Artificial expression constructs that selectively regulate gene expression in inhibitory neocortical neurons
Artificial expression constructs with enhancer elements like eHGT_089h and eHGT_064h address the limitations of recombinase driver strains by providing cost-effective and specific gene regulation in inhibitory neocortical GABAergic neurons, enhancing expression levels and applicability to humans.
Patent Information
- Application Number
- JP2025181548
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2020-06-04
- Filing Date
- 2025-10-28
- Publication Date
- 2026-08-25
- Estimated Expiration
- 2041-06-04
Smart Images
Figure 0007911131000011 
Figure 0007911131000012 
Figure 0007911131000013
Abstract
Description
[Technical Field]
[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 034,794, filed on 4 June 2020, and its entire contents are incorporated herein by reference as if they were fully described herein.
[0002] Statements relating to research or development funded by the federal government This invention was completed with the support of the United States Government under grants MH114126 and MH121274 from the National Institute of Health. The United States Government has certain rights to this invention.
[0003] References regarding sequence listings The sequence listing relating to this application is provided in text format instead of on paper, and is incorporated herein by reference as constituting a part of this specification. The name of the text file containing the sequence listing is A166-0023PCT_ST25.txt. This text file is 15.1 MB in size, was created on June 4, 2021, and was submitted electronically via EFS-Web.
[0004] Areas of disclosure This disclosure provides artificial expression constructs for selectively regulating gene expression in selected central nervous system cell types. These artificial expression constructs can be used to selectively express or modify the gene expression of synthetic genes in inhibitory neocortical GABAergic neurons, including somatostatin GABAergic neurons, parvalbumin GABAergic neurons, vasoactive enteropeptide GABAergic neurons, and Lamp5 GABAergic neurons, as well as in some examples astrocytes. [Background technology]
[0005] Background of Disclosure To fully understand brain biology, it is necessary to distinguish and define different cell types, and to further study them, to identify artificial expression constructs that can selectively label and disrupt them. In mice, recombinase driver strains have proven highly effective in labeling cell populations that share marker gene expression. However, the creation, maintenance, and use of such strains that label cell types with high specificity are expensive and often require triple transgenic crosses, resulting in a low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans. [Overview of the project]
[0006] Summary of Disclosure This disclosure provides artificial expression constructs that selectively drive gene expression in target central nervous system cell populations. Target central nervous system cell populations include inhibitory neocortical GABAergic neurons, including somatostatin (Sst) GABAergic neurons, parvalbumin (Pvalb) GABAergic neurons, vasoactive enteropeptide (Vip) GABAergic neurons, and Lamp5 GABAergic neurons.
[0007] Certain embodiments of the artificial expression construct utilize the following enhancers to selectively drive protein expression within target central nervous system cell populations (enhancer(s) / target cell populations) such as: eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_340m, eHGT_528h, eHGT_515h, eHGT_226h, eHGT_170h, eHGT_519h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, and eHGT_156h / Sst GABAergic neurons: eHGT_076h, eHGT_759m, and eHGT_064h / Pvalb / Sst; GABAergic neurons: eHGT_072h, eHGT_131hv1, eHGT_131hv2, and eHGT_130h / Pvalb; GABAergic neurons: eHGT_354h, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_107h, MGT_E81, MGT_E85, MGT_E88, and MGT_E83 / Vip GABAergic neurons; MGT_E36, MGT_E37, and MGT_E41 / Lamp5_Lhx6 GABAergic neurons; eHGT_354m, eHGT_060m, and eHGT_060h / VIP GABAergic neurons and astrocytes; eHGT_025h, eHGT_096h, eHGT_098h, and eHGT_104m / Lamp5 GABAergic neurons; eHGT_682h, eHGT_600m, eHGT_468m, eHGT_338m, eHGT_341m, and eHGT_339m / Sst and Choldl GABAergic neurons.
[0008] In certain embodiments, the artificial enhancer element includes a ligated core of the enhancer. Examples include the ligated cores of eHGT_226h and / or eHGT_064h. These artificial enhancer elements can result in higher levels of transgene expression and faster initiation of expression compared to a single full-length original (native) enhancer.
[0009] In certain embodiments, the enhancer core includes the sequence described in any one of SEQ ID NOs: 161, 163, and 165. In certain embodiments, these cores are concatenated and also have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. SEQ ID NOs: 162, 164, and 166 provide concatemers of three selected enhancer cores.
[0010] Certain embodiments of the artificial expression construct utilize 3xcore2_eHGT_226h and / or 3xcore3_eHGT_226h to selectively drive protein expression in Sst GABAergic neurons, and / or utilize 3xcore_eHGT_064h to selectively drive protein expression in Pvalb / Sst GABAergic neurons.
[0011] Specific embodiments include vectors: CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567, CN1626, CN1712, CN1700, CN1607, CN1605, CN1556, CN1526, CN1418, CN1404, AiV1173, AiV1174, AiV1177, CN1261, CN1542, CN1544, CN1598, CN1553, CN The present invention provides artificial expression constructs that include the characteristics of vectors described herein, such as CN2367, CN2357, CN2568, CN2569, CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309, CN2366, CN2257, CN1667, CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, and AiP1273.
[0012] Many of the drawings submitted herein are better understood through color. The applicant considers the colored drawings to be part of the original application documents and reserves the right to present images of the colored drawings by following the procedures described below. [Brief explanation of the drawing]
[0013] [Figure 1]This is an outline of enhancer discovery for viral tools. To construct cell type-specific labeling tools, cells derived from adult mouse cortex were isolated, and single-cell assays were performed on transposase-accessible chromatin using sequencing (scATAC-seq). Samples were clustered, and clusters could be identified by comparison with single-cell RNA sequencing (scRNA-seq) datasets. Single cells matching the same transcriptome type were then pooled, and the genomes were searched for type-specific putative enhancers. These regions were cloned upstream of the minimal promoter in the AAV genome backbone, which was used to generate self-complementary adeno-associated virus vectors (scAAV) or recombinant adeno-associated virus vectors (rAAV). These viral tools were delivered posterior orbital to label specific neocortical glutamatergic and GABAergic neuron populations. In cells with matching cell types, the enhancers recruit their homologous transcription factors to drive cell type-specific expression. In other cells, the viral genome is present, but the transcript is not expressed. [Figure 2-1]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 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 Chrna2 Glra3, 42 Sst Myh8 Fibin, 40 Sst Chrna2 Ptgdr, 39 Sst Tac2 Myh4, 37 Sst Hpse Sema3c, 36 Sst Hpse Cbln4, 34 Sst Crhr2 Efem1, 33 Sst Crh 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 Sema3e Kank4, 14 Pvalb Gpr149 Islr, 11 Pvalb Reln Itm2a, 10 Pvalb Tac1, 9 Pvalb Tpbg, 4 Pvalb Vipr2, Meis2 Adamts19, 170 Astro Aqp4, 171 OPC Pdgfr Grm5, Oligo Serpinb1a, 174 Oligo Synpr, VLMC Osr1 Cd74, VLMC Osr1 Mc5r, VLMC Spp1 Col15a1, Peri Kcnj8, SMC Acta2, Endo Ctla2a, and 181 MicrogliaSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 2-2]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 Kcne4, 58 Vip Col15a1 Pde1a, 54 Sst Chodl, 53 Sst Mme Fam114a1, 52 Sst Tac1 Htr1d, 5 &emSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 2-3]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 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 Chrna2 Glra3, 42 Sst Myh8 Fibin, 40 Sst Chrna2 Ptgdr, 39 Sst Tac2 Myh4, 37 Sst Hpse Sema3c, 36 Sst Hpse Cbln4, 34 Sst Crhr2 Efem1, 33 Sst Crh 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 Sema3e Kank4, 14 Pvalb Gpr149 Islr, 11 Pvalb Reln Itm2a, 10 Pvalb Tac1, 9 Pvalb Tpbg, 4 Pvalb Vipr2, Meis2 Adamts19, 170 Astro Aqp4, 171 OPC Pdgfr Grm5, Oligo Serpinb1a, 174 Oligo Synpr, VLMC Osr1 Cd74, VLMC Osr1 Mc5r, VLMC Spp1 Col15a1, Peri Kcnj8, SMC Acta2, Endo Ctla2a, and 181 MicrogliaSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 2-4]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 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 Chrna2 Glra3, 42 Sst Myh8 Fibin, 40 Sst Chrna2 Ptgdr, 39 Sst Tac2 Myh4, 37 Sst Hpse Sema3c, 36 Sst Hpse Cbln4, 34 Sst Crhr2 Efem1, 33 Sst Crh 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 Sema3e Kank4, 14 Pvalb Gpr149 Islr, 11 Pvalb Reln Itm2a, 10 Pvalb Tac1, 9 Pvalb Tpbg, 4 Pvalb Vipr2, Meis2 Adamts19, 170 Astro Aqp4, 171 OPC Pdgfr Grm5, Oligo Serpinb1a, 174 Oligo Synpr, VLMC Osr1 Cd7, VLMC Osr1 Mc5r, VLMC Spp1 Col15a1, Peri Kcnj8, SMC Acta2, Endo Ctla2a, and 181 MicrogliaSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 2-5]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 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 Chrna2 Glra3, 42 Sst Myh8 Fibin, 40 Sst Chrna2 Ptgdr, 39 Sst Tac2 Myh4, 37 Sst Hpse Sema3c, 36 Sst Hpse Cbln4, 34 Sst Crhr2 Efem1, 33 Sst Crh 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 Sema3e Kank4, 14 Pvalb Gpr149 Islr, 11 Pvalb Reln Itm2a, 10 Pvalb Tac1, 9 Pvalb Tpbg, 4 Pvalb Vipr2, Meis2 Adamts19, 170 Astro Aqp4, 171 OPC Pdgfr Grm5, Oligo Serpinb1a, 174 Oligo Synpr, VLMC Osr1 Cd74, VLMC Osr1 Mc5r, VLMC Spp1 Col15a1, Peri Kcnj8, SMC Acta2, Endo Ctla2a, and 181 MicrogliaSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 2-6]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 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 Chrna2 Glra3, 42 Sst Myh8 Fibin, 40 Sst Chrna2 Ptgdr, 39 Sst Tac2 Myh4, 37 Sst Hpse Sema3c, 36 Sst Hpse Cbln4, 34 Sst Crhr2 Efem1, 33 Sst Crh 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 CalbSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 2-7]Vector: CN2039 and enhancer: eHGT_354h are shown. (A~C) Animal: 554255. This is a fluorescence montage of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2039 virus packaged in a PHP.eB capsid. (D~E) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced by CN2039 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. This shows SYFP fluorescence (D) and markers of GABAergic cell subclasses (Sst (E) and Vip (F)) mRNA imaged by mFISH. The image is a montage. (G~I) Animal: 554255 and region: VISp. Mouse visual cortex (VISp) transduced with CN2039 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (G), SYFP fluorescence and VIP mFISH (H), and SYFP fluorescence only (I) are shown. The image is a montage. Quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping SYFP+ cells (blue circles with a small triangle in the upper right corner) is shown below. (J) Animal: 554255 and region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from VISp in the mouse cortex after post-orbital injection of CN2039 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data shows that eHGT_354h enhancer-driven reporter expression selectively occurs in VIP+ cells when VISp is evaluated. The letters at the bottom, from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmat, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISpWhrn Tox2、160 L5 IT VISp Batf3、158 L5 IT VISp Col6a1 Fezf2、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 C1qI2 Ptgfr、141 L5 PT VISp C1qI2 Cdh13、140 L5 PT VISp Krt80、134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L6 CT Nxph2 Sla, 130 L6 CT VISp Krt80 Sla, L6 CT VISp Nxph2 Vls, 127 L6 CT VISp Ctxn3 Brinp3, 126 L6 CT VISp Ctxn3 Sla, 122 L6 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, Lamp5 Fam19a1 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 Mab21I1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4Rxfp1 Chat, 71 Vip Lect1 Oxtr, 70 Vip Rspo1 Itga4, 67 Vip Chat Htr1f, 66 Vip Pygm C1qI1, 61 Vip Crispld2 Htr2c, 60 Vip Crispld2 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 Chrna2 Glra3, 42 Sst Myh8 Fibin, 40 Sst Chrna2 Ptgdr, 39 Sst Tac2 Myh4, 37 Sst Hpse Sema3c, 36 Sst Hpse Cbln4, 34 Sst Crhr2 Efem1, 33 Sst Crh 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 Sema3e Kank4, 14 Pvalb Gpr149 Islr, 11 Pvalb Reln Itm2a, 10 Pvalb Tac1, 9 Pvalb Tpbg, 4 Pvalb Vipr2, Meis2 Adamts19, 170 Astro Aqp4, 171 OPC Pdgfr Grm5, Oligo Serpinb1a, 174 Oligo Synpr, VLMC Osr1 Cd74, VLMC Osr1 Mc5r, VLMC Spp1 Col15a1, Peri Kcnj8, SMC Acta2, Endo Ctla2a, and 181 MicrogliaSiglech. (K~M) Animal: In vivo macaque and region: temporal cortex. Fluorescence images of macaque temporal cortex brain slices 51 days after in vivo injection of CN2039 virus. (K, L) Co-immunostaining of anti-GFP antibody and anti-calretinin antibody. (M) Reduplicate showing high on-target specificity of calretinin + cortical neurons, particularly in the upper layers. Virus was administered by stereotactic intraparenchymal injection of CN2039 virus packaged in a PHP.eB capsid. [Figure 3-1] Vector: CN2040, Animal: 554252, and Enhancer: eHGT_354m. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuronal morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2040 virus packaged in a PHP.eB capsid. (D~F) Region: VISp. Mouse visual cortex (VISp) transduced by CN2040 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows VIP mFISH (D), SYFP fluorescence and VIP mFISH (E), and SYFP fluorescence only (F). Images are montages. The quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping (blue circles with triangles) is shown, with 60 out of 73 cells being Vip+ (82% Vip+). [Figure 3-2]Vector: CN2040, Animal: 554252, and Enhancer: eHGT_354m. A montage of native SYFP2 fluorescence of sagittal sections of the whole mouse brain (A), caudal field (B), and visual field (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. Virus was administered to neonates after intracerebroventricular (ICV) injection of CN2040 virus packaged in the PHP.eB capsid. (D–F) Region: VISp. Mouse visual field (VISp) transduced by CN2040 virus packaged in the PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (D), SYFP fluorescence and VIP mFISH (E), and SYFP fluorescence only (F) are shown. Images are montages. Quantification of SYFP+ cells that overlap with Vip mFISH (red circles) or do not overlap (blue circles with triangles) is shown, with 60 out of 73 being Vip+ (82% Vip+). [Figure 3-3] Vector: CN2040, Animal: 554252, and Enhancer: eHGT_354m. A montage of native SYFP2 fluorescence of sagittal sections of the whole mouse brain (A), caudal field (B), and visual field (C), showing selective expression of SYFP2 in cells with bipolar neuron morphology. Virus was administered to neonates after intracerebroventricular (ICV) injection of CN2040 virus packaged in the PHP.eB capsid. (D–F) Region: VISp. Mouse visual field (VISp) transduced by CN2040 virus packaged in the PHP.eB capsid and delivered to neonates via ICV injection. VIP mFISH (D), SYFP fluorescence and VIP mFISH (E), and SYFP fluorescence only (F) are shown. Images are montages. Quantification of SYFP+ cells that overlap with Vip mFISH (red circles) or do not overlap (blue circles with triangles) is shown, with 60 out of 73 being Vip+ (82% Vip+). [Figure 3-4]Vector: CN2040, Animal: 554252, and Enhancer: eHGT_354m. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A), caudal cortex (B), and visual cortex (C), showing selective expression of SYFP2 in cells with bipolar neuronal morphology. The virus was administered to neonates after intraventricular (ICV) injection of CN2040 virus packaged in a PHP.eB capsid. (D~F) Region: VISp. Mouse visual cortex (VISp) transduced by CN2040 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows VIP mFISH (D), SYFP fluorescence and VIP mFISH (E), and SYFP fluorescence only (F). Images are montages. The quantification of SYFP+ cells overlapping with Vip mFISH (red circles) or not overlapping (blue circles with triangles) is shown, with 60 out of 73 cells being Vip+ (82% Vip+). [Figure 4-1] Vector: CN2569, Animal: 554257, and Enhancer: 3xCore3_eHGT_226h. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in scattered cells with non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN2569 virus packaged in a PHP.eB capsid. (C~E) Region: VISp. Mouse visual cortex (VISp) transduced with CN2569 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst (cyan) and Pvalb (yellow) mFISH (C), SYFP fluorescence only (D green), and SYFP fluorescence by Sst and Pvalb mFISH (E). The image is a montage. The quantification of SYFP+ cells that overlap with Sst mFISH (cyan circles) or do not overlap (red circles with stars) is shown, and of the 47 SYFP+ cells, 43 were Sst+ (91% Sst+). [Figure 4-2]Vector: CN2569, Animal: 554257, and Enhancer: 3xCore3_eHGT_226h. A montage of native SYFP2 fluorescence of sagittal sections of the whole mouse brain (A) and the visual cortex (B), showing the selective expression of SYFP2 in scattered cells with non - pyramidal neuron morphology. Virus was administered to neonates via intracerebroventricular (ICV) injection of the CN2569 virus packaged in the PHP.eB capsid. (C - E) Region: VISp. Mouse visual cortex (VISp) transduced by the CN2569 virus packaged in the PHP.eB capsid and delivered to neonates via ICV injection. Sst (cyan) and Pvalb (yellow) mFISH (C), SYFP fluorescence only (D green), and SYFP fluorescence by Sst and Pvalb mFISH (E) are shown. Images are montages. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or not overlapping (red circles with stars) is shown, with 43 out of 47 SYFP+ cells being Sst+ (91% Sst+). [Figure 4-3] Vector: CN2569, Animal: 554257, and Enhancer: 3xCore3_eHGT_226h. A montage of native SYFP2 fluorescence of sagittal sections of the whole mouse brain (A) and the visual cortex (B), showing the selective expression of SYFP2 in scattered cells with non - pyramidal neuron morphology. Virus was administered to neonates via intracerebroventricular (ICV) injection of the CN2569 virus packaged in the PHP.eB capsid. (C - E) Region: VISp. Mouse visual cortex (VISp) transduced by the CN2569 virus packaged in the PHP.eB capsid and delivered to neonates via ICV injection. Sst (cyan) and Pvalb (yellow) mFISH (C), SYFP fluorescence only (D green), and SYFP fluorescence by Sst and Pvalb mFISH (E) are shown. Images are montages. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or not overlapping (red circles with stars) is shown, with 43 out of 47 SYFP+ cells being Sst+ (91% Sst+). [Figure 5-1]Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-2] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-3] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-4] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-5] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-6] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-7] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 5-8] Vector: CN1567, Animal: 554248, and Enhancer: eHGT_121h. [ka] [Figure 6-1]Vector: CN2317, Animal: 554427, and Enhancer: eHGT_468m. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered neuronal morphology. In the caudal cortex, most labeled cells appear in the deep layers, while in the rostral cortex, labeled cells are not abundant in certain layers. The virus was administered to neonates via intraventricular (ICV) injection of CN2317 virus packaged in a PHP.eB capsid. (C~E) Region: VISp. Visual cortex (VISp) of a mouse transduced with CN2317 virus packaged in a PHP.eB capsid and delivered to a neonate via ICV injection. Shows Sst (yellow) mFISH (C), SYFP fluorescence (green) and Sst mFISH (D), as well as SYFP fluorescence only (green in E). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles with triangles) or not overlapping with Sst mFISH (red circles) shows that 10 out of 38 SYFP+ cells are Sst+ (26% Sst+). (F~H) Region: Mouse rostral area (ALM). ALM transduced with CN2317 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst (yellow) and Gad1 (cyan) mFISH (F), SYFP fluorescence only (green G), and SYFP fluorescence, Sst, and Gad1 mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or not overlapping with Sst mFISH (red circles with stars) shows that 32 out of 51 SYFP+ cells are Sst+ (63% Sst+). [Figure 6-2]Vector: CN2317, Animal: 554427, and Enhancer: eHGT_468m. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered neuronal morphology. In the caudal cortex, most labeled cells appear in the deep layers, while in the rostral cortex, labeled cells are not abundant in certain layers. The virus was administered to neonates via intraventricular (ICV) injection of CN2317 virus packaged in a PHP.eB capsid. (C~E) Region: VISp. Visual cortex (VISp) of a mouse transduced with CN2317 virus packaged in a PHP.eB capsid and delivered to a neonate via ICV injection. Shows Sst (yellow) mFISH (C), SYFP fluorescence (green) and Sst mFISH (D), as well as SYFP fluorescence only (green in E). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles with triangles) or not overlapping with Sst mFISH (red circles) shows that 10 out of 38 SYFP+ cells are Sst+ (26% Sst+). (F~H) Region: Mouse rostral area (ALM). ALM transduced with CN2317 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst (yellow) and Gad1 (cyan) mFISH (F), SYFP fluorescence only (green G), and SYFP fluorescence, Sst, and Gad1 mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or not overlapping with Sst mFISH (red circles with stars) shows that 32 out of 51 SYFP+ cells are Sst+ (63% Sst+). [Figure 6-3]Vector: CN2317, Animal: 554427, and Enhancer: eHGT_468m. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered neuronal morphology. In the caudal cortex, most labeled cells appear in the deep layers, while in the rostral cortex, labeled cells are not abundant in certain layers. The virus was administered to neonates via intraventricular (ICV) injection of CN2317 virus packaged in a PHP.eB capsid. (C~E) Region: VISp. Visual cortex (VISp) of a mouse transduced with CN2317 virus packaged in a PHP.eB capsid and delivered to a neonate via ICV injection. Shows Sst (yellow) mFISH (C), SYFP fluorescence (green) and Sst mFISH (D), as well as SYFP fluorescence only (green in E). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles with triangles) or not overlapping with Sst mFISH (red circles) shows that 10 out of 38 SYFP+ cells are Sst+ (26% Sst+). (F~H) Region: Mouse rostral area (ALM). ALM transduced with CN2317 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst (yellow) and Gad1 (cyan) mFISH (F), SYFP fluorescence only (green G), and SYFP fluorescence, Sst, and Gad1 mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or not overlapping with Sst mFISH (red circles with stars) shows that 32 out of 51 SYFP+ cells are Sst+ (63% Sst+). [Figure 6-4]Vector: CN2317, Animal: 554427, and Enhancer: eHGT_468m. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered neuronal morphology. In the caudal cortex, most labeled cells appear in the deep layers, while in the rostral cortex, labeled cells are not abundant in certain layers. The virus was administered to neonates via intraventricular (ICV) injection of CN2317 virus packaged in a PHP.eB capsid. (C~E) Region: VISp. Visual cortex (VISp) of a mouse transduced with CN2317 virus packaged in a PHP.eB capsid and delivered to a neonate via ICV injection. Shows Sst (yellow) mFISH (C), SYFP fluorescence (green) and Sst mFISH (D), as well as SYFP fluorescence only (green in E). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles with triangles) or not overlapping with Sst mFISH (red circles) shows that 10 out of 38 SYFP+ cells are Sst+ (26% Sst+). (F~H) Region: Mouse rostral area (ALM). ALM transduced with CN2317 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst (yellow) and Gad1 (cyan) mFISH (F), SYFP fluorescence only (green G), and SYFP fluorescence, Sst, and Gad1 mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or not overlapping with Sst mFISH (red circles with stars) shows that 32 out of 51 SYFP+ cells are Sst+ (63% Sst+). [Figure 7-1]Enhancer: eHGT_156h, Animal: 539841, Vector: CN1649, Region: VISp. Mouse visual cortex (VISp) transduced with CN1649 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. (A) SYFP fluorescence (green) and DAPI (gray), (B) Sst, Pvalb, Gad1 mFISH, (C) SYFP fluorescence only, and (D) SYFP fluorescence and Sst, Pvalb, Gad1 mFISH. Colors are shown. [Figure 7-2] Enhancer: eHGT_156h, Animal: 539841, Vector: CN1649, Region: VISp. Mouse visual cortex (VISp) transduced with CN1649 virus packaged in PHP.eB capsid and delivered to neonates via ICV injection. (A) SYFP fluorescence (green) and DAPI (gray), (B) Sst, Pvalb, Gad1 mFISH, (C) SYFP fluorescence only, and (D) SYFP fluorescence and Sst, Pvalb, Gad1 mFISH. Colors are shown. [Figure 8-1]Enhancer: eHGT_170h, Animal: 539842, and Vector: CN1663. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN1663 virus packaged in a PHP.eB capsid. (C~H) Region: VISp. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Showing SYFP fluorescence only (C), SYFP fluorescence and Sst mFISH (D), and Sst mFISH only (E). Images are montages. The image shows the quantification of SYFP+ cells overlapping with Sst mFISH (yellow circles) or not overlapping with Sst mFISH (red circles with stars), with 44 out of 48 cells being Sst+ (92% Sst+). F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence from Sst and Pvalb mFISH (H). The image is a montage. [Figure 8-2]Enhancer: eHGT_170h, Animal: 539842, and Vector: CN1663. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN1663 virus packaged in a PHP.eB capsid. (C~H) Region: VISp. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Showing SYFP fluorescence only (C), SYFP fluorescence and Sst mFISH (D), and Sst mFISH only (E). Images are montages. The image shows the quantification of SYFP+ cells overlapping with Sst mFISH (yellow circles) or not overlapping with Sst mFISH (red circles with stars), with 44 out of 48 cells being Sst+ (92% Sst+). F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence from Sst and Pvalb mFISH (H). The image is a montage. [Figure 8-3]Enhancer: eHGT_170h, Animal: 539842, and Vector: CN1663. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN1663 virus packaged in a PHP.eB capsid. (C~H) Region: VISp. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Showing SYFP fluorescence only (C), SYFP fluorescence and Sst mFISH (D), and Sst mFISH only (E). Images are montages. The image shows the quantification of SYFP+ cells overlapping with Sst mFISH (yellow circles) or not overlapping with Sst mFISH (red circles with stars), with 44 out of 48 cells being Sst+ (92% Sst+). F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence from Sst and Pvalb mFISH (H). The image is a montage. [Figure 8-4]Enhancer: eHGT_170h, Animal: 539842, and Vector: CN1663. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN1663 virus packaged in a PHP.eB capsid. (C~H) Region: VISp. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Showing SYFP fluorescence only (C), SYFP fluorescence and Sst mFISH (D), and Sst mFISH only (E). Images are montages. The image shows the quantification of SYFP+ cells overlapping with Sst mFISH (yellow circles) or not overlapping with Sst mFISH (red circles with stars), with 44 out of 48 cells being Sst+ (92% Sst+). F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN1663 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence from Sst and Pvalb mFISH (H). The image is a montage. [Figure 9-1]Enhancer: eHGT_526h, Animal: 554251, and Vector: CN2365. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN2365 virus packaged in a PHP.eB capsid. Strong expression is also observed in MSN neurons of the striatum and direct pathway axons. (C~E) Region: VISp. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows SYFP fluorescence by Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). Images are montages. F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence by Sst and Pvalb mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or non-overlapping SYFP+ cells (red circles with stars) shows that 31 out of 35 SYFP+ cells were Sst+ (89% Sst+). (I-K) Region: Hippocampus. Mouse hippocampus transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows SYFP fluorescence only (I), Sst mFISH (J), and Pvalb mFISH (K). The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH indicates that all 24 of the 24 SYFP+ cells are 100% Sst+. [Figure 9-2]Enhancer: eHGT_526h, Animal: 554251, and Vector: CN2365. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN2365 virus packaged in a PHP.eB capsid. Strong expression is also observed in MSN neurons of the striatum and direct pathway axons. (C~E) Region: VISp. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows SYFP fluorescence by Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). Images are montages. F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence by Sst and Pvalb mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or non-overlapping SYFP+ cells (red circles with stars) shows that 31 out of 35 SYFP+ cells were Sst+ (89% Sst+). (I-K) Region: Hippocampus. Mouse hippocampus transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows SYFP fluorescence only (I), Sst mFISH (J), and Pvalb mFISH (K). The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH indicates that all 24 of the 24 SYFP+ cells are 100% Sst+. [Figure 9-3]Enhancer: eHGT_526h, Animal: 554251, and Vector: CN2365. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN2365 virus packaged in a PHP.eB capsid. Strong expression is also observed in MSN neurons of the striatum and direct pathway axons. (C~E) Region: VISp. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows SYFP fluorescence by Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). Images are montages. F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence by Sst and Pvalb mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or non-overlapping SYFP+ cells (red circles with stars) shows that 31 out of 35 SYFP+ cells were Sst+ (89% Sst+). (I-K) Region: Hippocampus. Mouse hippocampus transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows SYFP fluorescence only (I), Sst mFISH (J), and Pvalb mFISH (K). The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH indicates that all 24 of the 24 SYFP+ cells are 100% Sst+. [Figure 9-4]Enhancer: eHGT_526h, Animal: 554251, and Vector: CN2365. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN2365 virus packaged in a PHP.eB capsid. Strong expression is also observed in MSN neurons of the striatum and direct pathway axons. (C~E) Region: VISp. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows SYFP fluorescence by Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). Images are montages. F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence by Sst and Pvalb mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or non-overlapping SYFP+ cells (red circles with stars) shows that 31 out of 35 SYFP+ cells were Sst+ (89% Sst+). (I-K) Region: Hippocampus. Mouse hippocampus transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows SYFP fluorescence only (I), Sst mFISH (J), and Pvalb mFISH (K). The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH indicates that all 24 of the 24 SYFP+ cells are 100% Sst+. [Figure 9-5]Enhancer: eHGT_526h, Animal: 554251, and Vector: CN2365. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing selective expression of SYFP2 in cells with scattered non-pyramidal neuron morphology. The virus was administered to neonates via intraventricular (ICV) injection of CN2365 virus packaged in a PHP.eB capsid. Strong expression is also observed in MSN neurons of the striatum and direct pathway axons. (C~E) Region: VISp. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in a PHP.eB capsid and delivered to neonates via ICV injection. Shows SYFP fluorescence by Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). Images are montages. F-H are insets of C-E. Mouse visual cortex (VISp) transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows Sst and Pvalb mFISH only (F), SYFP fluorescence only (G), and SYFP fluorescence by Sst and Pvalb mFISH (H). The image is a montage. Quantification of SYFP+ cells overlapping with Sst mFISH (cyan circles) or non-overlapping SYFP+ cells (red circles with stars) shows that 31 out of 35 SYFP+ cells were Sst+ (89% Sst+). (I-K) Region: Hippocampus. Mouse hippocampus transduced with CN2365 virus packaged in PHP.eB capsid and delivered to neonatal pups via ICV injection. Shows SYFP fluorescence only (I), Sst mFISH (J), and Pvalb mFISH (K). The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH indicates that all 24 of the 24 SYFP+ cells are 100% Sst+. [Figure 10A]Vector: CN1584 and enhancer: eHGT_090m. (A) Region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after post-orbital injection of CN1584 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_090m enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. (B-D) Species: Macaca and region: Left Occipital Lateral Cortex. Macaca transduced with CN1584 virus packaged in PHP.eB capsid and delivered by intraparenchymal injection. The entire cortical column is shown with SYFP and DAPI fluorescence (B), the inset shows only SYFP fluorescence (C), and the inset shows VIP, GAD1, and SST mFISH (D). Arrows indicate overlap between SYFP and SST, and asterisks indicate SYFP and no SST. The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH is shown, with 115 out of 159 SYFP+ cells being Sst+ (72% Sst+). The quantification at the bottom shows specificity in two different images. [Figure 10B]Vector: CN1584 and enhancer: eHGT_090m. (A) Region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after post-orbital injection of CN1584 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_090m enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. (B-D) Species: Macaca and region: Left Occipital Lateral Cortex. Macaca transduced with CN1584 virus packaged in PHP.eB capsid and delivered by intraparenchymal injection. The entire cortical column is shown with SYFP and DAPI fluorescence (B), the inset shows only SYFP fluorescence (C), and the inset shows VIP, GAD1, and SST mFISH (D). Arrows indicate overlap between SYFP and SST, and asterisks indicate SYFP and no SST. The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH is shown, with 115 out of 159 SYFP+ cells being Sst+ (72% Sst+). The quantification at the bottom shows specificity in two different images. [Figure 10C]Vector: CN1584 and enhancer: eHGT_090m. (A) Region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after post-orbital injection of CN1584 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_090m enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. (B-D) Species: Macaca and region: Left Occipital Lateral Cortex. Macaca transduced with CN1584 virus packaged in PHP.eB capsid and delivered by intraparenchymal injection. The entire cortical column is shown with SYFP and DAPI fluorescence (B), the inset shows only SYFP fluorescence (C), and the inset shows VIP, GAD1, and SST mFISH (D). Arrows indicate overlap between SYFP and SST, and asterisks indicate SYFP and no SST. The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH is shown, with 115 out of 159 SYFP+ cells being Sst+ (72% Sst+). The quantification at the bottom shows specificity in two different images. [Figure 10D]Vector: CN1584 and enhancer: eHGT_090m. (A) Region: VISp. Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after post-orbital injection of CN1584 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_090m enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. (B-D) Species: Macaca and region: Left Occipital Lateral Cortex. Macaca transduced with CN1584 virus packaged in PHP.eB capsid and delivered by intraparenchymal injection. The entire cortical column is shown with SYFP and DAPI fluorescence (B), the inset shows only SYFP fluorescence (C), and the inset shows VIP, GAD1, and SST mFISH (D). Arrows indicate overlap between SYFP and SST, and asterisks indicate SYFP and no SST. The image is a montage. Quantification of SYFP+ cells (cyan circles) overlapping with Sst mFISH is shown, with 115 out of 159 SYFP+ cells being Sst+ (72% Sst+). The quantification at the bottom shows specificity in two different images. [Figure 11-1]Vector: CN2367, Animal: 200826-09, and Enhancer: eHGT_528h. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing extremely sparse selective expression of SYFP2. The virus was administered via posterior orbital injection of the CN2367 virus packaged in a PHP.eB capsid. (C~F) Region: VISp. Mouse visual cortex (VISp) introduced by the CN2367 virus packaged in a PHP.eB capsid and delivered via posterior orbital injection. Shows Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). The image is a montage. Quantification of SYFP+ cells is shown in the lower right. All 11 SYFP-positive cells were Sst+. (F) Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after posterior orbital injection of CN2367 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_528h enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. [Figure 11-2]Vector: CN2367, Animal: 200826-09, and Enhancer: eHGT_528h. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing extremely sparse selective expression of SYFP2. The virus was administered via posterior orbital injection of the CN2367 virus packaged in a PHP.eB capsid. (C~F) Region: VISp. Mouse visual cortex (VISp) introduced by the CN2367 virus packaged in a PHP.eB capsid and delivered via posterior orbital injection. Shows Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). The image is a montage. Quantification of SYFP+ cells is shown in the lower right. All 11 SYFP-positive cells were Sst+. (F) Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after posterior orbital injection of CN2367 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_528h enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. [Figure 11-3]Vector: CN2367, Animal: 200826-09, and Enhancer: eHGT_528h. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing extremely sparse selective expression of SYFP2. The virus was administered via posterior orbital injection of the CN2367 virus packaged in a PHP.eB capsid. (C~F) Region: VISp. Mouse visual cortex (VISp) introduced by the CN2367 virus packaged in a PHP.eB capsid and delivered via posterior orbital injection. Shows Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). The image is a montage. Quantification of SYFP+ cells is shown in the lower right. All 11 SYFP-positive cells were Sst+. (F) Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after posterior orbital injection of CN2367 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_528h enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. [Figure 11-4]Vector: CN2367, Animal: 200826-09, and Enhancer: eHGT_528h. Fluorescent montages of native SYFP2 in sagittal sections of the entire mouse brain (A) and visual cortex (B), showing extremely sparse selective expression of SYFP2. The virus was administered via posterior orbital injection of the CN2367 virus packaged in a PHP.eB capsid. (C~F) Region: VISp. Mouse visual cortex (VISp) introduced by the CN2367 virus packaged in a PHP.eB capsid and delivered via posterior orbital injection. Shows Sst and Pvalb mFISH only (C), SYFP fluorescence only (D), and SYFP fluorescence by Sst and Pvalb mFISH (E). The image is a montage. Quantification of SYFP+ cells is shown in the lower right. All 11 SYFP-positive cells were Sst+. (F) Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after posterior orbital injection of CN2367 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_528h enhancer-driven reporter expression occurs selectively in SST+ cells when VISp is evaluated. The text below is a brief description of the drawing in Figure 2J. [Figure 12A] Vector: CN2689, Animal: 556452, Enhancer: eHGT_682h, and Region: Neocortex. (A) Fluorescence image of native SYFP2 in sagittal section of mouse brain, showing very sparse expression of SYFP2 in the neocortex. (B) Anti-GFP, (C) Anti-Nos1, and (D) Reduplicate images showing highly specific labeling in the target Nos1+ cell population. Nos1 is a marker gene for Sst-Chodl cell types. The virus was administered by posterior orbital injection of the CN2689 virus packaged in a PHP.eB capsid. [Figure 12B]Vector: CN2689, Animal: 556452, Enhancer: eHGT_682h, and Region: Neocortex. (A) Fluorescence image of native SYFP2 in sagittal section of mouse brain, showing very sparse expression of SYFP2 in the neocortex. (B) Anti-GFP, (C) Anti-Nos1, and (D) Reduplicate images showing highly specific labeling in the target Nos1+ cell population. Nos1 is a marker gene for Sst-Chodl cell types. The virus was administered by posterior orbital injection of the CN2689 virus packaged in a PHP.eB capsid. [Figure 12C] Vector: CN2689, Animal: 556452, Enhancer: eHGT_682h, and Region: Neocortex. (A) Fluorescence image of native SYFP2 in sagittal section of mouse brain, showing very sparse expression of SYFP2 in the neocortex. (B) Anti-GFP, (C) Anti-Nos1, and (D) Reduplicate images showing highly specific labeling in the target Nos1+ cell population. Nos1 is a marker gene for Sst-Chodl cell types. The virus was administered by posterior orbital injection of the CN2689 virus packaged in a PHP.eB capsid. [Figure 12D] Vector: CN2689, Animal: 556452, Enhancer: eHGT_682h, and Region: Neocortex. (A) Fluorescence image of native SYFP2 in sagittal section of mouse brain, showing very sparse expression of SYFP2 in the neocortex. (B) Anti-GFP, (C) Anti-Nos1, and (D) Reduplicate images showing highly specific labeling in the target Nos1+ cell population. Nos1 is a marker gene for Sst-Chodl cell types. The virus was administered by posterior orbital injection of the CN2689 virus packaged in a PHP.eB capsid. [Figure 13-1] This document provides an overview of the characteristics and components of the artificial expression construct. [Figure 13-2] This document provides an overview of the characteristics and components of the artificial expression construct. [Figure 14] This specification provides an overview of the target cell type specificity of the different artificial expression constructs disclosed herein. [Figure 15-1] Includes sequences supporting this disclosure. [Figure 15-2] Includes sequences supporting this disclosure. [Figure 15-3] Includes sequences supporting this disclosure. [Figure 15-4] Includes sequences supporting this disclosure. [Figure 15-5] Includes sequences supporting this disclosure. [Figure 15-6] Includes sequences supporting this disclosure. [Figure 15-7] Includes sequences supporting this disclosure. [Figure 15-8] Includes sequences supporting this disclosure. [Figure 15-9] Includes sequences supporting this disclosure. [Figure 15-10] Includes sequences supporting this disclosure. [Figure 15-11] Includes sequences supporting this disclosure. [Figure 15-12] Includes sequences supporting this disclosure. [Figure 15-13] Includes sequences supporting this disclosure. [Figure 15-14] Includes sequences supporting this disclosure. [Figure 15-15] Includes sequences supporting this disclosure. [Figure 15-16] Includes sequences supporting this disclosure. [Figure 15-17] Includes sequences supporting this disclosure. [Figure 15-18] Includes sequences supporting this disclosure. [Figure 15-19] Includes sequences supporting this disclosure. [Figure 15-20] Includes sequences supporting this disclosure. [Figure 15-21] Includes sequences supporting this disclosure. [Figure 15-22] Includes sequences supporting this disclosure. [Figure 15-23] Includes sequences supporting this disclosure. [Figure 15-24] Includes sequences supporting this disclosure. [Figure 15-25] Includes sequences supporting this disclosure. [Figure 15-26] Includes sequences supporting this disclosure. [Figure 15-27] Includes sequences supporting this disclosure. [Figure 15-28] Includes sequences supporting this disclosure. [Figure 15-29] Includes sequences supporting this disclosure. [Figure 15-30] Includes sequences supporting this disclosure. [Figure 15-31] Includes sequences supporting this disclosure. [Figure 15-32] Includes sequences supporting this disclosure. [Figure 15-33] Includes sequences supporting this disclosure. [Figure 15-34] Includes sequences supporting this disclosure. [Figure 15-35] Includes sequences supporting this disclosure. [Figure 15-36] Includes sequences supporting this disclosure. [Figure 15-37] Includes sequences supporting this disclosure. [Figure 15-38] Includes sequences supporting this disclosure. [Figure 15-39] Includes sequences supporting this disclosure. [Figure 15-40] Includes sequences supporting this disclosure. [Figure 15-41] Includes sequences supporting this disclosure. [Figure 15-42] Includes sequences supporting this disclosure. [Figure 15-43] Includes sequences supporting this disclosure. [Figure 15-44] Includes sequences supporting this disclosure. [Figure 15-45] Includes sequences supporting this disclosure. [Figure 15-46] Includes sequences supporting this disclosure. [Figure 15-47] Includes sequences supporting this disclosure. [Figure 15-48] Includes sequences supporting this disclosure. [Figure 15-49] Includes sequences supporting this disclosure. [Figure 15-50] Includes sequences supporting this disclosure. [Figure 15-51] Includes sequences supporting this disclosure. [Figure 15-52] Includes sequences supporting this disclosure. [Figure 15-53] Includes sequences supporting this disclosure. [Figure 15-54] Includes sequences supporting this disclosure. [Figure 15-55] Includes sequences supporting this disclosure. [Figure 15-56] Includes sequences supporting this disclosure. [Figure 15-57] Includes sequences supporting this disclosure. [Figure 15-58] Includes sequences supporting this disclosure. [Figure 15-59] Includes sequences supporting this disclosure. [Figure 15-60] Includes sequences supporting this disclosure. [Figure 15-61] Includes sequences supporting this disclosure. [Figure 15-62] Includes sequences supporting this disclosure. [Figure 15-63] Includes sequences supporting this disclosure. [Figure 15-64] Includes sequences supporting this disclosure. [Figure 15-65] Includes sequences supporting this disclosure. [Figure 15-66] Includes sequences supporting this disclosure. [Figure 15-67] Includes sequences supporting this disclosure. [Figure 15-68] Includes sequences supporting this disclosure. [Figure 15-69] Includes sequences supporting this disclosure. [Modes for carrying out the invention]
[0014] Detailed explanation To fully understand the biology of the brain, it is necessary to distinguish and define different cell types, and to identify artificial expression constructs that can selectively label and disrupt them in order to further study them. Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018), Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017). In mice, recombinase driver strains have been found to be highly effective in labeling cell populations that share marker gene expression. Daigle et al., Cell 174, 465-480. e22 (2018), Taniguchi et al., Neuron 71, 995-1013 (2011), Gong et al., J. Neurosci. 27, 9817-9823 (2007). However, the creation, maintenance, and use of such strains that label cell types with high specificity are expensive and often require triple transgenic crosses, resulting in a low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans.
[0015] This disclosure provides artificial expression constructs that selectively drive gene expression in target central nervous system cell populations. Target central nervous system cell populations include: somatostatin (Sst) GABAergic neurons, parvalbumin (Pvalb) GABAergic neurons, vasoactive enteropeptide (Vip) GABAergic neurons, and Lamp5 GABAergic neurons, as well as inhibitory neocortical GABAergic neurons, including in some cases astrocytes.
[0016] Specific embodiments of the artificial expression constructs include the following enhancers: eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_340m, eHGT_528h, eHGT_515h, eHGT_226h, eHGT_170h, eHGT_519h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, and eHGT_156hSst GABAergic neurons; eHGT_076h eHGT_759m, and eHGT_064h / Pvalb / Sst GABAergic neurons: eHGT_072h, eHGT_131hv1, eHGT_131hv2, and eHGT_130h / Pvalb; GABAergic neurons: eHGT_354h, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_107h, MGT_E81, MGT_E85, MGT_E88, and MGT_E83 / VIP; GABAergic neurons: MGT_E36, MGT_E37, and MGT_E41 / Lamp5_Lhx6 GABAergic neurons; eHGT_354m, eHGT_060m, and eHGT_060h / VIP GABAergic neurons and astrocytes, eHGT_025h, eHGT_096h, eHGT_098h, and eHGT_104m / Lamp5 GABAergic neurons; eHGT_682h, eHGT_600m, eHGT_468m, eHGT_338m, eHGT_341m, and eHGT_339m / Sst and Chold GABAergic neurons are used to selectively drive gene expression within the above target central nervous system cell populations (enhancer / target cell populations).
[0017] In certain embodiments, the artificial enhancer element includes a ligated core of the enhancer, for example, a ligated core of eHGT_226h and / or eHGT_064h. These artificial enhancer elements can provide higher levels of transgene expression and faster initiation of expression compared to a single full-length original (native) enhancer.
[0018] In certain embodiments, the enhancer core includes the sequence described in any one of SEQ ID NOs: 161, 163, and 165. In certain embodiments, these cores are concatenated and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. SEQ ID NOs: 162, 164, and 166 provide concatemers of three selected enhancer cores.
[0019] Certain embodiments of the artificial expression construct utilize 3xcore2_eHGT_226h and / or 3xcore3_eHGT_226h to selectively drive protein expression in Sst GABAergic neurons, and / or utilize 3xcore_eHGT_064h to selectively drive protein expression in Pvalb / Sst GABAergic neurons.
[0020] Specific embodiments include vectors: CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567, CN1626, CN1712, CN1700, CN1607, CN1605, CN1556, CN1526, CN1418, CN1404, AiV1173, AiV1174, AiV1177, CN1261, CN1542, CN1544, CN1598, CN1553, CN The present invention provides artificial expression constructs that include the characteristics of vectors described herein, such as CN2367, CN2357, CN2568, CN2569, CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309, CN2366, CN2257, CN1667, CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, and AiP1273.
[0021] Aspects of this disclosure are described in the following further options and details: (i) expression constructs and vectors for selective expression of genes in selected cell types, (ii) compositions for administration, (iii) cell lines containing the expression constructs, (iv) transgenic animals, (v) methods of use, (vi) kits and commercial products, (vii) exemplary embodiments, (viii) experimental examples, and (ix) concluding paragraphs.
[0022] (i) Expression constructs and vectors for the selective expression of genes in selected cell types. The expression constructs disclosed herein include (i) an enhancer sequence that induces the selective expression of a coding sequence in a target central nervous system cell type, (ii) the coding sequence to be expressed, and (iii) a promoter. The expression constructs may also include other regulatory elements if necessary or beneficial.
[0023] In certain embodiments, the “enhancer” or “enhancer element” is a cis-acting sequence that can function in either direction with respect to the promoter and coding sequence to enhance the level of transcription associated with the promoter, and can be located upstream or downstream of the promoter or coding sequence to be transcribed. Methods and techniques exist and are recognized in the art to measure the function(s) of enhancer element sequences. Specific examples of enhancer sequences used in the artificial expression constructs disclosed herein include: eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, e HGT_121h,eHGT_133h,eHGT_219h,eHGT_207h,eHGT_113m,eHGT_111m,eHGT_110h,eHGT_080h,e HGT_060m, eHGT_060h, MGT_E36, MGT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_104 m, eHGT_107h, eHGT_340m, eHGT_528h, eHGT_515h, eHGT_682h, eHGT_600m, eHGT_759m, eHGT_468 m, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT_131hv2, eHGT_130h, eHGT_527h, eHGT_470m, eHGT These include _174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E81, MGT_E85, MGT_E88, and MGT_E83, as well as linked cores, such as 3xCore2_eHGT_226h, 3xCore3_eHGT_226h, and 3xCore_eHGT_064h.
[0024] In certain embodiments, the target central nervous system cell type enhancer is an enhancer that is uniquely or primarily utilized by the target central nervous system cell type. The target central nervous system cell type enhancer enhances gene expression in the target central nervous system cell type but does not substantially induce gene expression in other non-target cell types, and therefore has cell type-specific transcriptional activity.
[0025] If a coding sequence is selectively expressed in selected cells and substantially not expressed in other cell types, the product of the coding sequence is preferentially expressed in the selected cell types. In certain embodiments, preferential expression is greater than 50% compared to the reference cell type; greater than 60% compared to the reference cell type; greater than 70% compared to the reference cell type; greater than 80% compared to the reference cell type; or greater than 90% compared to the reference cell type. In certain embodiments, the reference cell type refers to untargeted cells. Untargeted cells are located within the same anatomical structure as the targeted cells and / or appear in a common anatomical region. In certain embodiments, the reference cell type is located within an anatomical structure adjacent to the anatomical structure containing the target cell type. In certain embodiments, the reference cell type is untargeted cells having a different gene expression profile than the target cells.
[0026] In certain embodiments, the coding sequence product may be expressed in non-selective cell types at levels less than 1% of the level at which the product is expressed in selected cells, or at low levels of 1%, 2%, 3%, 5%, 10%, 15%, or 20%. In certain embodiments, the target central nervous system cell type is the only cell type that expresses the appropriate combination of transcription factors that bind to the enhancers disclosed herein to drive gene expression. Thus, in certain embodiments, expression occurs only within the target cell type.
[0027] In certain embodiments, target cell types (e.g., neurons and / or non-neurons) can be identified based on transcriptional profiles, such as those described in Tasic et al., Nature, 563, 72-78 (2018) and Hodge et al., Nature, 573, 61-68 (2019). For reference, a description of cell types and identifying features is provided below:
[0028] Neocortical GABAergic neuron subclass: ●All: Expresses the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2. ● Lamp5, Sncg, Serpinf1, and Vip GABAergic neurons: Developmentally derived from neuronal precursors originating from the caudal ganglion luminescence (CGE) or preoptic area (POA). ● Sst and Pvalb GABAergic neurons: These are developmentally induced from neuronal precursors in medial ganglion luminescence (MGE). ● Lamp5 GABAergic neurons: These are found in many cortical layers, especially the upper layers (L1-L2 / 3), and mainly have glial cell morphology and single plexus morphology. ● Lamp5_Lhx6 agonist neurons: A subset of Lamp5 GABAergic neurons that simultaneously express Lamp5 and Lhx6. ●Sncg GABAergic neurons: These are found in many cortical layers and have molecular overlap with Lamp5 and Vip cells, but Lamp5 or Vip expression is inconsistent, while Sncg expression is more consistent. ●Serpinf1 GABAergic neurons: These are found in many cortical layers and have molecular overlap with Sncg and Vip cells, but Sncg or Vip expression is inconsistent, while Serpinf1 expression is more consistent. ●Vip GABAergic neurons: These are found in many cortical layers, but are particularly abundant in the upper layers (L1-L4), and highly express the neurotransmitter vasoactive intestinal peptide (Vip). ● Sst GABAergic neurons: These are found in many cortical layers, but are particularly frequent in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic entry into postsynaptic neurons. This subclass includes sleep-activating Sst Chodl neurons (which also express Nos1 and Tacr1), which are quite different from other Sst neurons but express several co-marker genes including Sst. In humans, SST gene expression is often detected in the layer 1 LAMP5+ GABAergic neuron subtype. ● Pvalb GABAergic neurons: These are found in many cortical layers, but are particularly frequent in the lower layers (L5-L6). They highly express the calcium-binding protein parvalbumin (Pvalb), express the neuropeptide Tac1, and often attenuate postsynaptic neuronal output. Most GABAergic neurons strongly express Pvalb. This subclass includes chandelier cells that have a distinct chandelier-like morphology and express the markers Cpne5 and Vipr2 in mice, as well as NOG and UNC5B in humans. ●Meis2: Defined as a distinct subclass consisting only of neocortical GABAergic neurons expressing the Meis2 gene, and not expressing some other genes expressed by all other neocortical GABAergic neuron types (e.g., Thy1 and Scn2b). This type is found in L6b and subcortical white matter.
[0029] Neocortical glutamatergic neuron subclass: ● All: Express glutamate transmitters Slc17a6 and / or Slc17a7. They all express Snap25 and lack Gad1 / Gad2 expression. ● L2 / 3 IT glutamatergic neurons: These are mainly located in layers 2 and 3 and primarily possess telencephalic (corticocortical) processes. ● L4 IT glutamatergic neurons: These are mainly located in layer 4 and have projections that are either focal or intratelncemal (corticocortical). ● L5 IT glutamatergic neurons: Primarily located in layer 5, and mainly possessing telencephalic (corticocortical) processes. Also referred to as L5a. ● L5 PT glutamatergic neurons: Primarily located in layer 5 and possessing primarily corticospinal (pyramidal tract or corticofugal) processes. Also referred to as L5b or L5 CF (corticofugal), or L5 ET (extratelncenal). This subclass includes cells located in the primary motor cortex and adjacent regions that are corticospinal process neurons associated with motor neuron / motor disorders such as ALS. This subclass includes thick, tufted pyramidal neurons, including characteristic subtypes found only in specialized regions, such as Betz cells, Meynart cells, and von Economo cells. ●L5 NP glutamatergic neurons: These are mainly located in layer 5 and have projections in the vicinity. ●L6 CT glutamatergic neurons: Primarily located in layer 6 and primarily possessing corticothalamic processes. ● L6 IT glutamatergic neurons: These are mainly located in layer 6 and primarily possess intracerebral (corticocortical) processes. ●L6 IT Car3 glutamatergic neurons: These are most densely located in the claustrum and internal piriform nucleus, and are sparsely distributed throughout L6 in many cortical regions, including the primary visual cortex. These cells primarily have intracortical (intercortical) processes. Additional marker genes for claustrum-enriched neurons include Gnb4 and Ntng2. ● L6b glutamatergic neurons: These are mainly located in the neocortical subplate (L6b) and have focal (near the cell body) processes, several corticocortical processes from the VISp to the anterior cyst, and corticocortical processes to the thalamus. ●CR neurons: Cajal-Retzius cells, a distinct subclass defined as a single type in L1, express distinct molecular markers Lhx5 and Trp73.
[0030] Cerebellar Purkinje cells are large GABAergic neurons, the only protruding neurons and their only product originating from the cerebellum. Their cell bodies form a monolayer known as the "Purkinje cell layer," and they express parvalbumin.
[0031] Deep cerebellar nucleus neurons: Neurons located within the deep cerebellar nucleus structure. These include glutamatergic and GABAergic cells that express the Pvalb gene.
[0032] Non-neuronal subclass: ● Astrocytes: Glial cells derived from neuroectoderm that express the marker Aqp4 and often GFAP, but do not express the neuronal marker SNAP25. They can have a distinct star-shaped morphology and are involved in supporting the metabolism of other cells in the brain. Multiple star cell morphologies have been observed in mice and humans. ● Oligodendrocytes: Glial cells derived from the neuroectoderm that express the marker Sox10. This category includes oligodendrocyte vesicle precursor cells (OPCs). Oligodendrocyte cells are a subclass primarily involved in neuronal myelination. ●VLMC: Vascular leptomeninge cells (VLMCs) are a part of the meninges that surround the outer layer of the cortex and express the marker genes Lum and Col1a1. ● Pericytes: Blood vessel-related cells that express the marker genes Kcnj8 and Abcc9. Pericytes surround endothelial cells and are important for regulating blood flow in capillaries, and are involved in blood-brain barrier permeability. ● SMC: A special type of smooth muscle cell that expresses the marker gene Acta2 and is associated with blood vessels. SMCs cover the arteries in the brain and are involved in blood-brain barrier permeability. ● Endothelial cells: Cells that reinforce the inner lining of blood vessels in the brain. Endothelial cells express the markers Tek and PDGF-B. ● Microglia: Hematopoietic-derived immune cells that are resident macrophages and perivascular macrophages (PVMs) of the brain, which may be transitionally associated with brain tissue or may be included as a byproduct of brain dissection. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).
[0033] In certain embodiments, the coding sequence is a heterogeneous coding sequence that codes for an effector element. An effector element is a sequence that is expressed to achieve a desired effect, and that effect is actually achieved. Examples of effector elements include reporter genes / proteins and functional genes / proteins.
[0034] Exemplary reporter genes / proteins include those expressed by Addgene ID numbers 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), 83900 (pAAV-mDlx-GFP-Fishell-1), and 89897 (pcDNA3-FLAG-mTET2(N500)).Exemplary reporter genes include, in particular, those encoding expressible fluorescent proteins or expressible biotin: blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, Amcyan, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreen, Oregon Green (Trademark) (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric) Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red (Trademark) (Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.
[0035] GFP is composed of 238 amino acids (26.9 kDa) and was originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresce green when exposed to blue light. GFP from A. victoria has a large excitation peak at 395 nm and a smaller excitation peak at 475 nm. Its emission peak is at 509 nm, in the lower green part of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to its potential for widespread use and the evolving needs of researchers, many different variants of GFP have been created through genetic engineering. The first major improvement was a single-point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the primary excitation peak to 488 nm, with peak emission maintained at 509 nm. Adding a 37°C folding efficiency (F64L) point mutant to this skeleton yielded enhanced GFP (EGFP). EGFP is also cited as 55,000 L / (mol●cm), with a density of 9.13 × 10⁻²¹ m 2 It possesses an extinction factor (denoted by ε), also known as the optical cross-section of the molecule. Superfolder GFP, a series of mutations that rapidly fold and mature GFP even when fused to poorly folded peptides, was reported in 2006.
[0036] Yellow fluorescent protein (YFP) is a genetic variant of green fluorescent protein derived from the jellyfish Aequorea victoria. Its excitation peak is at 514 nm and its emission peak is at 527 nm.
[0037] Exemplary functional molecules include functional ion transporters, cell transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, microRNAs, or designer receptors (DREADDs) that are exclusively activated by designer drugs.
[0038] Ion transporters are transmembrane proteins that mediate the transport of ions across the cell membrane. These ion transporters permeate most cell types and are important for regulating cellular excitability and homeostasis. Ion transporters are involved in numerous cellular processes, including action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many important biological processes in living cells involve the movement of cations, such as calcium (Ca2+), potassium (K+), and sodium (Na+) ions, through such ion channels. In certain embodiments, ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).
[0039] Exemplary enzymes, transcription factors, receptors, membrane proteins, cell transport proteins, signaling molecules, and neurotransmitters include enzymes such as lactase, lipase, helicase, alpha-glucosidase, and amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAA-T / enhancer-binding protein), and Oct-1; receptors such as transforming growth factor receptor beta-1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, and interleukin-8 receptor alpha; membrane proteins and cell transport proteins, such as clathrin, dynamin, caveolin, Rab-4A, and Rab-11A; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPases, and HRas; and neurotransmitters such as cocaine and amphetamine regulatory transcripts, substance P, oxytocin, and somatostatin.
[0040] In certain embodiments, functional molecules include state-of-the-art cellular function reporters and calcium reporters. Intracellular calcium concentration is a key predictor of numerous cellular activities, including neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels is via genetically encoded calcium indicators (GECIs). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor called GCaMP is an efficient and widely used tool. GCaMP is formed by fusing the M13 and calmodulin proteins to the N-terminus and C-terminus of a cyclic mutant GFP. Some GCaMPs yield distinct fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs exhibiting green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Furthermore, GECIs exhibiting red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV8-CAG-GCaMP3 (catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog number: BS12-NXSAAV9), and AAV9-CAG-FLEX We provide AAV products including -jGCaMP7f-WPRE (catalog number: BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (catalog number: BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (catalog number: BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (catalog number: BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (catalog number: BS7-NXBAAV8).
[0041] In certain embodiments, calcium reporters include genetically encoded calcium indicators GECI, NTnC; myosin light chain kinase, GFP, calmodulin chimera; calcium indicator TN-XXL; BRET-type autoluminescent calcium indicator; and / or calcium indicator protein OeNL(Ca2+)-18u).
[0042] In certain embodiments, functional molecules include regulators of neuronal activity, such as channelrhodopsins (e.g., channelrhodopsin-1, channelrhodopsin-2, and their variants). Channelrhodopsins are a subfamily of retinilidene proteins (rhodopsins) that function as photosensitive ion channels. In addition to channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2), several channelrhodopsin variants have been developed. For example, Lin et al. (Biophys J, 2009, 96(5):1803-14) describe the creation of chimeras of the transmembrane domains of ChR1 and ChR2 in combination with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6):631-3) describe VChR1, a red-shifted channelrhodopsin variant. VChR1 exhibits low photosensitivity and poor membrane transport and expression. Other known channelrhodopsin variants include ChR2 variants described in Nagel et al., Proc Natl Acad Sci USA, 2003, 100(24):13940-5, ChR2 / H134R (Nagel, G. et al., Curr Biol, 2005, 15(24):2279-84), and ChD / ChEF / ChIEF (Lin, JY et al., Biophys J, 2009, 96(5):1803-14), which are activated by blue light (470 nm) but do not show sensitivity to orange / red light. Further variants are described in Lin, Experimental Physiology, 2010, 96.1:19-25, and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.
[0043] In certain embodiments, the functional molecule may include DNA and RNA editing tools such as CRISPR / CAS (e.g., guide RNA and nucleases such as Cas, Cas9, or cpf1). The functional molecule may also include genetically modified Cpf1, e.g., as described in US2018 / 0030425, US2016 / 0208243, WO / 2017 / 184768, and Zetsche et al. (2015) Cell 163:759-771; a single gRNA (see, e.g., Jinek et al. (2012) Science 337:816-821, Jinek et al. (2013) eLife 2:e00471, Segal (2013) eLife 2:e00563), or an editase, guide RNA molecule, microRNA, or homologous recombination donor cassette.
[0044] The sequences are publicly available. Examples include lactase (e.g., GenBank:EAX11622.1), lipase (e.g., GenBank:AAA60129.1), helicase (e.g., GenBank:AMD82207.1), amylase (e.g., GenBank:AAA51724.1), alpha-glucosidase (e.g., GenBank:ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot:P08047.3), transcription factor AP-1 (e.g., NP_002219.1), and heat shock factor proteins. 1 (e.g., UniProtKB / Swiss-Prot:Q00613.1), CCAAT / enhancer-binding protein (C / EBP) beta isoform A (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot:P14859.2), TGFβ (e.g., GenBank:CAF02096.2), platelet-derived growth factor receptor (e.g., GenBank:AAA60049.1), epidermal growth factor receptor (e.g., GenBank:CAA25240.1), vascular endothelial growth factor receptor Clathrin body (e.g., GenBank: AAC16449.2), interleukin-8 receptor alpha (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B isoform Ras-related protein Rab-a (e.g., NP_001825.1), Ras-related protein Rab-4A isoform 1 (e.g., NP_004569.2), Ras-related protein Rab-11A (e.g., UniProtKB / Swiss-Prot:P62491.3), platelet-derived growth factor (e.g., GenBank:AAA60552.1), transforming growth factor-beta-3 (e.g., GenBank:AAA61161.1), nerve growth factor (e.g., GenBank:CAA37703.1), EGF (e.g., GenBank:CAA34902.2) Cocaine and amphetamine-modulating transcripts (chain A) (e.g., PDB:1HY9_A), protachykinin-1 (e.g., UniProtKB-P20366), oxytocin-neurophysin-1 (e.g., UniProtKB-P01178), somatostatin (e.g., GenBank:AAH32625.1), genetically encoded green calcium indicator NTnC (chain A) [synthetic construct] (e.g., PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g., GenBank:ACF93133.1), BRET-based self-luminescent calcium indicator [synthetic construct] (e.g., GenBank ADF42668.1), calcium indicator protein OeNL(Ca2+)-18u [synthetic construct] ((e.g., GenBank BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (chain A) [synthetic construct] (e.g., PDB:3EKJ_A), channelopsin 1 (e.g., UniProtKB-F8UVI5), channelopsin 1 (e.g., GenBank:AER58217.1), channelrhodopsin-2 (e.g., UniProtKB-B4Y105), channelrhodopsin 2 [synthetic construct] (e.g., GenBank:ABO64386.1), CRISPR-related protein (Cas) (e.g., GenBank:AKG27598.1), Cas9 [synthetic construct] (e.g., GenBank:AST09977.1), CRISPR-related end These include nuclease Cpf1 (e.g., UniProtKB / Swiss-Prot:U2UMQ6.1), ribonuclease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease II beta (e.g., GenBank:AAF76893.1), sodium channel protein type 1 subunit alpha (e.g., UniProtKB-P35498), potassium voltage-gated channel subfamily KQT member 2 (e.g., UniProtKB-O43526), and voltage-gated L-type calcium channel subunit alpha-1C (e.g., UniProtKB-Q13936).
[0045] Further effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to codon-modified Cre. dgCre refers to an enhanced GFP / Cre recombinase fusion gene with an N-terminal fusion of the first 159 amino acids of the Escherichia coli K-12 strain chromosome dihydrofolate reductase gene (DHFR or folA) with the G67S mutation, and is modified to also include the R12Y / Y100I unstable domain mutation. FlpO refers to a codon-optimized form of FLPe that significantly increases protein expression and FRT recombination efficiency in mouse cells. Similar to the Cre / LoxP system, the FLP / FRT system is widely used for gene expression (and the generation of conditional knockout mice mediated by the FLP / FRT system). tTA2 refers to a tetracycline transactivator.
[0046] Exemplary expressible elements are expression products that do not contain effector elements, such as non-functional or defective proteins. In certain embodiments, expressible elements can provide a method for studying the effects of their functional counterparts. In certain embodiments, expressible elements are non-functional or defective based on genetically engineered mutations that render them non-functional. In these embodiments, non-expressible elements are as structurally similar as possible to their functional counterparts.
[0047] An exemplary self-cleaving peptide is the 2A peptide, which leads to the production of two proteins from a single mRNA. Because 2A sequences are short (e.g., 20 amino acids), they allow for greater use in size-restricted constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct includes an internal ribosome entry site (IRES) sequence. The IRES allows the ribosome to initiate translation at a second internal site on the mRNA molecule, leading to the production of two proteins from a single mRNA.
[0048] The coding sequences that encode the molecules described herein (e.g., RNA, proteins) can be obtained from publicly available databases and publications. The coding sequences may further include various sequence polymorphisms, mutations, and / or sequence variants, such modifications which do not affect the function of the encoded molecule. The term “encode” or “encoding” refers to the properties of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, or mRNA, that serves as a template for the synthesis of proteins and other molecules.
[0049] The term "gene" may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or terminal regions. The term may further include all introns and other DNA sequences spliced from mRNA transcripts, as well as variants arising from alternative splice sites. Sequences may also include degenerate codons of a reference sequence, or sequences that can be introduced to provide codon preference in a particular organism or cell type.
[0050] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can also be strong promoters, weak promoters, constitutive expression promoters, and / or inductive promoters. Inductive promoters induce expression in response to specific conditions, signals, or cellular events. For example, a promoter may be an inductive promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to produce transcription from the promoter. Specific examples of promoters include minBglobin, CMV, minCMV, and minCMV. * (minCMV * (minCMV is minCMV with the Sacl restriction site removed), minRho, minRho * (minRho *Examples include the minRho promoter (with the Sacl restriction site removed), the SV40 immediate early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous sarcoma virus (RSV) long-term repeat (LTR) promoter. Minimal promoters do not have the activity to drive gene expression on their own, but can be activated to drive gene expression when ligated to a proximal enhancer element.
[0051] In certain embodiments, the expression construct is provided within a vector. The term vector refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is generally ligated, for example, into the vector nucleic acid molecule, or inserted. The vector may contain sequences that direct autonomous replication within a cell, or sequences that enable integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
[0052] The term "viral vector" is widely used to refer to nucleic acid molecules containing virus-derived nucleic acid elements that facilitate the transfer and expression of non-native nucleic acid molecules into cells. The term "adeno-associated virus vector" primarily refers to viral vectors or plasmids containing structural and functional genetic factors, or parts thereof, derived from AAV. The term "retroviral vector" primarily refers to viral vectors or plasmids containing structural and functional genetic factors, or parts thereof, derived from retroviruses, etc. The term "lentiviral vector" primarily refers to viral vectors or plasmids containing structural and functional genetic factors, or parts thereof, derived from lentiviruses, etc. The term "hybrid vector" refers to a vector containing structural and / or functional genetic factors derived from two or more viral types.
[0053] An adenovirus vector is a construct that (a) supports the packaging of an artificial expression construct and (b) contains sufficient adenovirus sequences to express the cloned coding sequence in either sense or antisense direction. Recombinant adenovirus vectors contain genetically modified forms of adenoviruses. Knowledge of the genetic structure of adenoviruses, which are 36kb linear double-stranded DNA viruses, allows for the substitution of large elements of adenovirus DNA with foreign sequences of up to 7kb. In contrast to retroviruses, adenovirus DNA can replicate in an episomal manner without potential genotoxicity, so adenovirus infection of host cells does not lead to chromosomal integration. Furthermore, adenoviruses are structurally stable, and no genomic rearrangements have been detected after extensive amplification.
[0054] Adenoviruses are particularly well-suited for use as gene transfer vectors due to their medium genome size, ease of handling, high titer, broad target cell range, and high infectivity. The viral genome contains 100-200 base pair reverse repeats (ITRs) at both ends, which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in regulating the transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) leads to the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell blockade. The late gene product, which contains most of the viral capsid proteins, is expressed only after significant treatment of a single primary transcript derived from the major late promoter (MLP). MLP is particularly efficient in the later stages of infection, and all mRNA derived from this promoter has a 5'-tripartite leader (TPL) sequence, which is preferred for translation.
[0055] Aside from the requirement that the adenovirus vector is replication-deficient, or at least conditionally defective, the properties of the adenovirus vector are not considered important for the successful practice of the particular embodiments disclosed herein. The adenovirus may be any of the 42 different known serotypes or subgroups A-F. In certain embodiments, adenovirus type 5 of subgroup C is a preferred starting material for obtaining a conditionally replication-deficient adenovirus vector for use in those embodiments, namely because adenovirus type 5 is a human adenovirus for which much of the biochemical and genetic information is publicly known, and because it has been historically used in most constructs that use adenovirus as a vector.
[0056] As explicitly stated, a typical vector is a replication defect and lacks the adenovirus E1 region. Therefore, it would be most convenient to introduce the polynucleotide encoding the target gene at the location where the E1 coding sequence is missing. However, the insertion site of the construct within the adenovirus sequence is not critical. The polynucleotide encoding the target gene can also be inserted in place of the deleted E3 region in an E3 substitution vector or helper cell line, or in the E4 region where a helper virus complements the E4 deficiency.
[0057] Adeno-associated virus (AAV) is a parvovirus discovered as a contaminant of adenovirus strains. It is a ubiquitous virus not associated with any disease (antibodies are present in 85% of the US human population). It is also classified as a helper virus because its replication depends on the presence of helper viruses such as adenoviruses. Various serotypes have been isolated, of which AAV-2 is the most characterized. AAV has single-stranded linear DNA that is encapsulated in capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20-24 nm.
[0058] The AAV DNA is 4.7 kilobases long. It contains two open reading frames and two adjacent ITRs. The AAV genome has two main genes: rep and cap. The rep gene codes for proteins involved in viral replication, while cap codes for capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis-component of AAV for chromosomal integration. Therefore, AAV can be used as a vector with all viral coding sequences removed and replaced with a cassette of genes for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their mapped locations. Transcription from p5 and p19 results in the production of rep proteins, while transcription from p40 produces capsid proteins.
[0059] AAVs are noteworthy for use within this disclosure due to their superior safety profile, as well as the fact that their capsids and genomes can be modified to enable expression in selected cell populations. scAAV refers to self-complementary AAVs. pAAV refers to plasmid adeno-associated viruses. rAAV refers to recombinant adeno-associated viruses.
[0060] Other viral vectors can be used. For example, vectors derived from viruses such as vaccinia virus, poliovirus, and herpesvirus can be used. These offer several attractive properties to various mammalian cells.
[0061] Retroviruses are a common tool for gene delivery. A "retrovirus" is an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates that genomic DNA into the host genome. Once integrated into the host genome, the virus is called a "provirus." The provirus functions as a template for RNA polymerase II, inducing the expression of RNA molecules that encode structural proteins and enzymes necessary to produce new viral particles.
[0062] Exemplary retroviruses suitable for use in specific embodiments include: Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.
[0063] "Lentivirus" refers to a group (or genus) of compound retroviruses. Exemplary lentiviruses include: HIV (human immunodeficiency virus, including HIV types 1 and HIV 2); bisnamedivirus (VMV); capriculoarthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector skeleton (i.e., an HIV cis-action sequence element) may be used.
[0064] Improved safety for using certain vectors can be achieved by substituting the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used for this purpose include, for example, the promoters for virulent monkey virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase). Typical promoters can drive high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to produce a reproducible virus because the complete U3 sequence is not present in the viral production system. In certain embodiments, heterologous promoters have further advantages in controlling how the viral genome is transcribed. For example, heterologous promoters can be inducible so that transcription of all or part of the viral genome occurs only when an inducer is present. Inducing factors include one or more chemical compounds, or physiological conditions for culturing host cells, such as temperature or pH.
[0065] In certain embodiments, the viral vector includes a TAR element. The term "TAR" refers to the "transactivation response" gene element located in the R region of the lentiviral LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required in embodiments where the U3 region of the 5' LTR is replaced with a heterologous promoter.
[0066] The "R region" refers to a region within the retroviral LTR that begins at the start of the capping group (i.e., at the start of transcription) and ends immediately before the start of the poly(A) region. The R region is also defined as being adjacent to the U3 and U5 regions. The R region plays a role in enabling the transfer of nascent DNA from one end of the genome to the other during reverse transcription.
[0067] In certain embodiments, the expression of heterologous sequences in viral vectors is enhanced by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. Various post-transcriptional regulatory elements can enhance the expression of heterologous nucleic acids. Examples include the woodchuck virus post-transcriptional regulatory element (WPRE, Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and similar elements (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector includes a post-transcriptional regulatory element such as WPRE or HPRE. In certain embodiments, the vector lacks or does not include a post-transcriptional regulatory element such as WPRE or HPRE.
[0068] Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts can enhance heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector contains a 3' polyadenylation sequence of the polynucleotide encoding the molecule to be expressed (e.g., a protein). The term "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Certain embodiments may utilize BGHpA or SV40pA. In certain embodiments, a preferred embodiment of the expression construct includes a terminator element. These elements can help increase transcription levels and minimize reading from the construct to other plasmid sequences.
[0069] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may contribute to protecting viral vector expression sequences, such as effector elements or expressible elements, from integrated site effects (i.e., positional effects, see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002, and Zhan et al., Hum. Genet., 109:471, 2001), which may result in uncontrolled expression of the import sequence, mediated by cis-acting elements present in the genomic DNA. In certain embodiments, the viral import vector comprises one or more insulator elements in the 3'LTR, and upon integration of the provirus into the host genome, the provirus replicates the 3'LTR, thereby comprising one or more insulators in both the 5'LTR and 3'LTR. Suitable insulators for use in specific embodiments include chicken β-globin insulators (Chung et al. Cell 74:505, 1993, Chung et al., PNAS USA 94:575, 1997, and Bell et al. Cell 98:387, 1999), SP10 insulators (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF-recognizing sequences that function as enhancer-blocking insulators (Liu et al. Nature, Biotechnology, 33:198, 2015).
[0070] Beyond the foregoing description, a wide range of suitable expression vector types are known to those skilled in the art. These may include commercially available expression vectors designed for common recombination procedures, such as plasmids containing one or more reporter genes and regulatory elements necessary for the expression of a reporter gene in cells. Numerous vectors are commercially available from, for example, Invitrogen, Stratagene, Clontech, and others, and are described in many relevant guides. In certain embodiments, suitable expression vectors may include any plasmid, cosmid, or phage construct capable of supporting the expression of an encoding gene in mammalian cells, such as the pUC or Bluescript plasmid series.
[0071] Specific embodiments of the vectors disclosed herein include: [Table 1-1] [Table 1-2]
[0072] Those skilled in the art can readily identify the sequences of subcomponents within a large vector sequence, and can readily identify them based on the content of this disclosure (see Figure 15). The identifiable and enumerated nucleotides between the subcomponents reflect restriction enzyme recognition sites used in construct assembly (cloning), and in some cases, further nucleotides do not convey identifiable function. These segments of the complete vector sequence can be adjusted based on different cloning strategies and / or the use of the vector. Generally, short six-nucleotide palindromic sequences reflect vector construction artifacts that are not important to the vector function.
[0073] In certain embodiments, a vector having a capsid that crosses the blood-brain barrier (BBB) (e.g., AAV) is selected. In certain embodiments, the vector is modified to include a capsid that crosses the BBB. Examples of AAVs with viral capsids that can cross the blood-brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014;7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014;22(7): 1299-1309), AAV1R6, AAV1R7 (Albright et al., Mol Ther. 2018;26(2): 510), rAAVrh.8 (Yang, et al., supra), AAV-BR1 (Marchio et al., EMBO Mol Med. 2016;8(6): 592), AAV-PHP.S (Chan et al., Nat Neurosci. 2017;20(8): 1172), and AAV-PHP.B (Deverman et al., Nat Biotechnol. These include 2016;34(2): 204), AAV-PPS (Chen et al., Nat Med. 2009;15: 1215), and PHP.eB. In certain embodiments, the PHP.eB capsid, unlike AAV9, references AAV9, and the amino acid S-AQ-A (SEQ ID NO: 199), which begins at residue 586, is changed to S-DGTLAVPFK-A (SEQ ID NO: 200). In certain embodiments, PHP.eB references SEQ ID NO: 50.
[0074] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the blood-brain barrier (BBB) after intravenous injection. It transforms a large portion of the central nervous system (CNS) and thus enables minimally invasive treatments, as described in relation to clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and CLN3 gene-associated neuronal ceroid-lipofuse syndrome (NCT03770572) (Naso et al., BioDrugs. 2017;31(4):317).
[0075] AAVrh.10 was originally isolated from rhesus monkeys and, compared to other common serotypes used for gene delivery, exhibits low seropositivity in humans (see Selot et al., Front Pharmacol. 2017;8:441). It has been evaluated in clinical trials for LYS-SAF302, LYSOGENE, and NCT03612869.
[0076] Two mutants isolated from a library of chimeric AAV vectors, AAV1R6 and AAV1R7 (in which the AAV1 capsid domain was replaced with AAVrh.10), retained the ability to cross the blood-brain barrier and transduce the central nervous system (CNS), while showing a significant reduction in hepatic and vascular endothelial transduction.
[0077] Similarly, rAAVrh.8 isolated from rhesus macaques exhibits global transduction of glial and neuronal cell types in clinically important areas after peripheral administration, and also shows reduced peripheral tissue tropism compared to other vectors.
[0078] AAV-BR1 is an AAV2 variant that displays the NRGTEWD (SEQ ID NO: 201) epitope, isolated during in vivo screening of a random AAV display peptide library. It exhibits high specificity with high transgene expression in the brain and minimal off-target affinity (including in the liver) (Korbelin et al., EMBO Mol Med. 2016;8(6):609).
[0079] AAV-PHP.S (Addgene, Watertown, MA) is a mutant of AAV9 created using the CREATE method, encoding the 7-mer sequence QAVRTSL (sequence number 202), and transducing neurons in the enteric nervous system, thereby strongly transducing peripheral sensory afference into the spinal cord and brainstem.
[0080] AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method, encoding the 7-mer sequence TLAVPFK (SEQ ID NO: 203). It translocates genes across the entire CNS with higher efficiency than AAV9, transducing a large portion of astrocytes and neurons across multiple CNS regions.
[0081] AAV-PPS was created by inserting the DSPAHPS (SEQ ID NO: 204) epitope into the capsid of AAV2, and it shows dramatically improved brain tropism compared to AAV2.
[0082] For further information on capsids that cross the blood-brain barrier, see Chan et al., Nat. Neurosci, 2017 Aug:20(8):1172-1179.
[0083] (ii) Compositions for administration. The artificial expression constructs and vectors of this disclosure (hereinafter referred to as physiologically active components) can be formulated with carriers suitable for administration to cells, tissue slices, animals (e.g., mice, non-human primates), or humans. The physiologically active components in the compositions described herein can be prepared in a neutral form, as free bases or as pharmacokinetically acceptable salts.
[0084] As pharmaceutically acceptable salts, there are acid addition salts (formed from free amino groups of proteins), which are formed from inorganic acids, such as hydrochloric acid or phosphoric acid, or from organic acids, such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed from free carboxyl groups can be obtained from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as from organic bases, such as isopropylamine, trimethylamine, histidine, and procaine.
[0085] Carriers for physiologically active components can include solvents, dispersion media, vehicles, coatings, diluents, isotonic agents, and absorption retarders, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active components is well known in the art. Any conventional culture medium or agent can be used with the compositions described herein, except when the conventional medium or agent is incompatible with the physiologically active component.
[0086] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic or similar adverse reactions when administered to humans, and in certain embodiments, when administered intravenously (for example, into the posterior orbital plexus).
[0087] In certain embodiments, the composition may be formulated for intravenous, intraparenchymal, intraocular, intravitreous, parenteral, subcutaneous, intraventricular, intramuscular, subarachnoid, intraspinal, intraperitoneal, oral or intranasal inhalation, or for direct injection or application to one or more cells, tissues, or organs.
[0088] The composition may include liposomes, lipids, lipid complexes, microspheres, fine particles, nanospheres, and / or nanoparticles.
[0089] The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulating half-lives have been developed (see, for example, U.S. Patent No. 5,741,516). Furthermore, various methods for liposomes and liposome-like preparations as potential drug carriers have been described (see, for example, U.S. Patents No. 5,567,434; No. 5,552,157; No. 5,565,213; No. 5,738,868; and No. 5,795,587).
[0090] Furthermore, this disclosure provides pharmaceutically acceptable nanocapsule formulations of physiologically active components. Nanocapsules can generally encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al. Drug Dev Ind Pharm 24(12):1113-1128,1998; Quintanar-Guerrero et al. Pharm Res. 15(7):1056-1062,1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119,1998; Douglas et al. Crit, Rev Ther Drug Carrier Syst 3(3):233-261,1987). To avoid side effects due to intracellular polymer overload, such ultrafine particles can be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are planned for use in this disclosure. Such particles can be readily produced as described in Couvreur et al., J Pharm Sci 69(2):199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlenet al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998, and U.S. Patent No. 5,145,684.
[0091] The injectable composition may include a sterile aqueous solution, or a dispersion and sterile powder for preparing a sterile injectable solution or dispersion at the time of use (U.S. Patent No. 5,466,468). For delivery by injection, the form is sterile and fluid enough to be delivered by syringe. In certain embodiments, the composition is stable under manufacturing and storage conditions and optionally contains one or more preservative compounds to protect against microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, maintaining the required particle size in the case of a dispersion, and / or by using a surfactant. Prevention of microbial action can be achieved with various antimicrobial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In various embodiments, the preparation may include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectable composition can be achieved by including agents that delay absorption into the composition, such as aluminum monostearate and gelatin. The injectable composition may be appropriately buffered as needed, and the liquid diluent may first be made isotonic with sufficient saline or glucose.
[0092] The dispersion may be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. As shown, under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth.
[0093] Sterile compositions can be prepared by incorporating physiologically active components into an appropriate amount of a solvent containing other optional components (e.g., those described above), followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized physiologically active components into a sterile vehicle containing a basic dispersion medium and other necessary components (e.g., those described above). In the case of sterile powders for preparing sterile injection solutions, preferred preparation methods may be vacuum drying and freeze-drying techniques, from which powders of physiologically active components and any further desired components are obtained from a pre-sterilized filtered solution.
[0094] Oral compositions may be in liquid form, for example, as a solution, syrup, or suspension, or they may be presented as drug products to be reconstituted with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoic acid or sorbic acid). The composition may take the form of tablets or capsules prepared with pharmaceutically acceptable excipients such as, for example, binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art.
[0095] The inhalable composition may be delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer, using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dose unit may be determined by providing a valve for delivering the measured amount. Capsules and cartridges, for example, gelatin capsules and cartridges for use in inhalers or fillers, may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.
[0096] The composition also includes a microchip device (U.S. Patent No. 5,797,898), an ophthalmic formulation (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), a transdermal matrix (U.S. Patents No. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).
[0097] Furthermore, auxiliary active ingredients can also be incorporated into the composition.
[0098] Typically, a composition may contain at least 0.1% of a physiologically active component, although the proportion of the physiologically active component can certainly vary, and conveniently, this could be 1 or 2% to 70% or 80% or more, or 0.5% to 99%, of the total weight or volume of the composition. Naturally, the amount of physiologically active component in each physiologically useful composition can be prepared in such a way that a suitable dose is obtained for any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are planned by those skilled in the art who prepare such pharmaceutical formulations, and therefore, various compositions and dosages may be desirable.
[0099] In certain embodiments, for administration to humans, the composition should meet sterility, pyrogenicity, and general safety and purity standards required by the U.S. Food and Drug Administration (FDA) or other applicable regulatory bodies in the country.
[0100] (iii) Cell lines containing the artificial expression construct. This disclosure includes cells containing the artificial expression construct described herein. Cells transformed with the artificial expression construct can be used for a number of purposes, including neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to evaluate the regulatory properties of enhancers.
[0101] Various host cell lines can be used, but in certain embodiments, the cells are mammalian cells. In certain embodiments, the artificial expression constructs are eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_060m, eHGT_060h , MGT_E36, MGT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_104m, eHGT_107h, eHGT_340m, eHGT_528h, eHGT_515h, 3xCore2_eHGT_2 26h, 3xCore3_eHGT_226h, eHGT_682h, eHGT_600m, eHGT_759m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT_131 hv2, eHGT_130h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E81, MGT_E85, MGT_E88, and / or MGT_E83 and / or CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567, CN1626, CN1 712, CN1700, CN1607, CN1605, CN1556, CN1526, CN1418, CN1404, AiV1173, AiV1174, AiV1177, CN1261, CN1542, CN1544, CN1598, CN1553, CN1992, CN2367, CN2357, CN2568, CN2569, CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309, CN2366, CN2257, CN1667,The cell lines include CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, and / or AiP1273, and the cell lines are human, primate, or mouse cells. The cell lines available for transformation in this disclosure also include primary cell lines derived from living tissues such as rat or mouse brains, and organ-type cell cultures containing brain slices derived from animals such as rats or mice. The PC12 cell line (available from American Type Culture Collection, ATCC, Manassas, VA) has been shown to express numerous neuronal marker proteins in response to neuronal growth factor (NGF). The PC12 cell line is considered a neuronal cell line and is available for use in this disclosure. JAR cells (available from ATCC) are platelet-derived cell lines expressing certain neuronal genes, such as the serotonin transporter gene, and may be used in conjunction with the embodiments described herein.
[0102] WO91 / 13150 describes various cell lines, including neuronal cell lines, and methods for producing them. Similarly, WO97 / 39117 describes neuronal cell lines and methods for producing such cell lines. The neuronal cell lines disclosed in these patent applications are applicable to use in this disclosure.
[0103] In certain embodiments, “neuronal” means a nerve cell, related to a nerve cell, or containing a nerve cell. A nerve cell is defined by the presence of an axon and dendrites. The term “neuronal-specific” means an activity found or occurring in nerve cells or cells derived from nerve cells that is not found, does not occur, or substantially not found or substantially not occurring in non-nerve cells or cells not derived from nerve cells, such as glial cells like astrocytes or oligodendrocytes.
[0104] In certain embodiments, non-neuronal cell lines, including mouse embryonic stem cells, may be used. Using cultured mouse embryonic stem cells, gene construct expression can be analyzed using transient transfection with plasmid constructs. Mouse embryonic stem cells are pluripotent and undifferentiated. These cells can be maintained in this undifferentiated state by leukemia inhibitors (LIFs). LIF withdrawal induces differentiation of embryonic stem cells. In culture, stem cells form various differentiated cell types. Differentiation is triggered by the expression of tissue-specific transcription factors, and the function of enhancer sequences can be evaluated. (See, for example, Fiskerstrand et al., FEBS Lett 458:171-174, 1999.)
[0105] Methods for differentiating stem cells into different cell types include replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF) heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenate), laminin, and polyornithine. The process for producing myelinated oligodendrocyte cells from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. Bibel, et al., 2007, Nat. Protoc. 2:1034-43 describes a protocol for producing glutamatergic neurons from stem cells, while Chatzi, et al., 2009, Exp. Neurol. 217:407-16 describes a procedure for producing GABAergic neurons. This procedure involves exposing stem cells to total trans RA for 3 days. Subsequently, after culturing in serum-free neuronal induction medium containing Neurobasal medium supplemented with B27, bFGF, and EGF, 95% of the GABA neurons grow.
[0106] U.S. Publication No. 2012 / 0329714 describes increasing the number of neural stem cells using prolactin, and U.S. Publication No. 2012 / 0308530 describes a culture surface with amino groups that promotes neuronal differentiation into neurons, astrocytes, and oligodendrocytes. Therefore, the fate of neural stem cells can be controlled by various extracellular factors. Commonly used factors include brain-derived growth factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425); fibroblast growth factor (bFGF; U.S. Patent No. 5,766,948; FGF-1, FGF-2); neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4); Caldwell, et al., 2001, Nat. Biotechnol. 1; 19:475-9); ciliary neurotrophic factor (CNTF); BMP-2 (U.S. Patent Nos. 5,948,428 and 6,001,654); and isobutyl These include 3-methylxanthine; leukemia-suppressing growth factor (LIF; U.S. Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transformation of growth factor-α and TGF-β (U.S. Patents No. 5,851,832 and 5,753,506).
[0107] In certain embodiments, a yeast one-hybrid system is used for eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, eHGT_121h, eHGT_1 33h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_060m, eHGT_060h , MGT_E36, MGT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_104m, eHGT_107h, eHGT_340 m, eHGT_528h, eHGT_515h, 3xCore2_eHGT_226h, 3xCore3_eHGT_226h, eHGT_682h, eHGT_600m, eHGT_ 759m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT_131hv2, eHGT_13 Compounds that inhibit specific protein / DNA interactions, such as the transcription factors of their cores, may also be identified, including MGT_E81, MGT_E85, MGT_E88, or MGT_E83.
[0108] Transgenic animals are described below. Cell lines may also be derived from such transgenic animals. For example, primary tissue cultures derived from transgenic mice (as described below, for example) can provide cell lines that have artificial expression constructs already incorporated into the genome (see, for example, MacKenzie and Quinn, Proc Natl Acad Sci USA 96:15251-15255, 1999).
[0109] (iv) Transgenic animals. Another aspect of the present disclosure includes transgenic animals whose genomes are operably linked to heterologous coding sequences eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHG T_354m, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT _080h, eHGT_060m, eHGT_060h, MGT_E36, MGT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eH GT_104m, eHGT_107h, eHGT_340m, eHGT_528h, eHGT_515h, 3xCore2_eHGT_226h, 3xCore3_eHGT_22 6h, eHGT_682h, eHGT_600m, eHGT_759m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, Includes artificial expression constructs containing eHGT_519h, eHGT_131hv2, eHGT_130h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E81, MGT_E85, MGT_E88, and / or MGT_E83. Concatemers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core may also be used. In certain embodiments, the genomes of transgenic animals are CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567, CN1626, CN1712, CN1700, CN1607, CN1605, CN1556, CN1526, CN1418, CN1404, AiV1173, AiV1174, AiV1177, CN1261, CN1542, CN1544, CN1598, CN1553, CN1992, CN2367, CN2357, CN2568, CN2569,Including CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309, CN2366, CN2257, CN1667, CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, and / or AiP1273. In certain embodiments, when non-embedded vectors are used, the transgenic animal expresses artificial constructs eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, eHGT_121h, eHGT_133h, eHGT_21 9h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_060m, eHGT_060h, MGT_E36, MGT_E37, MGT_E41, eHGT_0 25h, eHGT_096h, eHGT_098h, eHGT_104m, eHGT_107h, eHGT_340m, eHGT_528h, eHGT_515h, 3xCore2_eHGT_226h, 3xCore3_eHG T_226h, eHGT_682h, eHGT_600m, eHGT_759m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT_13 1hv2, eHGT_130h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E81, MGT_E85, MGT_E88, and / or MGT_E83, and / or CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567, CN1626, CN1712, CN1700, CN1607, CN1605, CN1556, CN1526, CN1418, CN1404, AiV1173, AiV1174,Includes AiV1177, CN1261, CN1542, CN1544, CN1598, CN1553, CN1992, CN2367, CN2357, CN2568, CN2569, CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309, CN2366, CN2257, CN1667, CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, and / or AiP1273. Concatenators of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core can also be used.
[0110] Detailed methods for producing transgenic animals are described in U.S. Patent No. 4,736,866. Transgenic animals may be any non-human species, but preferably include non-human primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens, and rodents, such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.
[0111] In certain embodiments, the construction of a transgenic animal results in an organism having a genetically modified construct present in all cells within the same genomic integration site. Therefore, cell lines derived from such transgenic animals will be as consistent in all cells as if the genetically modified construct were located in the same genomic integration site, and thus will undergo the same position-effect mutations. In contrast, introducing a gene into a cell line or primary cell culture can result in heterologous expression of the construct. A drawback of this approach is that the expression of the introduced DNA may be influenced by the specific genetic background of the host animal.
[0112] As shown above in relation to cell lines, the artificial expression constructs of this disclosure 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. The transformed ES cells are then injected into a blastocyst from a host mother, and the host embryo is re-transplanted into the mother. This results in a chimeric mouse whose tissues are composed of cells derived from both embryonic stem cells present in the cultured cell line and embryonic stem cells present in the host embryo. Typically, the mice from which the cultured ES cells used for introduction originate are selected to have a different coat color from the host mouse into which the transformed cells are injected into its embryo. The chimeric mice then have a variety of coat colors. At least some of these can be crossed with a suitable strain to produce offspring that carry the transgene, as long as the germline tissues are derived from genetically modified cells.
[0113] In addition to the delivery methods described above, the following techniques are also intended as alternative methods for delivering artificial expression constructs to target cells or selected tissues and organs of animals, particularly cells, organs, or tissues of vertebrates: ultrasound (e.g., ultrasound as described in U.S. Patent No. 5,656,016); intraosseous injection (U.S. Patent No. 5,779,708); microchip devices (U.S. Patent No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrices (U.S. Patents No. 5,770,219 and U.S. Patent No. 5,783,208); feedback-controlled delivery (U.S. Patent No. 5,697,899); and other delivery methods available and / or described elsewhere in this disclosure.
[0114] (v) Method of use. In certain embodiments, compositions containing the physiologically active ingredients described herein are administered to a subject to produce a physiological effect.
[0115] In certain embodiments, this disclosure includes the use of the artificial expression constructs described herein to modulate the expression of heterologous genes partially or completely encoded at a downstream position of an enhancer in a genetically engineered sequence. Thus, methods of using the disclosed artificial expression constructs in the research, clinical trials, and potential development of pharmaceuticals for preventing, treating, or mitigating symptoms of disease, dysfunction, or impairment are provided herein.
[0116] Specific embodiments are described herein as eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_060m, eHGT _060h, MGT_E36, MGT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_104m, eHGT_107h, eHGT_340m, eHGT_528h, eHGT_515h, 3xCore 2_eHGT_226h, 3xCore3_eHGT_226h, eHGT_682h, eHGT_600m, eHGT_759m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519 h, eHGT_131hv2, eHGT_130h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E81, MGT_E85, MGT_E88, and / or MGT_E83, and / or CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567,CN1626,CN1712,CN1700,CN1607,CN1605,CN1556,CN1526,CN1418,CN1404,AiV1173,AiV1174,AiV1177,CN1261,CN1542,CN1544,C N1598, CN1553, CN1992, CN2367, CN2357, CN2568, CN2569, CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309Administering to an artificial expression construct comprising CN2366, CN2257, CN1667, CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, and / or AiP1273, and comprising a method of driving selective expression of a gene in a selected cell type. One, two, four, five, six, seven, eight, nine, or ten copies of the disclosed enhancer core concatemer 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 art, the dosage of any one subject depends on many factors, including the size, surface area, age, particular compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously, of the subject. The dosages of the compounds of the present disclosure vary, but in certain embodiments, the dosage can be 10 5 ~10 100 copies of the artificial expression construct of the present disclosure. In certain embodiments, a patient receiving intravenous, intrasubstantial, intraspinal, retroorbital, or intrathecal administration can be injected with 10 6 ~10 22 copies of the artificial expression construct.
[0118] An "effective amount" is the amount of a composition necessary to effect a desired physiological change in a subject. An effective amount is often administered for research purposes. The effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay.
[0119] The amount and duration of administration of such expression constructs will be within the scope of those skilled in the art who benefit from this teaching. However, administration of an effective amount of the disclosed composition is likely to be achieved by a single dose, such as a single injection of a sufficient number of infectious particles to produce an effect on the subject. Alternatively, in some situations, it may be desirable to provide multiple or consecutive doses of the artificial expression construct composition or other gene construct over a relatively short or relatively long period, as can be determined by the individual supervising the administration of such composition. For example, the number of infectious particles administered to a mammal may be given as a single dose or divided into two or more doses, as required to achieve the intended effect. 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or even higher infectivity particles / ml. In fact, in certain embodiments, it may be desirable to administer two or more different expression constructs in combination to achieve the desired effect.
[0120] In certain circumstances, it may be desirable to deliver the artificial expression construct in a suitably formulated composition disclosed herein by any of the following means: pipetting, posterior orbital injection, subcutaneous, intraocular, intravitreous, parenteral, subcutaneous, intravenous, intraparenchymal, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or direct application or injection into one or more cells, tissues, or organs. The methods of administration may also include those forms as described in U.S. Patents 5,543,158, 5,641,515, and 5,399,363.
[0121] (vi) Kits and commercially available packages. Kits and commercially available packages contain the artificial expression constructs described herein. The artificial expression constructs can be isolated. In certain embodiments, the components of the expression product can be isolated from each other. In certain embodiments, the expression product may be in a vector, in a viral vector, in an intracellular, in a tissue slice or sample, and / or in a transgenic animal. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or means of delivering compositions such as syringes or injectable preparations.
[0122] Embodiments of the kit or commercially available package also include, for example, a description of the use of the components contained herein in basic research, electrophysiological research, neuroanatomical research, and / or research and / or treatment of disorders, diseases or conditions.
[0123] The following exemplary embodiments and experimental examples are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art will understand that many modifications can be made to the specific embodiments disclosed herein in light of the Disclosure, and that similar or comparable results can be obtained without departing from the spirit and scope of the Disclosure.
[0124] (vii) Exemplary embodiments. 1. A concatenated core containing 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence numbers 161, 163, and 165. 2. The linked enhancer or enhancer core according to Embodiment 1, wherein the linked enhancer or enhancer core includes sequence number 162, sequence number 164, sequence number 166, or 166. 3.(i)eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHG T_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, eHGT_121h,eHGT_133h, eHGT_219h, e HGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_060m, eHGT_060h, MGT_E36, M GT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_104m, eHGT_107h, eHGT_340m, eHGT _528h, eHGT_515h, 3xCore2_eHGT_226h, 3xCore3_eHGT_226h, eHGT_682h, eHGT_600m, eHGT_7 59m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT_131hv2, eHGT (ii) promoters; and (iii) artificial expression constructs containing heterologous coding sequences. 4. The artificial expression construct according to Embodiment 3, wherein the heterogeneous code sequence codes for an effector element or an expressible element. 5. The artificial expression construct according to Embodiment 3 or 4, wherein the effector element comprises a reporter protein or a functional molecule. 6. The artificial expression construct according to Embodiment 5, wherein the reporter protein comprises a fluorescent protein. 7. An artificial expression construct according to Embodiment 5 or 6, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or a designer receptor (DREADD) that is exclusively activated by a designer drug. 8. An artificial expression construct according to any of Embodiments 4, wherein the expressible element includes a non-functional molecule. 9. The artificial expression construct according to Embodiment 8, wherein the non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD. 10. The artificial expression construct according to any one of embodiments 3 to 9, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier. 11. The artificial expression construct according to Embodiment 10, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 12. An artificial expression construct according to any one of embodiments 3 to 11, wherein the artificial expression construct includes or encodes a skipping element. 13. The artificial expression construct according to Embodiment 12, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 14. The artificial expression construct according to Embodiment 13, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 15. The artificial expression constructs are: eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_354h, eHGT_354m, eHGT_121h, eHGT_133h, eHGT_219h , eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_060m, eHGT_060h, MGT_E36, MG T_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_104m, eHGT_107h, eHGT_340m, eHGT_528 h, eHGT_515h, 3xCore2_eHGT_226h, 3xCore3_eHGT_226h, eHGT_682h, eHGT_600m, eHGT_759m, eHG T_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT_131hv2, eHGT_130h, eHGT _527h, eHGT_470m, eHGT_174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E 81, MGT_E85, MGT_E88, MGT_E83, hsA2, AAV, scAAV, rAAV, minBglobin, CMV, minCMV, minRho, minRho * An artificial expression construct according to any of Embodiments 2 to 14, comprising or encoding a set of features selected from fluorescent proteins (e.g., EGFP, SYFP, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), WPRE, WPRE3, hGHpA, and / or BGHpA. 16. The artificial expression constructs are: hsA2-eHGT_089h-minRho-[heterogeneous coding sequence]-WPRE3-BGHpA;hsA2-eHGT_087h-minRho-[heterogeneous coding sequence]--WPRE3-BGHpA;hsA2-eHGT_154h-minRho-[heterogeneous coding sequence]--WPRE3-BGHpA;hsA2-eHGT_226h-minRho-[heterogeneous coding sequence]--WPRE3-BGHpA;eHGT_526h-minBglobin-[heterogeneous coding sequence]--WPRE3-BGHpA;eHGT _512h-minBglobin-[heterogeneous code sequence]--WPRE3-BGHpA;hsA2-eHGT_283h-minRho-[heterogeneous code sequence]--WPRE3-BGHpA;hsA2-eHGT_090m-minRho-[heterogeneous code sequence]--WPRE3-BGHpA;eHGT_076h-minBglobin-[heterogeneous code sequence]--WPRE3-BGHpA;eHGT_072h-minBglobin-[heterogeneous code sequence]--WPRE3-BGHpA;3xSP10ins-eHGT_354h-min Rho*-[different code sequence]--WPRE3-BGHpA;3xSP10ins-eHGT_354m-minRho*-[different code sequence]--WPRE3-BGHpA;hsA2-eHGT_121h-minRho-[different code sequence]--WPRE3-BGHpA;hsA2-eHGT_133h-minRho-[different code sequence]--WPRE3-BGHpA;hsA2-eHGT_219h-minRho-[different code sequence]--WPRE3-BGHpA;hsA2-eHGT_207h-minRho-[different code sequence] [Code Array]--WPRE3-BGHpA;hsA2-eHGT_113m-minRho-[Different Code Array]--WPRE3-BGHpA;hsA2-eHGT_111m-minRho-[Different Code Array]--WPRE3-BGHpA;hsA2-eHGT_110h-minRho-[Different Code Array]--WPRE3-BGHpA;hsA2-eHGT_080h-minRho-[Different Code Array]--WPRE3-BGHpA;eHGT_060m-minBglobin-[Different Code Array]--WPRE3-BGHpA;eHGT_060h-minBglobin-[heterogeneous code sequence]--WPRE3-BGHpA;MGT_E36-minBglobin-[heterogeneous code sequence]--WPRE-hGHpA;MGT_E37-minBglobin-[heterogeneous code sequence]--WPRE-hGHpA;MGT_E41-minBglobin-[heterogeneous code sequence]--WPRE-hGHpA;eHGT_025h-minBGlobin-[heterogeneous code sequence]-WPRE3-BGHpA;hsA2-eHGT_096h-minRho-[heterogeneous code sequence]-W PRE3-BGHpA;hsA2-eHGT_098h-minRho-[heterogeneous code sequence]-WPRE3-BGHpA;hsA2-eHGT_104m-minRho-[heterogeneous code sequence]-WPRE3-BGHpA;hsA2-eHGT_107h-minRho-[heterogeneous code sequence]-WPRE3-BGHpA;3xSP10ins-eHGT_340m-minRho*-[heterogeneous code sequence]-WPRE3-BGHpA;eHGT_528h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA;eHGT_51 5h-minBglobin-[heterogeneous code array]-WPRE3-BGHpA;3xCore2_eHGT_226h-minBglobin-[heterogeneous code array]-WPRE3-BGHpA;3xCore3_eHGT_226h-minBglobin-[heterogeneous code array]-WPRE3-BGHpA;eHGT_682h-minBglobin-[heterogeneous code array]-WPRE3-BGHpA;eHGT_600m-minBG-[heterogeneous code array]-WPRE3-BGHpA;eHGT_759m-minBglobin-[heterogeneous [Code Sequence]-WPRE3-BGHpA;eHGT_468m-minBglobin-[Different Code Sequence]-WPRE3-BGHpA;3xCore_eHGT_064h_minBglobin-[Different Code Sequence]-WPRE3-BGHpA;hsA2-eHGT_170h-minRho-[Different Code Sequence]-WPRE3-BGHpA;eHGT_131hv1-minBglobin-[Different Code Sequence]-WPRE3-BGHpA;eHGT_519h-minBglobin-[Different Code Sequence]-WPRE3-BGHpA;hsA2-eHGT_131hv2-minRho-[different code sequence]-WPRE3-BGHpA;eHGT_130h-minBglobin-[different code sequence]-WPRE3-BGHpA;eHGT_527h-minBglobin-[different code sequence]-WPRE3-BGHpA;eHGT_470m-minBglobin-[different code sequence]-WPRE3-BGHpA;hsA2-eHGT_174h-minRho-[different code sequence]-WPRE3-BGHpA;hsA2-eHGT_087m-minRho-[different code [Code Array]-WPRE3-BGHpA;hsA2-eHGT_156h-minRho-[Different Code Array]-WPRE3-BGHpA;pAAV-eHGT_338m-minBGprom-[Different Code Array]-WPRE-hGHpA;pAAV-eHGT_341m-monBGprom-[Different Code Array]-WPRE-hGHpA;pAAV-eHGT_339m-minBGprom-[Different Code Array]-WPRE-hGHpA;pAAV-MGT_E81-minBGprom-[Different Code Array]-WPRE3-bGHpA;pA AV-MGT_E85-minBGprom-[heterogeneous code sequence]-WPRE3-bGHpA;pAAV-MGT_E88-minBGprom-[heterogeneous code sequence]-WPRE3-bGHpA;pAAV-MGT_E83-minBGprom-[heterogeneous code sequence]-WPRE3-bGHpA;eHGT_089h-promoter-[heterogeneous code sequence]-[regulatory region (multiple possible)];eHGT_087h-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_154h-promoter-[heterogeneous code sequence]--[regulatory region ( (Multiple entries possible)]; eHGT_226h-promoter-[heterogeneous code sequence]--[regulatory region (multiple entries possible)]; eHGT_526h-promoter-[heterogeneous code sequence]--[regulatory region (multiple entries possible)]; eHGT_512h-promoter-[heterogeneous code sequence]--[regulatory region (multiple entries possible)]; eHGT_283h-promoter-[heterogeneous code sequence]--[regulatory region (multiple entries possible)]; eHGT_090m-promoter-[heterogeneous code sequence]--[regulatory region (multiple entries possible)]; eHGT_076h-promoter-[heterogeneous code sequence]--[regulatory region (multiple entries possible)];eHGT_072h-promoter-[heterogeneous code sequence]--[modulatory region (multiple possible)];3xSP10ins-eHGT_354h-promoter-[heterogeneous code sequence]--[modulatory region (multiple possible)];3xSP10ins-eHGT_354m-v-[heterogeneous code sequence]--[modulatory region (multiple possible)];eHGT_121h-promoter-[heterogeneous code sequence]--[modulatory region (multiple possible)];eHGT_133h-promoter-[heterogeneous code sequence]--[modulatory region (multiple possible)];eHGT_219h-v-[heterogeneous code sequence]--[modulatory region (multiple possible)] ;eHGT_207h-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_113m-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_111m-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_110h-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_080h-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_060m-promoter-[heterogeneous code sequence]--[regulatory region (multiple possible)];eHGT_060h -Promoter-[Heterogeneous Code Sequence]--[Adjustment Region (Multiple)];MGT_E36-Promoter-[Heterogeneous Code Sequence]--[Adjustment Region (Multiple)];MGT_E37-Promoter-[Heterogeneous Code Sequence]--[Adjustment Region (Multiple)];MGT_E41-Promoter-[Heterogeneous Code Sequence]--[Adjustment Region (Multiple)];eHGT_025h-minBGlobin-[Heterogeneous Code Sequence]-[Adjustment Region (Multiple)];hsA2-eHGT_096h-minRho-[Heterogeneous Code Sequence]-[Adjustment Region (Multiple)];hsA2-eHGT_098h-min Rho-[heterogeneous code sequence]-[adjustment region (multiple possible)];hsA2-eHGT_104m-minRho-[heterogeneous code sequence]-[adjustment region (multiple possible)];hsA2-eHGT_107h-minRho-[heterogeneous code sequence]-[adjustment region (multiple possible)];3xSP10ins-eHGT_340m-minRho*-[heterogeneous code sequence]-[adjustment region (multiple possible)];eHGT_528h-minBglobin-[heterogeneous code sequence]-[adjustment region (multiple possible)];eHGT_515h-minBglobin-[heterogeneous code sequence]-[adjustment region (multiple possible)];3xCore2_eHGT_226h-minBglobin-[heterogeneous code array]-[adjustable area (multiple possible)];3xCore3_eHGT_226h-minBglobin-[heterogeneous code array]-[adjustable area (multiple possible)];eHGT_682h-minBglobin-[heterogeneous code array]-[adjustable area (multiple possible)];eHGT_600m-minBG-[heterogeneous code array]-[adjustable area (multiple possible)];eHGT_759m-minBglobin-[heterogeneous code array]-[adjustable area (multiple possible)];eHGT_468m-minBglobin- [Heterogeneous code sequence]-[Adjustment region (multiple possible)];3xCore_eHGT_064h_minBglobin-[Heterogeneous code sequence]-[Adjustment region (multiple possible)];hsA2-eHGT_170h-minRho-[Heterogeneous code sequence]-[Adjustment region (multiple possible)];eHGT_131hv1-minBglobin-[Heterogeneous code sequence]-[Adjustment region (multiple possible)];eHGT_519h-minBglobin-[Heterogeneous code sequence]-[Adjustment region (multiple possible)];hsA2-eHGT_131hv2-minRho-[Heterogeneous code sequence]-[Adjustment region (multiple possible)] )];eHGT_130h-minBglobin-[heterogeneous code sequence]-[adjustment region (multiple possible)];eHGT_527h-minBglobin-[heterogeneous code sequence]-[adjustment region (multiple possible)];eHGT_470m-minBglobin-[heterogeneous code sequence]-[adjustment region (multiple possible)];hsA2-eHGT_174h-minRho-[heterogeneous code sequence]-[adjustment region (multiple possible)];hsA2-eHGT_087m-minRho-[heterogeneous code sequence]-[adjustment region (multiple possible)];hsA2-eHGT_156h-minRho-[heterogeneous code sequence] [Code Sequence]-[Adjustment Region (Multiple)];pAAV-eHGT_338m-minBGprom-[Different Code Sequence]-[Adjustment Region (Multiple)];pAAV-eHGT_341m-monBGprom-[Different Code Sequence]-[Adjustment Region (Multiple)];pAAV-eHGT_339m-minBGprom-[Different Code Sequence]-[Adjustment Region (Multiple)];pAAV-MGT_E81-minBGprom-[Different Code Sequence]-[Adjustment Region (Multiple)];pAAV-MGT_E85-minBGprom-[Different Code Sequence]-[Adjustment Region (Multiple)];pAAV-MGT_E88-minBGprom-[heterogeneous code sequence]-[adjustment region (multiple possible)];pAAV-MGT_E83-minBGprom-[heterogeneous code sequence]-[adjustment region (multiple possible)];hsA2-eHGT_089h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_087h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_154h-minRho-SYFP2-WPRE3-BGHpA;h; sA2-eHGT_226h-minRho-SYFP2-WPRE3-BGHpA;eHGT_526h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_512h-minBglobin-SYFP2-WPRE3-BGHpA;hsA2-eHGT_283h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_090m-minRho-SYFP2-WPRE3-BGHpA;eHGT_076h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_072h-minBglobin-SYFP2-WPRE3-BGHpA;3xSP10ins-eHGT_354h-minRho*-SYFP2-WPRE3-BGHpA;3xSP10ins-eHGT_354m-minRho*-SYFP2-WPRE3-BGHpA;hsA2-eHGT_121h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_133h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_219h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_207h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_113m-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_111m-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_110h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_080h-minRho-SYFP2-WPRE3-BGHpA;eHGT_060m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_060h-minBglobin-SYFP2-WPRE3-BGHpA;MGT_E36-minBglobin-FlpO-WPRE-hGHpA;MGT_E37-minBglobin-FlpO-WPRE-hGHpA;MGT_E41-minBglobin-FlpO-WPRE-hGHpA;eHGT_025h-minBGlobin-SYFP2-WPRE3-BGHpA;hsA2-eHGT_096h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_098h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_104m-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_107h-minRho-SYFP2-WPRE3-BGHpA;3xSP10ins-eHGT_340m-minRho*-SYFP2-WPRE3-BGHpA;eHGT_528h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_515h-minBglobin-SYFP2-WPRE3-BGHpA;3xCore2_eHGT_226h-minBglobin-SYFP2-WPRE3-BGHpA;3xCore3_eHGT_226h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_682h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_600m-minBG-SYFP2-WPRE3-BGHpA;eHGT_759m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_468m-minBglobin-SYFP2-WPRE3-BGHpA;3xCore_eHGT_064h_minBglobin-SYFP2-WPRE3-BGHpA;hsA2-eHGT_170h-minRho-SYFP2-WPRE3-BGHpA;eHGT_131hv1-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_519h-minBglobin-SYFP2-WPRE3-BGHpA;hsA2-eHGT_131hv2-minRho-SYFP2-WPRE3-BGHpA;eHGT_130h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_527h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_470m-minBglobin-SYFP2-WPRE3-BGHpA;hsA2-eHGT_174h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_087m-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_156h-minRho-SYFP2-WPRE3-BGHpA;pAAV-eHGT_338m-minBGprom-FlpO-WPRE-hGHpA;pAAV-eHGT_341m-monBGprom-FlpO-WPRE-hGHpA;An artificial expression construct according to any of Embodiments 2 to 15, comprising or encoding a set of features selected from pAAV-eHGT_339m-minBGprom-FlpO-WPRE-hGHpA;pAAV-MGT_E81-minBGprom-SYFP2-WPRE3-bGHpA;pAAV-MGT_E85-minBGprom-SYFP2-WPRE3-bGHpA;pAAV-MGT_E88-minBGprom-SYFP2-WPRE3-bGHpA; or pAAV-MGT_E83-minBGprom-SYFP2-WPRE3-bGHpA. 17. A vector comprising an artificial expression construct according to any of Embodiments 2 to 16. 18. The vector according to Embodiment 17, wherein the vector includes a viral vector. 19. The vector according to Embodiment 17 or 18, wherein the viral vector comprises a recombinant adeno-associated virus (AAV) vector. 20. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is a promoter and eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_076h, eHGT_072h, eHGT_35 4h, eHGT_354m, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHG T_080h, eHGT_060m, eHGT_060h, MGT_E36, MGT_E37, MGT_E41, eHGT_025h, eHGT_096h, eHGT_098h, eHGT_1 04m, eHGT_107h, eHGT_340m, eHGT_528h, eHGT_515h, 3xCore2_eHGT_226h, 3xCore3_eHGT_226h, eHGT_6 82h, eHGT_600m, eHGT_759m, eHGT_468m, 3xCore_eHGT_064h, eHGT_170h, eHGT_131hv1, eHGT_519h, eHGT The adeno-associated virus (AAV) vector under the control of an enhancer selected from _131hv2, eHGT_130h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, eHGT_156h, eHGT_338m, eHGT_341m, eHGT_339m, MGT_E81, MGT_E85, MGT_E88, and MGT_E83. 21. Transgenic cells comprising an expression construct or vector as described in any of the prior embodiments. 22. Transgenic cells of Embodiment 21, wherein the transgenic cells are somatostatin (Sst) GABAergic neurons, parvalbumin (Pvalb) GABAergic neurons, pvalb / Sst GABAergic neurons, vasoactive intestinal peptide (VIP) GABAergic neurons, Lamp5 GABAergic neurons, or astrocytes. 23. Transgenic cells of Embodiment 22, wherein the transgenic cells are Lamp5_Lhx6 GABAergic neurons. 24. A non-human transgenic animal comprising an artificial expression construct, vector, or transgenic cell as described in any of the prior embodiments. 25. The non-human transgenic animal according to Embodiment 24, wherein the non-human transgenic animal is a mouse or a non-human primate. 26. An administerable composition comprising an expression construct, vector, or transgenic cell as described in any of the prior embodiments. 27. A kit comprising an artificial expression construct, vector, transgenic cells, transgenic animals, and / or an administerable composition as described in any of the prior embodiments. 28. A method for selectively expressing a heterologous gene in a cell population in vivo or in vitro, comprising providing a sample or subject containing a cell population with an administerable composition according to Embodiment 26 in a sufficient dose and over a sufficient period of time, thereby selectively expressing the gene in the cell population. 29. The method according to Embodiment 28, wherein the heterogene encodes an effector element or an expressible element. 30. The method according to Embodiment 29, wherein the effector element comprises a reporter protein or a functional molecule. 31. The method according to Embodiment 30, wherein the reporter protein includes a fluorescent protein. 32. The method according to Embodiment 30 or 31, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD. 33. The method according to Embodiment 29, wherein the expressible element includes a non-functional molecule. 34. The method according to Embodiment 33, wherein the non-functional molecule includes a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD. 35. The method according to any one of embodiments 28 to 34, wherein the provision includes pipetting. 36. The method according to embodiment 35, wherein the pipetting is performed on brain slices. 37. The method according to Embodiment 36, wherein the brain slice includes Sst GABAergic neurons, pvalb GABAergic neurons, pvalb / Sst GABAergic neurons, VIP GABAergic neurons, LAMP5 GABAergic neurons, and / or astrocytes. 38. The method according to 36 of Embodiment 37, wherein the brain slice includes Lamp5_Lhx6 GABAergic neurons. 39. The method according to any one of embodiments 36 to 38, wherein the brain slice is from a mouse, a human, or a non-human primate. 40. The method according to any one of Embodiments 28 to 34, wherein the provision includes administration to a living organism. 41. The method according to Embodiment 40, wherein the organism is a human, a non-human primate, or a mouse. 42. The method according to Embodiment 40 or 41, wherein the administration to the living organism is performed by injection. 43. The method according to Embodiment 42, wherein the injection includes intravenous injection, intraparenchymal injection into brain tissue, intraventricular (ICV) injection, intracisional (ICM) injection, or subarachnoid injection. 44.CN1535, CN1533, CN1647, CN1719, CN2365, CN2355, CN1797, CN1584, CN1455, CN1451, CN2039, CN2040, CN1567, CN1626, CN171 2, CN1700, CN1607, CN1605, CN1556, CN1526, CN1418, CN1404, AiV1173, AiV1174, AiV1177, CN1261, CN1542, CN1544, CN1598, CN1 Artificial expression constructs including 553, CN1992, CN2367, CN2357, CN2568, CN2569, CN2689, CN2408, CN2596, CN2317, CN2571, CN1663, CN2310, CN2360, CN1624, CN2309, CN2366, CN2257, CN1667, CN1581, CN1649, AiP1099, AiP1102, AiP1100, AiP1270, AiP1271, AiP1272, or AiP1273. 45. Any of Embodiments 3 to 44 that utilize the core or linked core described in Embodiment 1 or 2.
[0125] (ix) Closing paragraph This includes variants of the sequences disclosed and referenced herein. Guidance is available using computer programs well known in the art, such as DNASTAR® (Madison, Wisconsin) software, to determine which amino acid residues can be substituted, inserted, or deleted without impairing biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one amino acid family associated with their side chains.
[0126] Suitable conservative amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into the following conserved substitution families: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (Acidic): Aspartic acid (Asp) and Glutamic acid (Glu); Group 3: (Acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (Basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (Major aliphatic, nonpolar residues): Isoleucine (I Group 10: (Polyliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (Sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.
[0127] When making such modifications, the hydrophilicity index of amino acids may be considered. The importance of hydrothermal amino acid indexes in conferring biological functions that interact with proteins is generally understood in the art (Kyte and Doolittle, 1982, J.Mol.Biol.157(1),105-32). Each amino acid has been assigned a hydrophobic index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values are as follows: Ile (+4.5), Val (+4.2), Leu (+3.8), Phe (+2.8), Cys (+2.5), Met (+1.9), Ala (+1.8), Gly (-0.4), Thr (-0.7), Ser (-0.8), Trp (-0.9), Tyr (-1.3), Pro (-1.6), His (-3.2), Glutamate (-3.5), Gln (-3.5), Aspartate (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5).
[0128] In the art, it is known that certain amino acids may be substituted with other amino acids having similar hydrophilicity indices or scores, resulting in proteins with similar biological activity, i.e., proteins that are still biologically functionally equivalent. When making such changes, substitutions of amino acids with hydrophilicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more preferred. It is also understood in the art that substitutions of similar amino acids can be effectively carried out based on hydrophilicity.
[0129] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: Arg (+3.0), Lys (+3.0), aspartic acid (+3.0±1), glutamic acid (+3.0±1), Ser (+0.3), Asn (+0.2), Gln (+0.2), Gly (0), Thr (-0.4), Pro (-0.5±1), Ala (-0.5), His (-0.5), Cys (-1.0), Met (-1.3), Val (-1.5), Leu (-1.8), Ile (-1.8), Tyr (-2.3), Phe (-2.5), Trp (-3.4). It is understood that amino acids can be substituted with other amino acids having similar hydrophilicity values, and that a biologically, and especially immunologically, equivalent protein can still be obtained. In such changes, substitutions of amino acids with hydrophilicity values of ±2 are preferred, those of ±1 are particularly preferred, and those of ±0.5 are even more preferred.
[0130] As described above, amino acid substitutions may be based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc.
[0131] As shown elsewhere, gene sequence variants may include codon-optimizing variants, sequence polymorphisms, splice variants, and / or mutations that do not statistically significantly affect the function of the encoded product.
[0132] The variants of proteins, nucleic acids, and gene sequences disclosed herein also include sequences having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the proteins, nucleic acids, or gene sequences disclosed herein.
[0133] "% sequence identity" refers to the relationship between two or more sequences, as determined by comparing them. In the art, "identity" also means the degree of sequence correlation between protein, nucleic acid, or gene sequences, as determined by the matching of such sequence strings. "Identity" (often referred to as "similarity") can be readily calculated using known methods; see Computational Molecular Biology (Lesk, AM ed.) Oxford University Press, NY (1988), Biocomputing: Informatics and Genome Projects (Smith, DW ed.) Academic Press, NY (1994), Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG ed.) Humana Press, NJ (1994), Sequence Analysis in Molecular Biology (Von Heijne, G. ed.) Academic Press (1987), and Sequence Analysis Primer (Gribskov, M. and Devereux, J. ed.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are coded into publicly available computer programs. Sequence alignment and identity percentage calculation may be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignments can also be performed using the Clustal alignment method (with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10, Higgins and Sharp CABIOS, 5, 151-153 (1989))).Related programs also include the GCG suite programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J.Mol.Biol.215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992, 111-20. Editors: Suhai, Sandor. Publisher: Plenum, New Including York, NY. In the context of this disclosure, if sequence analysis software is used for analysis, it will be understood that the results of the analysis are based on the “default values” of the referenced program. As used herein, “default values” means any set of values or parameters that are initially loaded by the software when it is first initialized.
[0134] The variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and provide the same functionality as the reference sequences. Exemplary stringent hybridization conditions may involve overnight incubation at 42°C in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt solution, 10% dextran sulfate, and 20 μg / ml denatured shear salmon sperm DNA, followed by washing the filter in 0.1×SSC at 50°C. Changes in the stringency of hybridization and signal detection are primarily achieved by formamide concentration (lower percentages of formamide result in lower stringency), salt conditions, or temperature manipulation. For example, moderately high stringency conditions include overnight incubation at 37°C in a solution containing 6×SSPE (20×SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm blocking DNA, followed by washing at 50°C with 1×SSPE and 0.1% SDS. In addition, to achieve even lower stringency, washing performed after stringent hybridization can be done with a higher salt concentration (e.g., 5×SSC). Modifications in the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may necessitate modifications to the hybridization conditions described above due to compatibility issues.
[0135] As will be understood by those skilled in the art, each embodiment disclosed herein includes, essentially consists of, or may consist of, the specific elements, steps, ingredients, or components described herein. Therefore, the terms “include” or “including” should be interpreted as “include, consist of, or essentially consist of.” The transitional terms “comprise” or “comprises” mean that the embodiment will include, but not limited to, an unspecified number of, elements, steps, ingredients, or components. The transitional term “consists of” excludes any unspecified elements, steps, ingredients, or components. The transitional term “essentially consists of” limits the scope of the embodiment to specific elements, steps, ingredients, or components, and those that do not substantially affect the embodiment.The substantial impact is on the target cell population defined by scRNA-Seq, as well as the following enhancer / target cell population pairs: eHGT_089h, eHGT_087h, eHGT_154h, eHGT_226h, eHGT_526h, eHGT_512h, eHGT_283h, eHGT_090m, eHGT_340m, eHGT_528h, eHGT_515h, eHGT_226h, eHGT_170h, eHGT_519h, eHGT_527h, eHGT_470m, eHGT_174h, eHGT_087m, and eHGT_156h / Sst GABAergic neurons; eHGT_076h, eHGT_759m, and eHGT_064h / Pvalb / Sst GABAergic neurons: eHGT_072h, eHGT_131hv1, eHGT_131hv2, and eHGT_130h / Pvalb; GABAergic neurons: eHGT_354h, eHGT_121h, eHGT_133h, eHGT_219h, eHGT_207h, eHGT_113m, eHGT_111m, eHGT_110h, eHGT_080h, eHGT_107h, MGT_E81, MGT_E85, MGT_E88, and MGT_E83 / VIP; GABAergic neurons: MGT_E36, MGT_E37, and MGT_E41 / Lamp5_Lhx6 This would result in a statistically significant decrease in selective expression of GABAergic neurons; and eHGT_354m, eHGT_060m, eHGT_060h / VIP GABAergic neurons, as well as astrocytes; eHGT_025h, eHGT_096h, eHGT_098h, and eHGT_104m / Lamp5 GABAergic neurons; eHGT_682h, eHGT_600m, eHGT_468m, eHGT_338m, eHGT_341m, and eHGT_339m / Sst and Chold GABAergic neurons.
[0136] In certain embodiments, artificial means mean that they do not exist in nature.
[0137] Unless otherwise indicated, all numbers expressing properties such as the amount, molecular weight, and reaction conditions of components used herein and in the claims should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties obtained by the invention. At the very least, and not as an attempt to limit the application of the principle of value to the claims, each numerical parameter should be interpreted in light of the number of significant digits reported and by applying common rounding techniques. If further clarification is needed, the term “approximately” has the meaning reasonably assigned by a person skilled in the art to be within the range of the stated number or range, i.e., ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value.
[0138] Although the numerical ranges and parameters describing the broad scope of the present invention are approximations, the numerical values described in specific examples are reported as accurately as possible. However, each numerical value inherently contains certain errors that inevitably arise from the standard deviation found in each test measurement.
[0139] In the context describing the present invention (particularly in the context of the following claims), “a,” “an,” “the,” and similar references should be construed to encompass both singular and plural unless otherwise indicated herein or unless explicitly stated otherwise or the context expressly contradicts it. The enumeration of value ranges herein is intended solely as a shorthand for individually referring to each distinct value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually listed herein. All methods described herein may be carried out in any preferred order unless otherwise indicated herein or the context expressly contradicts it. Any and all examples provided herein, or the use of exemplary language (e.g., “etc.”), are intended merely to better illuminate the present invention and do not impose any limitation on the scope of the present invention as separately claimed. No language herein should be construed to indicate any element not described in any of the claims that is essential to the practice of the present invention.
[0140] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, it is anticipated that one or more members of a group may be included in or removed from a group. In the event of such inclusion or removal, this specification shall be deemed to include the modified group, thereby satisfying all descriptions of the Markush groups used in the appended claims.
[0141] Specific embodiments of the present invention are described herein, including the best modes known to the inventors for carrying out the invention. Of course, variations relating to these described embodiments will be obvious to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will adopt such modifications as needed, and the inventors intend that the invention may be carried out in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equalities of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless clearly inconsistent with the context, any combination of the elements described above in all possible variations thereof is incorporated into the invention.
[0142] Furthermore, numerous references are made throughout this specification to patents, printed publications, journal articles, and other written texts (materials referenced herein). Each of these referenced materials is individually incorporated herein by reference in its entirety for reference teaching purposes.
[0143] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be adopted are within the scope of the invention. Therefore, alternative configurations of the invention may be used in accordance with the teachings herein, but not limiting them, for example. Thus, the invention is not limited to those precisely shown and described.
[0144] The details provided herein are presented, for example, solely for illustrative purposes of a preferred embodiment of the present invention and are intended to provide what is considered to be the most useful and readily understandable description of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt has been made to show the structural details of the present invention; rather, an attempt is needed to understand the present invention fundamentally, and an attempt is needed to make it clear to those skilled in the art, through the description taken together with the drawings and / or examples, how some forms of the present invention can actually be embodied.
[0145] The definitions and descriptions used in this disclosure are intended to be controlled in any future interpretation unless expressly and expressly modified in the following examples, or unless the application of the meaning would render any interpretation meaningless or essentially meaningless. Where the interpretation of a term would render it meaningless or essentially meaningless, the definition should be taken from dictionaries known to those skilled in the art, such as Webster's Dictionary, 3rd Edition, or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).
Claims
1. An artificial expression construct comprising (i) an enhancer having a sequence having at least 95% sequence identity with sequence number 140 or sequence number 140; (ii) a promoter; and (iii) a coding sequence.
2. The artificial expression construct according to claim 1, wherein the coding sequence codes for a fluorescent protein or a neurotransmitter.
3. The artificial expression construct according to claim 1, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier.
4. The artificial expression construct according to claim 3, wherein the capsid comprises PHP. eB, AAV9, AAVrh. 10, AAV-BR1, AAV-PHP. S, AAV-PHP. B, or AAV-PPS.
5. The artificial expression construct according to claim 1, wherein the artificial expression construct includes or encodes a skipping element.
6. The artificial expression construct according to claim 5, wherein the skipping element comprises a T2A peptide, a P2A peptide, an E2A peptide, an F2A peptide, or an internal ribosome entry site (IRES).
7. The artificial expression construct according to claim 1, wherein the artificial expression construct is located within a viral vector.
8. The artificial expression construct according to claim 7, wherein the viral vector comprises a recombinant adeno-associated virus (AAV) vector.
9. A composition for use in a method for selectively expressing a coding sequence in a cell population in vivo or in vitro, wherein the composition comprises the artificial expression construct described in Claim 1, and the method comprises providing the composition to a sample or subject comprising the cell population in a sufficient dose and for a sufficient amount of time, thereby selectively expressing the coding sequence in the cell population.
10. The composition according to claim 9, wherein the coding sequence codes for a fluorescent protein or a neurotransmitter.
11. The composition according to claim 9, wherein the provision comprises pipetting onto brain slices.
12. The composition according to claim 11, wherein the brain slice comprises SstGABAergic neurons, PvalbGABAergic neurons, Pvalb / SstGABAergic neurons, VipGABAergic neurons, Lamp5GABAergic neurons, and / or astrocytes.
13. The composition according to claim 11, wherein the brain slice comprises Lamp5_Lhx6 GABAergic neurons.
14. The composition according to claim 9, wherein the provision includes administration to a living organism.
15. The composition according to claim 14, wherein the organism is a human, a non-human primate, or a mouse.
16. The composition according to claim 14, wherein the administration to the living organism is performed by injection.
17. The composition according to claim 16, wherein the injection includes intravenous injection, intraparenchymal injection into brain tissue, intraventricular (ICV) injection, intracisional (ICM) injection, or subarachnoid injection.
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