredient and any additional desired ingredients from the solution previously sterile-filtered.

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

[0095] Inhalable compositions can be delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. Capsules and cartridges, for example, of gelatin, for use in an inhaler or filler can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0096] Compositions also include microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Pat. No. 5,697,899).

[0097] Supplementary active ingredients can also be incorporated into the compositions.

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

[0099] In certain embodiments, for human administration, compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by the U.S. Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.

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

[0101] Although a variety of host cell lines can be used, in certain embodiments the cells are mammalian cells.In certain embodiments, the artificial expression constructs are selected from the group consisting of 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_606m, eHGT_607m, eHGT_608m, eHGT_609m, eHGT_610m, eHGT_611m, eHGT_612m, eHGT_613m, eHGT_614m, eHGT_615m, eHGT_616m, eHGT_617m, eHGT_618m, eHGT_619m, eHGT_620m, eHGT_621m, eHGT_622m, eHGT_623m, eHGT_624m, eHGT_625m, eHGT_626m, eHGT_627m, eHGT_628m, eHGT_629m, eHGT_630m, eHGT_631m, eHGT_632m, eHGT_633m, eHGT_634m, eHGT_635m, eHGT_636m, 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 and the cell line is a human, primate, or mouse cell line.Concatamers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core can also be used. Cell lines that can be transformed with the present disclosure include primary cell lines derived from living tissues such as rat or mouse brain, and organotypic cell cultures, including brain slices 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 multiple neuronal marker proteins in response to neuron growth factor (NGF). The PC12 cell line is considered a neuronal cell line and can also be used with the present disclosure. JAR cells (available from ATCC) are a platelet-derived cell line that express some neuronal genes, such as the serotonin transporter gene, and can be used with the embodiments described herein.

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

[0103] In certain embodiments, "neuronal" refers to something that is, relates to, or includes a nerve cell. A nerve cell is defined by the presence of an axon and a dendrite. The term "neuronal-specific" refers to activity that is found or occurs in a nerve cell or a cell derived from a nerve cell, but that is not found, occurs, or is substantially not found, or does not occur, in a non-neuronal cell or a cell not derived from a nerve cell, e.g., astrocytes or glial cells such as oligodendrocytes.

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

[0105] A method for differentiating stem cells into different cell types involves replacing stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenate), laminin, and polyornithine. A process for producing myelinating oligodendrocyte cells from stem cells is described in Hu et al., 2009, Nat. Protoc. 4:1614-22. 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 all-trans RA for 3 days. After subsequent culture in serum-free neuronal induction medium containing Neurobasal medium supplemented with B27, bFGF, and EGF, 95% of GABA neurons develop.

[0106] U.S. Publication No. 2012 / 0329714 describes the use of prolactin to increase neural stem cell numbers, and U.S. Publication No. 2012 / 0308530 describes culture surfaces with amino groups that promote neuronal differentiation into neurons, astrocytes, and oligodendrocytes. Thus, 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); isobutyl methylcellulose (BMP-2); 3-methylxanthine; leukemia inhibitory growth factor (LIF; U.S. Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (a glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transforming growth factor-α and TGF-β (U.S. Patent Nos. 5,851,832 and 5,753,506).

[0107] In certain embodiments, the yeast one-hybrid system is used to identify 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, eHGT_583m, and their core transcription factors may also be identified.

[0108] Transgenic animals are described below. Cell lines can also be derived from such transgenic animals. For example, primary tissue cultures from transgenic mice (e.g., as also described below) can provide cell lines that have artificial expression constructs already integrated into their genomes (see, e.g., MacKenzie and Quinn, Proc Natl Acad Sci USA 96:15251-15255, 1999).

[0109] (iv) Transgenic Animals. Another aspect of the present disclosure includes transgenic animals, the genome of which comprises one or more of the following: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_465m, eHGT_466m, eHGT_467m, eHGT_468m, eHGT_469m, eHGT_470m, eHGT_471m, eHGT_472m, eHGT_473m, eHGT_474m, eHGT_475m, eHGT_476m, eHGT_477m, eHGT_478m, eHGT_479m, eHGT_480m, eHGT_481m, eHGT_482m, eHGT_483m, eHGT_484m, eHGT_485m, eHGT_486m, eHGT_487m, eHGT_488m, eHGT_489m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT_494m, eHGT_495m, eHGT_496m, eHGT_497m, eHGT_498m, eHGT_499m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT , 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 Concatamers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer cores can also be used.In certain embodiments, the genome of the transgenic animal is selected from the group consisting of CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN2411, CN2412, CN2413, CN2414, CN2415, CN2416, CN2417, CN2418, CN2419, CN2510, CN2511, CN2512, CN2513, CN2514, CN2515, CN2516, CN2517, CN2518, CN2519, CN2620, CN2621, CN2622, CN2623, CN2624, CN2625, CN2626, CN2627, CN2628, CN2629, CN2730, CN2731, CN2732, CN2733, CN2734, CN2735, CN2736, CN2737, CN2738, CN2739, CN2740, CN2741, CN2742, CN2743, CN2744, CN2745, CN2746, CN2747, CN2748, CN2 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 a non-integrating vector is utilized, the transgenic animal contains in one or more cells thereof the artificial expression constructs eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_465m, eHGT_466m, eHGT_467m, eHGT_468m, eHGT_469m, eHGT_470m, eHGT_471m, eHGT_472m, eHGT_473m, eHGT_474m, eHGT_475m, eHGT_476m, eHGT_477m, eHGT_478m, eHGT_479m, eHGT_480m, eHGT_481m, eHGT_482m, eHGT_483m, eHGT_484m, eHGT_485m, eHGT_486m, eHGT_487m, eHGT_488m, eHGT_489m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT_494m, eHGT_495m, eHGT_496m, eHGT_497m, eHGT_498m, eHGT_499m, eHGT_500m, eHGT_501m, eHGT_502m, eHGT_503m, eHGT_504m, eHGT_505m, eHGT_506m, eHGT_507m, eHGT_ 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 including 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.Concatamers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer cores can also be used.

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

[0111] In certain embodiments, the construction of transgenic animals results in organisms with genetically engineered constructs present in all cells within the same genomic integration site.Therefore, cell lines derived from such transgenic animals will be as consistent as if the genetically engineered constructs were present in all cells at the same genomic integration site, and therefore will undergo the same position effect mutations.In contrast, introducing genes into cell lines or primary cell cultures can result in heterologous expression of constructs.The disadvantage of this approach is that the expression of introduced DNA can be affected by the specific genetic background of the host animal.

[0112] As noted above in connection with cell lines, the artificial expression constructs of the present disclosure can be used to genetically modify mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression construct is introduced into cultured mouse embryonic stem cells. Transformed ES cells are then injected into a blastocyst from a host mother, and the host embryo is reimplanted into the mother. This results in chimeric mice whose tissues are composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Typically, the mice from which the cultured ES cells used for transfer are derived are selected to have a coat color different from that of the host mouse whose embryo is injected with the transformed cells. The chimeric mice then have a variety of coat colors. The chimeric mice are bred with an appropriate strain to produce offspring carrying the transgene, as long as at least some of their germline tissue is derived from the genetically modified cells.

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

[0114] (v) Methods of Use. In certain embodiments, compositions comprising physiologically active ingredients described herein are administered to a subject to produce a physiological effect.

[0115] In certain embodiments, the present disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a heterologous gene partially or completely encoded at a position downstream of an enhancer in an engineered sequence. Accordingly, provided herein are methods of using the disclosed artificial expression constructs in the research, clinical trials, and potential development of pharmaceuticals to prevent, treat, or alleviate symptoms of a disease, dysfunction, or disorder.

[0116] Certain embodiments are directed to 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_606m, eHGT_607m, eHGT_608m, eHGT_609m, eHGT_610m, eHGT_611m, eHGT_612m, eHGT_613m, eHGT_614m, eHGT_615m, eHGT_616m, eHGT_617m, eHGT_618m, eHGT_619m, eHGT_620m, eHGT_621m, eHGT_622m, eHGT_623m, eHGT_624m, eHGT_625m, eHGT_626m, eHGT_627m, eHGT_628m, eHGT_629m, eHGT_630m, eHGT_631m, eHGT_632m, eHGT_633m, eHGT_634m, eHGT_635m, eHGT_636m, eHGT_637m, eH 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 , 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 subject to drive selective expression of a gene in a selected cell type.Concatamers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer cores 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 arts, the dosage for any one subject will depend on many factors, including the subject's size, surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. Doses of compounds of the present disclosure will vary, but in certain embodiments, the dosage will be approximately 10 times the dosage of an artificial expression construct of the present disclosure. 5 ~10 100 In certain embodiments, patients receiving intravenous, intraparenchymal, intraspinal, retro-orbital, or intrathecal administration may receive 10 6 ~10 22 A copy of the artificial expression construct can be injected.

[0118] An "effective amount" is the amount of a composition required to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amounts disclosed herein may produce statistically significant effects in animal models or in vitro assays.

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

[0120] In certain circumstances, it may be desirable to deliver the artificial expression construct in an appropriately formulated composition as disclosed herein either by pipette, retro-orbital injection, subcutaneous, intraocular, intravitreal, parenteral, subcutaneous, intravenous, intraparenchymal, intracerebroventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or by direct application or injection into one or more cells, tissues, or organs. Methods of administration may also include those forms described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363.

[0121] (vi) Kits and Commercial Packages. Kits and commercial packages contain the artificial expression constructs described herein. The artificial expression constructs can be sequestered. In certain embodiments, components of the expression product can be sequestered from one another. In certain embodiments, the expression product can be in a vector, a viral vector, a cell, a tissue slice or sample, and / or a transgenic animal. Such kits can further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or a means of delivery of the composition, such as a syringe, injection, etc.

[0122] Kit or commercial package embodiments also include instructions for use of the included components, for example, in basic research, electrophysiological studies, neuroanatomical studies, and / or in the study and / or treatment of disorders, diseases, or conditions.

[0123] The following exemplary embodiments are included to demonstrate specific embodiments of the present disclosure. Those of ordinary skill in the art will, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0124] (vii) Exemplary embodiments. 1. The linking core of the enhancer disclosed herein. 2. Concatenated core of eHGT_459m, eHGT_453m, eHGT_462m, eHGT_461m, eHGT_464m, eHGT_452m, eHGT_451m, or eHGT_460m enhancer. 3. The linked core of embodiment 1 or 2, wherein the linked core comprises the sequence set forth in 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, and / or SEQ ID NO:52. 4. The tethered core of embodiment 1 or 2, wherein said tethered core comprises the sequence set forth in 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. 5. The linked core of any of embodiments 1-4, wherein said linked core comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of said enhancer core. 6. The linked core of embodiment 5, comprising 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, and / or SEQ ID NO:52. 7. The linked core of embodiment 5, comprising 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 SEQ ID NO:45; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:46; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:47; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:48; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:49; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:50; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 51; or 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 52; The linked core of embodiment 5, comprising: 9. The linked core of embodiment 5, comprising 3 copies of SEQ ID NO:45; 3 copies of SEQ ID NO:46; 3 copies of SEQ ID NO:47; 3 copies of SEQ ID NO:48; 3 copies of SEQ ID NO:49; 3 copies of SEQ ID NO:50; 3 copies of SEQ ID NO:51; or 3 copies of SEQ ID NO:52. 10. The linked core of embodiment 5, wherein said linked core comprises 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 eHGT_662m, and eHGT_583m. 12. An artificial expression construct comprising: (i) an enhancer or linked core thereof according to any of embodiments 1 to 11; (ii) a promoter; and (iii) a heterologous coding sequence. 13. The artificial expression construct of embodiment 12, wherein the heterologous coding sequence encodes an effector element or an expressible element. 14. The artificial expression construct of embodiment 13, wherein the effector element comprises a reporter protein or functional molecule. 15. The artificial expression construct of embodiment 14, wherein the reporter protein comprises a fluorescent protein. 16. The artificial expression construct of embodiment 13 or 14, wherein said functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, designer receptor exclusively activated by designer drugs (DREADD), growth factor, or pro-survival gene. 17. An artificial expression construct according to any one of embodiments 13 to 16, wherein the expressible element comprises a non-functional molecule. 18. The artificial expression construct of embodiment 17, wherein said non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, DREADD, growth factor, or pro-survival gene. 19. An 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 of 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 comprises or encodes a skipping element. 22. The artificial expression construct of embodiment 21, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 23. The artificial expression construct of embodiment 22, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 24. The artificial expression construct is: 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 * 24. An artificial expression construct according to any one of embodiments 12 to 23, comprising or encoding a set of features selected from: a fluorescent protein (e.g., EGFP, SYFP, SYFP2, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10, WPRE, WPRE3, and / or BGHpA. 25. The artificial expression construct comprises: eHGT_450m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_451m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_452m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_453m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_457m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_458m-minBglobin-heterologous coding sequence-W PRE3-BGHpA;eHGT_459m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_460m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_462m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_464m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_461m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_463m-minBglobin-heterologous coding sequence-WPRE3 -BGHpA;eHGT_692h-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_693h-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_694h-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_695h-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_667m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_668m-minBglobin-heterologous coding sequence-WPRE3-BG HpA;eHGT_696m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_455m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_457h-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_603m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_604m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_605m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_661m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_766m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_767m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_768m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_663m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_660m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_665m-minB globin-heterologous coding sequence-WPRE3-BGHpA;eHGT_700m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_456m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_698m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_699m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_647m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_648m-minBglobin-heterologous coding sequence WPRE3-BGHpA;eHGT_649m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_670m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_697m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_662m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_583m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_4 59m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_453m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_462m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_461m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_464m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_452m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_451m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_460m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_450m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_451m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_452m-minimal promoter ter-heterologous coding sequence-WPRE3-BGHpA; eHGT_453m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_457m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_458m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_459m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_460m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_462m-minimal promoter-heterologous coding Sequence-WPRE3-BGHpA;eHGT_464m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_461m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_463m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_692h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_693h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_694h-minimal promoter-heterologous coding sequence-WPRE 3-BGHpA;eHGT_695h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_667m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_668m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_696m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_455m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_457h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_603m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_604m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_605m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_661m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_766m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_767m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_ 768m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_663m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_660m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_665m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_700m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_456m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_698m-minimal small-promoter-heterologous-coding-sequence-WPRE3-BGHpA;eHGT_699m-minimal-promoter-heterologous-coding-sequence-WPRE3-BGHpA;eHGT_647m-minimal-promoter-heterologous-coding-sequence-WPRE3-BGHpA;eHGT_648m-minimal-promoter-heterologous-coding-sequence-WPRE3-BGHpA;eHGT_649m-minimal-promoter-heterologous-coding-sequence-WPRE3-BGHpA;eHGT_670m-minimal-promoter-heterologous-coding-sequence-WPRE3-BGHpA;eHGT_697m-minimal-promoter -heterologous coding sequence-WPRE3-BGHpA;eHGT_662m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_583m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_459m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_453m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_462m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;25. An artificial expression construct according to any of embodiments 12 to 24, comprising or encoding a set of features selected from: 3xcore2_eHGT_461m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_464m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_452m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_451m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; or 3xcore2_eHGT_460m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA. 26. A vector comprising an artificial expression construct according to any one of embodiments 12 to 25. 27. The vector of embodiment 26, wherein the vector comprises a viral vector. 28. The vector of embodiment 26 or 27, wherein the viral vector comprises a recombinant adeno-associated viral (AAV) vector. 29. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence comprises a promoter and one of: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_465m, eHGT_466m, eHGT_467m, eHGT_468m, eHGT_469m, eHGT_470m, eHGT_471m, eHGT_472m, eHGT_473m, eHGT_474m, eHGT_475m, eHGT_476m, eHGT_477m, eHGT_478m, eHGT_479m, eHGT_480m, eHGT_481m, eHGT_482m, eHGT_483m, eHGT_484m, eHGT_485m, eHGT_486m, eHGT_487m, eHGT_488m, eHGT_489m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT_494m, eHGT_495m, eHGT_496m, eHGT_497m, eHGT_498m, eHGT_499m, eHGT_499m, eHGT_500m, eHGT_501m, eHGT_502m, eHGT_503m, eHGT_504m, eHGT_505m, eH _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 is 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. A transgenic cell comprising an expression construct or vector according to any of the preceding embodiments. 31. The transgenic cell of embodiment 30, wherein said transgenic cell is an L5 glutamatergic neuron. 32. The transgenic cell of embodiment 31, wherein said L5 glutamatergic neuron is an L5 ET neuron. 33. A non-human transgenic animal comprising an artificial expression construct, vector, or transgenic cell according to any of the preceding embodiments. 34. The non-human transgenic animal of embodiment 33, wherein said non-human transgenic animal is a mouse or a non-human primate. 35. An administrable composition comprising an expression construct, vector, or transgenic cell according to any of the preceding embodiments. 36. A kit comprising an artificial expression construct, vector, transgenic cell, transgenic animal, and / or administrable composition according to any of the preceding embodiments. 37. A method for selectively expressing a heterologous gene in a cell population in vivo or in vitro, comprising providing to a sample or subject containing the cell population an administrable composition of embodiment 35 in a sufficient dosage and for a sufficient period of time, thereby selectively expressing said gene in the cell population. 38. The method of embodiment 37, wherein the heterologous gene encodes an effector element or an expressible element. 39. The method of embodiment 38, wherein the effector element comprises a reporter protein or a functional molecule. 40. The method of embodiment 39, wherein the reporter protein comprises a fluorescent protein. 41. The method of embodiment 39 or 40, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, DREADD, growth factor, or pro-survival gene. 42. The method of any of embodiments 37-41, wherein the expressible element comprises a non-functional molecule. 43. The method of embodiment 42, wherein the nonfunctional molecule comprises a nonfunctional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, DREADD, growth factor, or pro-survival gene. 44. The method of any one of embodiments 37-43, wherein said providing comprises pipetting. 45. The method of embodiment 44, wherein the pipetting is performed on a brain slice. 46. ​​The method of embodiment 45, wherein the brain slice comprises L5 ET neurons. 47. The method of embodiment 45 or 46, wherein the brain slice is mouse, human, or non-human primate. 48. The method of any one of embodiments 37-43, wherein said providing comprises administering to a living organism. 49. The method of embodiment 48, wherein the organism is a human, a non-human primate, or a mouse. 50. The method of embodiment 48 or 49, wherein the administration to the organism is by injection. 51. The method of embodiment 50, wherein said injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection. 52. The method of any one of embodiments 48 to 51, wherein the organism has a neurodegenerative disease. 53. The method of any of embodiments 48-52, 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 , 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 Also included are variants of the sequences disclosed and referenced herein. Computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software, can be used to guide determining which amino acid residues can be substituted, inserted, or deleted without disrupting biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains.

[0126] Suitable conservative substitutions of amino acids in a peptide or protein are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families as follows: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, nonpolar residues): isoleucine (I Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (minor aliphatic, nonpolar, or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0127] In making such changes, the hydrophilicity index of amino acids may be taken into consideration. The importance of the hydrophilic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are as follows: Ile (+4.5), Val (+4.2), Leu (+3.8), Phe (+2.8), Cys (+2.5), Met (+1.9), Ala (+1.8), Gly (-0.4), Thr (-0.7), Ser (-0.8), Trp (-0.9), Tyr (-1.3), Pro (-1.6), His (-3.2), glutamate (-3.5), Gln (-3.5), aspartate (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5).

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

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

[0130] As discussed above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0131] As noted elsewhere, variants of a gene sequence can include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of the encoded product to a statistically significant extent.

[0132] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to a protein, nucleic acid, or gene sequence disclosed herein.

[0133] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated using known methods, see, for example, Computational Molecular Biology (Lesk, A. M. ed.), Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. Wed.), Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G. ed.), Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G. ed.), Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. ed.), Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignment and percent identity calculations may be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY = 10, GAP LENGTH PENALTY = 10).Related programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin), and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New Within the context of this disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" refers to any set of values ​​or parameters that are initially loaded into the software when first initialized.

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

[0135] The term "ligation" is used broadly to describe joining together into a chain or series. It is used to describe joining nucleotide or amino acid sequences into a single nucleotide or amino acid sequence, respectively. The terms "concatamerize" and "concatenate" can be used interchangeably.

[0136] As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its specifically described element, step, ingredient, or component. Accordingly, the terms "include" or "including" should be interpreted as describing "comprising, consisting of, or essentially consisting of." The transitional terms "comprise" or "comprises" mean to allow for the inclusion of unspecified elements, steps, ingredients, or components, even if in major amounts, without limitation. The transitional phrase "consisting of" excludes any unspecified element, step, ingredient, or component. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified element, step, ingredient, or component, and those that do not have a substantial effect on the embodiment. A substantial effect would cause a statistically significant decrease in selective expression in L5 glutamatergic neurons, such as L5 ET neurons.

[0137] In certain embodiments, artificial means means not occurring in nature.

[0138] Unless otherwise indicated, all numbers used in the specification and claims expressing properties such as quantities of ingredients, molecular weights, reaction conditions, and the like, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of equivalence principles to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where further clarity is needed, the term "about" has the meaning reasonably assigned by a person of ordinary skill in the art to a stated value or range, i.e., ±20% of the stated value, ±19% of the stated value, ±18% of the stated value, ±17% of the stated value, ±16% of the stated value, ±15% of the stated value, ±14% of the stated value, ±13% of the stated value, ±12% of the stated value, ±11% of the stated value, ±10% of the stated value, ±9% of the stated value, ±8% of the stated value, ±7% of the stated value, ±6% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1% of the stated value.

[0139] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0140] As used in the context of describing the present invention (particularly in the context of the claims below), "a," "an," "the," and similar referents should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0141] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the modified group, thereby satisfying the description of all recited Markush groups used in the appended claims.

[0142] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect skilled artisans to adopt such variations as necessary, and the inventors intend the invention to be practiced other than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

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

[0144] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0145] The details set forth herein are presented by way of example only for illustrative purposes of discussion of preferred embodiments of the present invention, and to provide what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt is made to show structural details of the present invention, but rather an attempt is made to provide a basic understanding of the present invention and, taken together with the drawings and / or examples, to make clear to those skilled in the art how some forms of the present invention may be embodied in practice.

[0146] The definitions and explanations used in this disclosure are meant and intended to control in any future interpretations unless clearly and unambiguously modified in the examples below, or unless application of the meaning would render any interpretation meaningless or essentially meaningless. If interpretation of a term renders it meaningless or essentially meaningless, the definition should be taken from a dictionary known to those skilled in the art, such as Webster's Dictionary, 3rd Edition, or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. 1. An artificial expression construct comprising: (i) eHGT_453m, eHGT_450m, eHGT_451m, eHGT_452m, 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, eH GT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eH GT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, e HGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcor 3xcore2_eHGT_451m, 3xcore2_eHGT_460m; (ii) an enhancer selected from 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, or 3xcore2_eHGT_460m; (iii) a promoter; and (iv) a heterologous coding sequence.

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

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

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

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

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

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

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

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

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

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

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

13. wherein said artificial expression constructs are: eHGT_453m, eHGT_450m, eHGT_451m, eHGT_452m, 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, eHG T_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, e HGT_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, 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 ＊ , a fluorescent protein (e.g., EGFP, SYFP, SYFP2, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10, WPRE, WPRE3, and / or BGHpA, and / or a set of features selected from the group consisting of:

14. The artificial expression constructs are: eHGT_453m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_450m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_451m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_452m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_457m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_458m-minBglobin-heterologous coding sequence-WPR E3-BGHpA; eHGT_459m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_460m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_462m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_464m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_461m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_463m-minBglobin-heterologous coding sequence-WPRE3- BGHpA; eHGT_692h-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_693h-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_694h-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_695h-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_667m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_668m-minBglobin-heterologous coding sequence-WPRE3-BGH pA; eHGT_696m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_455m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_457h-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_603m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_604m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_605m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;eHGT_661m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_766m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_767m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_768m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_663m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_660m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_665m-minB globin-heterologous coding sequence-WPRE3-BGHpA; eHGT_700m-minB globin-heterologous coding sequence-WPRE3-BGHpA; eHGT_456m-minB globin-heterologous coding sequence-WPRE3-BGHpA; eHGT_698m-minB globin-heterologous coding sequence-WPRE3-BGHpA; eHGT_699m-minB globin-heterologous coding sequence-WPRE3-BGHpA; eHGT_647m-minB globin-heterologous coding sequence-WPRE3-BGHpA; eHGT_648m-minB globin-heterologous coding sequence WPRE3-BGHpA; eHGT_649m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_670m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_697m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_662m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_583m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_4 59m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_453m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_462m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_461m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_464m-minBglobin-heterologous coding sequence-WPRE3-BGHpA;3xcore2_eHGT_452m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_451m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_460m-minBglobin-heterologous coding sequence-WPRE3-BGHpA; eHGT_453m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_450m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_451m-minimal promoter eHGT_452m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_457m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_458m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_459m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_460m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_462m-minimal promoter-heterologous coding Sequence-WPRE3-BGHpA; eHGT_464m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_461m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_463m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_692h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_693h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_694h-minimal promoter-heterologous coding sequence-WPRE eHGT_695h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_667m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_668m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_696m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_455m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_457h-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;eHGT_603m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_604m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_605m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_661m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_766m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_767m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_768m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_663m-minimal promoter-heterologous coding sequence-WPRE3 -BGHpA; eHGT_660m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_665m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_700m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_456m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_698m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_699m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_647m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_648m-minimal promoter-heterologous coding sequence WPRE3-BGHpA; eHGT_649m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_670m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_697m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_662m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; eHGT_5 83m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_459m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_453m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_462m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA;2. The artificial expression construct of claim 1, comprising or encoding a set of features selected from: 3xcore2_eHGT_461m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_464m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_452m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_451m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA; or 3xcore2_eHGT_460m-minimal promoter-heterologous coding sequence-WPRE3-BGHpA.

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

16. The vector of claim 15 , wherein the vector comprises a viral vector.

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

18. 1. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, said heterologous coding sequence comprising a promoter and one of: eHGT_453m, eHGT_450m, eHGT_451m, eHGT_452m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_465m, eHGT_466m, eHGT_467m, eHGT_468m, eHGT_469m, eHGT_470m, eHGT_471m, eHGT_472m, eHGT_473m, eHGT_474m, eHGT_475m, eHGT_476m, eHGT_477m, eHGT_478m, eHGT_479m, eHGT_480m, eHGT_481m, eHGT_482m, eHGT_483m, eHGT_484m, eHGT_485m, eHGT_486m, eHGT_487m, eHGT_488m, eHGT_489m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT_494m, eHGT_495m, eHGT_496m, eHGT_497m, eHGT_498m, eHGT_499m, eHGT_490m, eHGT_491m, eHGT_492m, eHGT_493m, eHGT_494m, eHGT_495m, eHGT_496m, eHGT_ 61m, 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_647 m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcore2_eHGT_459m, 3xco The adeno-associated virus (AAV) vector is 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.

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

20. 20. The transgenic cell of claim 19, wherein the transgenic cell is an L5 ET neuron.

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

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

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

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

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

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

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

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

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

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

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

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

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

34. 34. The method of claim 33, wherein the brain slice comprises L5 ET neurons.

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

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

37. 37. The method of claim 36, wherein the organism is a human, a non-human primate, or a mouse.

38. 37. The method of claim 36, wherein the administration to the living body is by injection.

39. 39. The method of claim 38, wherein the injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection.

40. 37. The method of claim 36, wherein the living organism has a neurodegenerative disease.

41. 37. The method of claim 36, wherein the living organism has amyotrophic lateral sclerosis (ALS) or multiple sclerosis.

42. CN2251, CN2248, CN2249, CN2250, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690 , CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, 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, or CN3024.

43. The linking core of the eHGT_459m, eHGT_453m, eHGT_462m, eHGT_461m, eHGT_464m, eHGT_452m, eHGT_451m, or eHGT_460m enhancer.

44. 44. The linked core of claim 43, wherein the linked core has the sequence set forth in 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.

45. 44. The linked core of claim 43, wherein the linked core has 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the enhancer core.

46. 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 45; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 46, SEQ ID NO: 47, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 48; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:49; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 50; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 51, or 44. The linked core of claim 43 having 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO:

52.

47. The linked core of claim 43 having three copies of SEQ ID NO: 45; three copies of SEQ ID NO: 46; three copies of SEQ ID NO: 47; three copies of SEQ ID NO: 48; three copies of SEQ ID NO: 49; three copies of SEQ ID NO: 50; three copies of SEQ ID NO: 51; or three copies of SEQ ID NO:

52.

48. The linking core has the sequence set forth in 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. The linked core according to claim 43.

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