Artificial expression constructs for regulating gene expression in cells within the spinal cord

Abstract

This invention describes artificial expression constructs for regulating gene expression in target types of central nervous system cells. By using the artificial expression constructs of this disclosure, it is possible to express or modify the expression of synthetic genes in spinal motor neurons, including SpP1 spinal motor neurons, Parg spinal motor neurons, Ogdh1 spinal motor neurons, and ChAT spinal motor neurons; α motor neurons, including Chodl spinal motor neurons; γ motor neurons; spinal excitatory motor neurons, including Mafa excitatory neurons, Esrrg Trhr excitatory neurons, and Slc17a6 spinal excitatory neurons; spinal inhibitory neurons, including Slc6a5 spinal inhibitory neurons; whole spinal neurons, including Esrrg spinal motor neurons and types of neurons found throughout the spinal cord; cerebrospinal fluid contact neurons, including Poln spinal motor neurons; and non-neuronal cells of the spinal cord, including astrocytes and oligodendrocytes.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 482,939, filed on 2 February 2023, which is incorporated herein by reference in its entirety as described herein.

[0002] Statement on research and development funded by the federal government This invention was made with government support under grant number MH114830, awarded by the National Institutes of Health. The U.S. government reserves certain rights with respect to this invention.

[0003] Sequence listing reference The sequence listing accompanying this application is provided in XML format, not as a hard copy, and is incorporated herein by reference. The file containing this sequence listing is named 44780372.xml. The file is 458,687 bytes in size, was created on January 27, 2024, and was submitted electronically via the Patent Center.

[0004] This disclosure provides artificial expression constructs for regulating gene expression in target types of central nervous system cells. By using the artificial expression constructs of this disclosure, synthetic gene expression or regulation of gene expression can be performed in the spinal cord, including spinal motor neurons including Spp1 spinal motor neurons, Parg spinal motor neurons, Ogdh1 spinal motor neurons, ChAT spinal motor neurons and Poln spinal motor neurons; α motor neurons including Choldl spinal motor neurons; γ motor neurons; spinal excitatory motor neurons including Mafa excitatory neurons, Esrrg Trhr excitatory neurons and Slc17a6 spinal excitatory neurons; spinal inhibitory neurons including Slc6a5 spinal inhibitory neurons; whole spinal neurons including Esrrg spinal motor neurons and types of neurons found throughout the spinal cord; cerebrospinal fluid contact neurons; and non-neuronal cells of the spinal cord, including astrocytes and oligodendrocytes. [Background technology]

[0005] A complete understanding of the biology of the central nervous system requires distinguishing, defining, and thoroughly investigating various cell types, as well as identifying artificial expression constructs that can label and perturb these cells. In mice, successful labeling of cell populations sharing marker gene expression has been achieved using driver strains expressing recombinases. However, creating, maintaining, and using such strains capable of labeling specific cell types with high specificity is costly, often requires the crossbreeding of transgenic animals between three species, and results in infrequent acquisition of the desired experimental animals. Furthermore, these tools cannot be applied to humans because they require germline transgenic animals. [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides an artificial expression construct for inducing gene expression in a target population of central nervous system cells, including a population of spinal cord cells.

[0007] In certain embodiments of the artificial expression constructs of this disclosure, the following enhancers are used to induce gene expression in a target central nervous system cell population within the spinal cord. The enhancer-target cell population combinations used in this disclosure are shown below in enhancer / target cell population order. Spinal motor neurons: eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, and eHGT_1137m / spinal motor neurons; eHGT_1141m and eHGT_1142m / Spp1 spinal motor neurons; eHGT_1049m and eHGT_1052m / Parg spinal motor neurons; eHGT_1051m / Ogdh1 spinal motor neuron; eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m and eHGT_1050m / ChAT Spinal motor neurons; and eHGT_1056m / Poln spinal motor neuron; Alpha motor neuron: eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m and eHGT_1185m / α motor neurons; and eHGT_1139m and eHGT_1140m / Chodl spinal motor neurons; Gamma motor neurons: eHGT_1186m, eHGT_1187m, and eHGT_1188m / γ motor neurons; Spinal cord excitatory neurons: eHGT_1158m / Mafa excitatory neuron; eHGT_1136m / Esrrg, Trhr excitatory neuron; eHGT_1053m and eHGT_1054m / Slc17a6 spinal cord excitatory neurons; and MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, and eHGT_743m / Spinal cord excitatory neurons; Spinal inhibitory neurons: eHGT_1055m / Slc6a5 spinal inhibitory neuron; and MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, and eHGT_743m / Spinal cord inhibitory neurons; Entire spinal cord neurons: eHGT_1143m and eHGT_1144m / Esrrg spinal motor neurons; eHGT_1159m / entire spinal neuron; and eHGT_1160m / All types of neurons found in the spinal cord; Neurons in contact with cerebrospinal fluid: eHGT_1144m / Cerebrospinal fluid contact neuron (CSF-cN); and Non-neuronal cells in the spinal cord: eHGT_380h, eHGT_387m, eHGT_385m and eHGT_386m / astrosite; and eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, and eHGT_641m / oligodendrocyte.

[0008] In certain embodiments, the artificial enhancer elements of the present disclosure include the core of an enhancer or the core of a concatenated enhancer. Examples of such enhancer cores or cores of concatenated enhancers include the cores of eHGT_390m, eHGT_410m, eHGT_1139m, eHGT_1140m, eHGT_1137m, eHGT_1138m, hI56i, eHGT_367h, eHGT_453m, eHGT_779m, eHGT_140h, eHGT_121h, and / or eHGT_450h, or concatenated cores thereof. By using such artificial enhancer elements, a transgene can be rapidly expressed to obtain high expression, compared to using the full length of the original (natural) enhancer alone.

[0009] In certain embodiments, the enhancer core includes the sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. In certain embodiments, these enhancer cores are concatenated and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, a concatenated sequence containing 3 copies of the selected enhancer core includes the sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29.

[0010] In certain embodiments of the artificial expression constructs of the present disclosure, 3xCore2_eHGT_390m is utilized to induce gene expression in astrocytes.

[0011] In certain embodiments of the artificial expression constructs of the present disclosure, 3xCore-eHGT_410m is utilized to induce gene expression in oligodendrocytes.

[0012] In certain embodiments of the artificial expression constructs of the present disclosure, 3Xcore_eHGT_1139m and 3Xcore-eHGT_1140m are utilized to induce gene expression within alpha motor neurons.

[0013] In certain embodiments of the artificial expression constructs of the present disclosure, 3Xcore_eHGT_1137m and 3Xcore_eHGT_1138m are utilized to induce gene expression throughout spinal motor neurons.

[0014] In certain embodiments of the artificial expression constructs of the present disclosure, 3Xcore2_eHGT_743m is utilized to induce gene expression within Tac2 excitatory neurons.

[0015] In certain embodiments of the artificial expression constructs of the present disclosure, 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, and 3xcore3_eHGT_450h are utilized to induce gene expression within spinal excitatory neurons.

[0016] In certain embodiments of the artificial expression constructs of the present disclosure, 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, and 3xcore3_eHGT_450h are utilized to induce gene expression within spinal inhibitory neurons.

[0017] In certain embodiments of the artificial expression constructs of the present disclosure, hI56i(core) is utilized to induce gene expression within GABAergic neurons.

[0018] In certain embodiments, the artificial enhancer element of the present disclosure includes a combination of concatemerized enhancers. In certain embodiments, the combination of concatemerized enhancers includes a core of enhancer selected from eHGT_390m and hI56i. In certain embodiments, the core of eHGT_390m (eHGT_390m(core2)) includes the sequence shown in Sequence ID No. 3. In certain embodiments, the core of hI56i (hI56i(core)) includes the sequence shown in Sequence ID No. 1.

[0019] In certain embodiments, concatemerized enhancer combinations include eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core), as shown in SEQ ID NO: 5. In certain embodiments, eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core) induces gene expression in astrocytes and GABAergic neurons.

[0020] Certain embodiments provide an artificial expression construct that includes the characteristics of the vectors described herein, such as AiP1425, CN2724, CN1390, AiP1365, CN3038, CN2102, CN2951, CN3323, CN2237, CN2514, CN3018, CN3044, CN2229, CN2787, CN1528, CN2609, CN2360, CN2847, CN1457, CN3317, CN3318, CN3184, CN4388, CN4262, CN4263, CN4264, CN4265, CN2043, HCT 1, HCT 2, HCT 3, HCT 4, HCT 5, HCT 6, HCT 7, HCT 8, HCT 9, HCT 10, HCT 11, HCT 12, HCT 13, HCT 14, HCT 15, HCT 16, HCT 17, HCT 18, HCT Examples include 19, CN3098, CN2122, CN2088, CN2162, CN2499, CN3062, CN2109, CN2845, CN2979, HCT32, HCT33, HCT34, HCT39, HCT40, HCT41, HCT42, HCT43, HCT44, HCT45, HCT46, CN3406, CN2253, CN2416, AiP1427, CN2786, CN2251, CN2913, CN2631, HCT47, HCT48, HCT49, HCT50, HCT69, and CN1389. [Brief explanation of the drawing]

[0021] [Figure 1] The enhancer eHGT_1137m induces potent expression of SYFP2 in spinal motor neurons. The viral vector HCT1 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In spinal cord sections, SYFP2+ cell bodies were large, located in the ventral horn, and had axons projecting ventrally.

[0022] [Figure 2]The enhancer eHGT_1139m induces potent expression of SYFP2 in alpha spinal motor neurons. The viral vector HCT3 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In spinal cord sections, SYFP2+ cell bodies were very large, located in the ventral horn, and had axons projecting ventrally.

[0023] [Figure 3] The optimized enhancer 3xcore2_eHGT_743m induces potent SYFP2 expression in Tac2 excitatory neurons. The viral vector CN3038 was packaged in PHP.eB capsid and delivered to one cerebral hemisphere of rats by intracerebroventricular administration. SYFP2+ cells were observed in layers 2–4 of the dorsal horn in spinal cord sections.

[0024] [Figure 4] The optimized enhancer 3xcore2_eHGT_779m induces potent SYFP2 expression in neurons. The viral vector CN3044 was packaged in PHP.eB capsid and delivered to one cerebral hemisphere of rats by intracerebroventricular administration. SYFP2+ cells were observed superficially in the dorsal horn in spinal cord sections.

[0025] [Figure 5] The optimized enhancer 3xcore2_eHGT_453m induces potent expression of SYFP2 in neurons. The viral vector CN3018 was packaged in PHP.eB capsid and delivered to one cerebral hemisphere of rats by intracerebroventricular administration. In spinal cord sections, SYFP2+ cells were observed superficially in the dorsal horn and sparsely in the ventral horn.

[0026] [Figure 6] The enhancer eHGT_779m induces potent expression of SYFP2 in neurons. The viral vector CN2609 was packaged in PHP.eB capsid and delivered to one cerebral hemisphere of rats by intracerebroventricular administration. SYFP2+ cells were observed throughout the dorsal horn in a spinal cord section.

[0027] [Figure 7] The enhancer eHGT_453m induces potent SYFP2 expression in neurons. The viral vector CN2251 was packaged in PHP.eB capsid and delivered to mice via postorbital administration. SYFP2+ cells were observed throughout the dorsal and ventral horns in spinal cord sections.

[0028] [Figure 8] The enhancer eHGT_078h induces potent SYFP2 expression in neurons. The viral vector CN1457 was packaged in PHP.eB capsid and delivered to mice via postorbital administration. SYFP2+ cells were observed mainly in the second and third layers of the dorsal horn in spinal cord sections.

[0029] [Figure 9] The enhancer eHGT_380h induces potent SYFP2 expression in spinal cord astrocytes. The viral vector CN3098 was packaged in PHP.eB capsid and delivered to mice via post-orbital administration. In spinal cord sections, SYFP2+ cells exhibit a characteristic astrocyte morphology.

[0030] [Figure 10] The enhancer eHGT_386m induces SYFP2 expression in spinal cord astrocytes. The viral vector CN2088 was packaged in PHP.eB capsid and delivered to mice via post-orbital administration. In spinal cord sections, SYFP2+ cells exhibit a characteristic astrocyte morphology.

[0031] [Figure 11] The enhancer eHGT_403m induces SYFP2 expression in oligodendrocytes of the spinal cord. The viral vector CN2499 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In spinal cord sections, SYFP2+ cells exhibit a characteristic oligodendrocyte morphology.

[0032] [Figure 12] The enhancer eHGT_409h induces SYFP2 expression in oligodendrocytes of the spinal cord. The viral vector CN3062 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In spinal cord sections, SYFP2+ cells exhibit a characteristic oligodendrocyte morphology.

[0033] [Figure 13] The enhancer eHGT_410m induces SYFP2 expression in oligodendrocytes of the spinal cord. The viral vector CN2109 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In spinal cord sections, SYFP2+ cells showed a characteristic oligodendrocyte distribution, but did not exhibit the rich cellular processes seen with other oligodendrocyte-selective vectors.

[0034] [Figure 14] The enhancer eHGT_641m induces SYFP2 expression in oligodendrocytes of the spinal cord. The viral vector CN2845 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In the cross-section of half of the spinal cord shown in the photograph, SYFP2+ cells exhibit a characteristic oligodendrocyte morphology.

[0035] [Figure 15] The enhancer eHGT_361h induces SYFP2 expression in oligodendrocytes of the spinal cord. The viral vector CN2979 was packaged in PHP.eB capsid and delivered to mice by postorbital administration. In spinal cord sections, SYFP2+ cells exhibit a characteristic oligodendrocyte morphology.

[0036] [Figure 16]The enhancer eHGT_1137m induces potent expression of mTFP1 in spinal motor neurons of the rhesus monkey spinal cord. The viral vector HCT69 was packaged in PHP.eB capsid and delivered to rhesus monkeys via the cisterna magna (ICM) administration route. In a cross-section of the cervical spinal cord, mTFP1+ cell bodies were large, located in the ventral horn, and had axons projecting ventrally.

[0037] [Figure 17]The sequences supporting this disclosure are shown below: hI56i(core)(SEQ ID NO: 1);3xhI56i(core)(SEQ ID NO: 2);Core2_eHGT_390m(eHGT_390m(core2);250bp length)(SEQ ID NO: 3);3xCore2_eHGT_390m(SEQ ID NO: 4);eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)(SEQ ID NO: 5);core2_eHGT_743m(SEQ ID NO: 6);3xcore2_eH GT_743m (SEQ ID NO: 7); Core-eHGT_410m (SEQ ID NO: 8); 3xCore-eHGT_410m (SEQ ID NO: 9); core2_eHGT_367h (SEQ ID NO: 10); core2_eHGT_453m (SEQ ID NO: 11); 3xcore2_eHGT_453m (SEQ ID NO: 12); core2_eHGT_779m (SEQ ID NO: 13); 3xcore2_eHGT_779m (SEQ ID NO: 14); Core_eHGT_140h (SEQ ID NO: 15); 3xCore_eHGT_140h (SEQ ID NO: 16); Core_eHGT _121h (SEQ ID NO: 17); 3xCore_eHGT_121h (SEQ ID NO: 19); core3_eHGT_450h (SEQ ID NO: 20); 3xcore3_eHGT_450h (SEQ ID NO: 21); core_eHGT_1138m (SEQ ID NO: 22); 3Xcore_eHGT_1138m (SEQ ID NO: 23); core_eHGT_1139m (SEQ ID NO: 24); 3Xcore_eHGT_1139m (SEQ ID NO: 25); core-eHGT_1140m (SEQ ID NO: 26); 3Xcore-eHGT_1140m (SEQ ID NO: 27); cor e_eHGT_1137m(sequence number 28);3Xcore_eHGT_1137m(sequence number 29);MGT_E132(sequence number 30);eHGT_638m(sequence number 31);MGT_E136(sequence number 32);eHGT_387m(sequence number 33);eHGT_452h(sequence number 34);eHGT_441h(sequence number 35);eHGT_082h(sequence number 36);eHGT_779m(sequence number 37);eHGT_519h(sequence number 38);eHGT_647m(sequence number 39);eHGT_078h(sequence number 40);eHGT_641m (Sequence ID 41); eHGT_1131h (Sequence ID 42); eHGT_1132h (Sequence ID 43); eHGT_1133h (Sequence ID 44); eHGT_1134h (Sequence ID 45); eHGT_1135h (Sequence ID 46); eHGT_356h (Sequence ID 47); eHGT_1137m (Sequence ID 48); eHGT_1138m (Sequence ID 49); eHGT_1139m (Sequence ID 50); eHGT_1140m (Sequence ID 51); eHGT_1136m (Sequence ID 52); eHGT_1141m (Sequence ID 41); eHGT_1131h (Sequence ID 42); eHGT_1141m (Sequence ID 44); eHGT_1134h (Sequence ID 45); eHGT_1135h (Sequence ID 46); eHGT_356h (Sequence ID 47); eHGT_1137m (Sequence ID 48); eHGT_1138m (Sequence ID 49); eHGT_1139m (Sequence ID 50); eHGT_1140m (Sequence ID 51); eHGT_1136m (Sequence ID 52); eHGT_1141m (Sequence ID 41); eHGT_1131h (Sequence ID 42); eHGT_1132h (Sequence ID 43); eHGT_1133h (Sequence ID 44); eHGT_1134h (Sequence ID 45); eHGT_1135h ( Column number 53); eHGT_1142m (Sequence number 54); eHGT_1143m (Sequence number 55); eHGT_1144m (Sequence number 56); eHGT_1145m (Sequence number 57); eHGT_1048m (Sequence number 58); eHGT_1049m (Sequence number 59); eHGT_1050m (Sequence number 60); eHGT_1051m (Sequence number 61); eHGT_1052m (Sequence number 62); eHGT_1053m (Sequence number 63); eHGT_1054m (Sequence number 64); eHGT_1055m (Sequence number 65); eHG T_1056m(SEQ ID NO: 66);eHGT_380h(SEQ ID NO: 67);eHGT_385m(SEQ ID NO: 68);eHGT_386m(SEQ ID NO: 69);eHGT_400h(SEQ ID NO: 70);eHGT_403h(SEQ ID NO: 71);eHGT_409h(SEQ ID NO: 72);eHGT_410m(SEQ ID NO: 73);eHGT_361h(SEQ ID NO: 74);eHGT_1158m(SEQ ID NO: 75);eHGT_1159m(SEQ ID NO: 76);eHGT_1160m(SEQ ID NO: 77);eHGT_1181m(SEQ ID NO: 78);eH GT_1182m (SEQ ID NO: 79); eHGT_1183m (SEQ ID NO: 80); eHGT_1184m (SEQ ID NO: 81); eHGT_1185m (SEQ ID NO: 82); eHGT_1186m (SEQ ID NO: 83); eHGT_1187m (SEQ ID NO: 84); eHGT_1188m (SEQ ID NO: 85); eHGT_888m (SEQ ID NO: 86); eHGT_458m (SEQ ID NO: 87); eHGT_577h (SEQ ID NO: 88); MGT_E135 (SEQ ID NO: 89); eHGT_453m (SEQ ID NO: 90); eHGT_743m (SEQ ID NO: 91);Minimal β-globin promoter (pBGmin / minBGlobin / minBGprom) (SEQ ID NO: 92); minCMV promoter (SEQ ID NO: 93); variant minCMV promoter (SacI RE site removed) (SEQ ID NO: 94); minRho promoter (SEQ ID NO: 95); minRho* promoter (SEQ ID NO: 96); minimal Hsp68 promoter (proHsp68) (SEQ ID NO: 97); SYFP2 (SEQ ID NO: 98); EGFP (SEQ ID NO: 99); mTFP1 (SEQ ID NO: 100); optimized Flp recombinase (FlpO) (SEQ ID NO: 101); improved Cre recombinase (iCre) (SEQ ID NO: 102); SP10 insulator (SP10ins) (SEQ ID NO: 103); 3xSP10ins (SEQ ID NO: 104); 4X2C (SEQ ID NO: 105); miR128 recognition sequence (SEQ ID NO: 106); miR221 recognition sequence (SEQ ID NO: 107); 3 XFLAG(SEQ ID NO: 108); 10aa(SEQ ID NO: 109); H2B(SEQ ID NO: 110); H2B*(SEQ ID NO: 111); WPRE3(SEQ ID NO: 112); WPRE(SEQ ID NO: 113); BGHpA(SEQ ID NO: 114); hGHpA(SEQ ID NO: 115); P2A(SEQ ID NO: 116); T2A(SEQ ID NO: 117); E2A(SEQ ID NO: 118); F2A(SEQ ID NO: 119); Exemplary plasmid skeleton 1 - left ITR(SEQ ID NO: 120); Exemplary plasmid skeleton 1 - right ITR(SEQ ID NO: 121); Exemplary plasmid skeleton 2 - left ITR(SEQ ID NO: 122); Exemplary plasmid skeleton 2 - right ITR(SEQ ID NO: 123); PHP.eB capsid(SEQ ID NO: 124); AAV9 VP1 capsid protein (SEQ ID NO: 125); tet-transactivator version 2 (tTA2) (SEQ ID NO: 126); GTPase HRas [Homo sapiens] (SEQ ID NO: 127); Substance P (SEQ ID NO: 128); Oxytocin (SEQ ID NO: 129); HA tag code sequence (SEQ ID NO: 131); GCaMP6m (SEQ ID NO: 132); GCaMP6s (SEQ ID NO: 133); GCaMP6f (SEQ ID NO: 134); AiP1425 (SEQ ID NO: 135); CN2724 (SEQ ID NO: 136); CN1390 (SEQ ID NO: 137); AiP1365 (SEQ ID NO: 138); CN3038 (SEQ ID NO: 139); CN2102 (SEQ ID NO: 140);CN2951(SEQ ID NO: 141);CN3323(SEQ ID NO: 142);CN2237(SEQ ID NO: 143);CN2514(SEQ ID NO: 144);CN3018(SEQ ID NO: 145);CN3044(SEQ ID NO: 146);CN2229(SEQ ID NO: 147);CN2787(SEQ ID NO: 148);CN1528(SEQ ID NO: 149);CN2609(SEQ ID NO: 150);CN2360(SEQ ID NO: 151);CN2847(SEQ ID NO: 152);CN1457(SEQ ID NO: 153);CN3317(SEQ ID NO: 154);CN3318(SEQ ID NO: 155);CN3184(SEQ ID NO: 156);CN4388(SEQ ID NO: 157);CN4262(SEQ ID NO: 158);CN4263(SEQ ID NO: 159);CN4264(SEQ ID NO: 160);CN4265(SEQ ID NO: 161);CN2043(SEQ ID NO: 162);HCT 1 (SEQ ID NO: 163); HCT 2 (SEQ ID NO: 164); HCT 3 (SEQ ID NO: 165); HCT 4 (SEQ ID NO: 166); HCT 5 (SEQ ID NO: 167); HCT 6 (SEQ ID NO: 168); HCT 7 (SEQ ID NO: 169); HCT 8 (SEQ ID NO: 170); HCT 9 (SEQ ID NO: 171); HCT 10 (SEQ ID NO: 172); HCT 11 (SEQ ID NO: 173); HCT 12 (SEQ ID NO: 174); HCT 13 (SEQ ID NO: 175); HCT 14 (SEQ ID NO: 176); HCT 15 (SEQ ID NO: 177); HCT 16 (SEQ ID NO: 178); HCT 17 (SEQ ID NO: 179); HCT 18 (SEQ ID NO: 180); HCT 19 (SEQ ID NO: 181); CN3098 (SEQ ID NO: 182); CN2122 (SEQ ID NO: 183); CN2088 (SEQ ID NO: 184); CN2162 (SEQ ID NO: 185); CN2499 (SEQ ID NO: 186); CN3062 (SEQ ID NO: 187); CN2109 (SEQ ID NO: 188); CN2845 (SEQ ID NO: 189); CN2979 (SEQ ID NO: 190); HCT32 (SEQ ID NO: 191); HCT33 (SEQ ID NO: 192); HCT34 (SEQ ID NO: 193); HCT39 (SEQ ID NO: 194); HCT40 (SEQ ID NO: 195); HCT41 (SEQ ID NO: 196); HCT42 (SEQ ID NO: 197); HCT43 (SEQ ID NO: 198); HCT44 (SEQ ID NO: 199); HCT45 (SEQ ID NO: 200); HCT46 (SEQ ID NO: 201); CN3406 (SEQ ID NO: 202); CN2253 (SEQ ID NO: 203);CN2416 (sequence number 204); AiP1427 (sequence number 205); CN2786 (sequence number 206); CN2251 (sequence number 207); CN2913 (sequence number 208); CN2631 (sequence number 209); HCT47 (sequence number 210); HCT48 (sequence number 211); HCT49 (sequence number 212); HCT50 (sequence number 213); HCT69 (sequence number 214); and CN1389 (sequence number 18). [Modes for carrying out the invention]

[0038] To fully understand the biology of the central nervous system, it is necessary to distinguish, define, and investigate various types of cells in detail, as well as to identify artificial expression constructs that can label and perturb these cells (Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018); Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017)). In mice, successful labeling of cell populations sharing marker gene expression has been achieved using driver strains expressing recombinase (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, creating, maintaining, and using such cell lines capable of labeling specific cell types with high specificity is costly, often requires crossbreeding transgenic animals among the three species, and results in infrequent acquisition of the desired experimental animals. Furthermore, these tools cannot be applied to humans because they require germline transgenic animals.

[0039] This disclosure provides an artificial expression construct for inducing gene expression in a target population of central nervous system cells, including a population of spinal cord cells.

[0040] In certain embodiments of the artificial expression constructs of this disclosure, the following enhancers are used to induce gene expression in a target central nervous system cell population within the spinal cord. The enhancer-target cell population combinations used in this disclosure are shown below in enhancer / target cell population order. Spinal motor neurons: eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, and eHGT_1137m / spinal motor neurons; eHGT_1141m and eHGT_1142m / Spp1 spinal motor neurons; eHGT_1049m and eHGT_1052m / Parg spinal motor neurons; eHGT_1051m / Ogdh1 spinal motor neuron; eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m and eHGT_1050m / ChAT spinal motor neurons; and eHGT_1056m / Poln spinal motor neuron; Alpha motor neuron: eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m and eHGT_1185m / α motor neurons; and eHGT_1139m and eHGT_1140m / Chodl spinal motor neurons; Gamma motor neurons: eHGT_1186m, eHGT_1187m, and eHGT_1188m / γ motor neurons; Spinal cord excitatory neurons: eHGT_1158m / Mafa excitatory neuron; eHGT_1136m / Esrrg, Trhr excitatory neuron; eHGT_1053m and eHGT_1054m / Slc17a6 spinal cord excitatory neurons; and MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, and eHGT_743m / Spinal cord excitatory neurons; Spinal inhibitory neurons: eHGT_1055m / Slc6a5 spinal inhibitory neuron; and MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, and eHGT_743m / Spinal cord inhibitory neurons; Entire spinal cord neurons: eHGT_1143m and eHGT_1144m / Esrrg spinal motor neurons; eHGT_1159m / entire spinal neuron; and eHGT_1160m / All types of neurons found in the spinal cord; Neurons in contact with cerebrospinal fluid: eHGT_1144m / Cerebrospinal fluid contact neuron (CSF-cN); and Non-neuronal cells in the spinal cord: eHGT_380h, eHGT_387m, eHGT_385m and eHGT_386m / astrosite; and eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, and eHGT_641m / oligodendrocyte.

[0041] In certain embodiments, the artificial enhancer elements of this disclosure include an enhancer core or a concatemerized enhancer core. Examples of such enhancer cores or concatemerized enhancer cores include the cores of eHGT_390m, eHGT_410m, eHGT_1139m, eHGT_1140m, eHGT_1137m, eHGT_1138m, hI56i, eHGT_367h, eHGT_453m, eHGT_779m, eHGT_140h, eHGT_121h and / or eHGT_450h, or concatemerized cores of these. By using such artificial enhancer elements, transgene expression can be accelerated and high expression can be achieved more rapidly than when the full length of the original (natural) enhancer is used alone.

[0042] In certain embodiments, the enhancer core includes the sequence shown in any of SEQ ID NOs: 1, 3, 6, 8, 10, 11, 13, 15, 17, 20, 22, 24, 26, and 28. In certain embodiments, these enhancer cores are concatenated and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, a concatemer containing 3 copies of a selected enhancer core includes the sequence shown in any of SEQ ID NOs: 2, 4, 7, 9, 12, 14, 16, 19, 21, 23, 25, 27, and 29.

[0043] In certain embodiments of the artificial expression constructs of this disclosure, 3xCore2_eHGT_390m is used to induce gene expression within astrocytes.

[0044] In certain embodiments of the artificial expression constructs of this disclosure, 3xCore-eHGT_410m is used to induce gene expression within oligodendrocytes.

[0045] In certain embodiments of the artificial expression constructs of this disclosure, 3Xcore_eHGT_1139m and 3Xcore-eHGT_1140m are used to induce gene expression within alpha motor neurons.

[0046] In certain embodiments of the artificial expression constructs of this disclosure, 3Xcore_eHGT_1137m and 3Xcore_eHGT_1138m are used to induce gene expression throughout spinal motor neurons.

[0047] In certain embodiments of the artificial expression constructs of this disclosure, 3Xcore2_eHGT_743m is used to induce gene expression within Tac2 excitatory neurons.

[0048] In certain embodiments of the artificial expression constructs of this disclosure, 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, and 3xcore3_eHGT_450h are used to induce gene expression in spinal cord excitatory neurons.

[0049] In certain embodiments of the artificial expression constructs of this disclosure, gene expression is induced in spinal cord inhibitory neurons using 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, and 3xcore3_eHGT_450h.

[0050] In certain embodiments of the artificial expression constructs of this disclosure, hI56i(core) is used to induce gene expression within GABAergic neurons.

[0051] In certain embodiments, the artificial enhancer element of the present disclosure includes a combination of concatemerized enhancers. In certain embodiments, the combination of concatemerized enhancers includes a core of enhancer selected from eHGT_390m and hI56i. In certain embodiments, the core of eHGT_390m (eHGT_390m(core2)) includes the sequence shown in Sequence ID No. 3. In certain embodiments, the core of hI56i (hI56i(core)) includes the sequence shown in Sequence ID No. 1.

[0052] In certain embodiments, concatemerized enhancer combinations include eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core), as shown in SEQ ID NO: 5. In certain embodiments, eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core) induces gene expression in astrocytes and GABAergic neurons.

[0053] Certain embodiments provide an artificial expression construct that includes the characteristics of the vectors described herein, such as AiP1425, CN2724, CN1390, AiP1365, CN3038, CN2102, CN2951, CN3323, CN2237, CN2514, CN3018, CN3044, CN2229, CN2787, CN1528, CN2609, CN2360, CN2847, CN1457, CN3317, CN3318, CN3184, CN4388, CN4262, CN4263, CN4264, CN4265, CN2043, HCT 1, HCT 2, HCT 3, HCT 4, HCT 5, HCT 6, HCT 7, HCT 8, HCT 9, HCT 10, HCT 11, HCT 12, HCT 13, HCT 14, HCT 15, HCT 16, HCT 17, HCT 18, HCT Examples include 19, CN3098, CN2122, CN2088, CN2162, CN2499, CN3062, CN2109, CN2845, CN2979, HCT32, HCT33, HCT34, HCT39, HCT40, HCT41, HCT42, HCT43, HCT44, HCT45, HCT46, CN3406, CN2253, CN2416, AiP1427, CN2786, CN2251, CN2913, CN2631, HCT47, HCT48, HCT49, HCT50, HCT69, and CN1389.

[0054] Various aspects of this disclosure, along with further options, are described in more detail below. These various aspects of this disclosure are described in accordance with the following sections: (i) artificial expression constructs and vectors for targeted expression of genes in target cell types; (ii) compositions for administration; (iii) cell lines containing artificial expression constructs; (iv) transgenic animals; (v) methods of use; (vi) kits and commercial packaging; (vii) exemplary embodiments; and (viii) conclusion. These headings are provided for systematic purposes only and do not limit the scope or interpretation of this disclosure.

[0055] (i) Artificial expression constructs and vectors for targeted gene expression in target cell types. The artificial expression constructs disclosed herein include (i) an enhancer sequence that induces targeted expression of a coding sequence in target central nervous system cells, (ii) the coding sequence to be expressed, and (iii) a promoter. The artificial expression constructs disclosed herein may further include other regulatory elements as they are necessary or beneficial.

[0056] In certain embodiments, the “enhancer” or “enhancer element” is a cis-acting sequence that increases the transcription amount associated with the promoter, can function in either the forward or reverse direction with respect to the promoter and the transcribed coding sequence, and can be positioned upstream or downstream of the promoter or the transcribed coding sequence. Various methods or techniques for measuring the function of enhancer element sequences are known in the art. Specific examples of enhancer sequences used in the artificial expression constructs disclosed herein include eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, and eHGT_387m. , eHGT_385m, eHGT_386m, eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, eHGT_641m, eHGT_743m, eHGT_11 58m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_1185m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_11 87m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052m, eHGT_1 051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, e HGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, 3xcore2_eHGT_743m, 3xCore2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m,Examples include 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, hI56i(core), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, 3xcore3_eHGT_450h, and eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core).

[0057] In certain embodiments, the enhancer used in a target type of central nervous system (CNS) cells is either an enhancer utilized exclusively in the target type of CNS cells, or an enhancer primarily utilized in the target type of CNS cells. The enhancer used in a target type of CNS cells is an enhancer that enhances gene expression in the target type of CNS. In certain embodiments, the enhancer used in a target type of CNS cells enhances gene expression in the target type of CNS but does not substantially induce gene expression in other non-target cells, and is therefore also a target CNS enhancer with cell type-specific transcriptional activity.

[0058] When a heterologous coding sequence operably linked to an enhancer disclosed herein is expressed in a target cell type, the administered heterologous coding sequence is expressed in the intended cell type.

[0059] When a heterologous coding sequence is selectively expressed in selected cells, the administered heterologous coding sequence is expressed in the intended cell type but substantially not in other cell types. This is explained in more detail below. In certain embodiments, substantially not being expressed in other cell types means that the expression in reference cells is less than 50% compared to target cells; less than 40% compared to target cells; less than 30% compared to target cells; less than 20% compared to target cells; or less than 10% compared to target cells. In certain embodiments, “reference cells” refer to non-target cells. Non-target cells may be located within the same anatomical structure as the target cells and / or project to a common anatomical region. In certain embodiments, reference cells are located within an anatomical structure adjacent to the anatomical structure containing the target cells. In certain embodiments, reference cells are non-target cells that have a different gene expression profile than the target cells.

[0060] In certain embodiments, the coding sequence transcript may be expressed at low levels in unselected cell types, for example, at less than 1% of the transcript expression level in selected cells, or at 1%, 2%, 3%, 5%, 10%, 15%, or 20%. In certain embodiments, the target type of central nervous system cells is the only type of cell capable of expressing the correct combination of various transcription factors that can bind to the enhancers disclosed herein and induce gene expression. Therefore, in certain embodiments, expression occurs only in the target type of cell.

[0061] In certain embodiments, the target cell types (e.g., neuronal and / or non-neuronal cells) can be identified based on transcriptional profiles described in Tasic et al., Nature 563, 72-78 (2018) and Hodge et al., Nature 573, 61-68 (2019), among others. For reference, various cell types and their notable characteristics are described below.

[0062] Subclassification of motor neurons: Motor neurons are specialized neurons located in the spinal cord and brain that integrate signals from the central nervous system and sensory systems to control voluntary and involuntary movements. Motor neurons in the spinal cord receive input from intracortical neurons and relay the received information to control muscles throughout the body. Alpha motor neurons express relatively high levels of Choldl, Poln, and Spp1. Alpha motor neurons selectively stimulate the extrafusal fibers of muscles, which are the primary force generators. - Spp1 spinal motor neurons express Spp1. - Poln spinal motor neurons express Poln. Gamma motor neurons express relatively high levels of Esrrg and Htr1f. Gamma motor neurons selectively stimulate intrafusal fibers of muscles. - Esrrg spinal motor neurons express Essrg. • Choldl spinal motor neurons express Choldl. • Spinal motor neurons express Parg. Ogdh1 spinal motor neurons express Ogdh1.

[0063] Subclassification of excitatory neurons: Tac2-excitatory neurons express Tac2. Mafa-excitatory neurons express Mafa. • Esrrg and Trhr excitatory neurons express Esrrg and Trhr. • Spinal excitatory neurons express Slc17a6.

[0064] Subclassification of inhibitory (GABAergic) neurons: Slc6a5-inhibitory neurons express Slc6a5.

[0065] Cerebrospinal fluid contact neurons (CSF-cNs) are often identified by Pkd2l1 and Pkd1l2. CSF-cNs are inhibitory and express the early neuronal marker Sox2 and the V2b cell lineage markers Gata2 and Gata3, suggesting an immature phenotype.

[0066] Subclassification of non-neuronal cells: Astrocytes are glial cells derived from the neuroectoderm that express the Aqp4 marker and often the GFAP marker, but do not express the neuronal marker SNAP25. Astrocytes may have a characteristic star-shaped morphology and are involved in supporting the metabolism of other cells in the central nervous system. Numerous types of astrocyte morphology have been observed in mice and humans. Oligodendrocytes are glial cells derived from the neuroectoderm that express the Sox10 marker. This classification includes oligodendrocyte progenitor cells (OPCs). Oligodendrocytes are a subgroup primarily responsible for myelin formation in neurons.

[0067] In certain embodiments, the coding sequence is a heterogeneous coding sequence that codes for an effector element. The effector element is a sequence that is expressed to achieve a desired effect, and this effector element is what actually achieves the desired effect. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0068] Exemplary reporter genes / proteins include those expressed by Addgene's ID No. 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), ID No. 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), ID No. 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), ID No. 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), ID No. 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), ID No. 83900 (pAAV-mDlx-GFP-Fishell-1), and ID No. 89897 (pcDNA3-FLAG-mTET2(N500)). Exemplary reporter genes include, in particular, expressible fluorescent proteins or expressible biotin; blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise, mTFP1); and green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreen1, Oregon Green). TM (Thermo Fisher Scientific Inc.); Luciferase; Orange fluorescent protein (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); Red fluorescent protein (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red) TMExamples include reporter genes encoding complexes of far-infrared fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellow1); or tandem-linked complexes.

[0069] GFP, composed of 238 amino acids (26.9 kDa), was first isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresces green when exposed to blue light. GFP isolated from A. victoria has a major excitation peak at 395 nm and a smaller excitation peak at 475 nm. Its emission peak is at 509 nm, in the low wavelength range of green light in the visible spectrum. GFP obtained from the sea slug (Renilla reniformis) has one major excitation peak at 498 nm. Due to its wide range of applications and the demand for further improvements from researchers, various GFP variants have been created. The first major improvement was a single point mutation (S65T) reported in Nature by Roger Tsien in 1995. This mutation dramatically improved the spectral characteristics of GFP, increasing fluorescence and photostability, and shifting the primary excitation peak to 488 nm while maintaining the emission peak at 509 nm. Enhanced GFP (EGFP) was obtained by adding a point mutation (F64L) to GFP that improved folding efficiency at 37°C. EGFP has an extinction coefficient (denoted by ε) of 55,000 L / mol·cm, which corresponds to 9.13 × 10¹⁶ molecules per molecule. -21 m 2 It is also known as the optical cross-section. Furthermore, in 2006, Superfolder GFP was reported as a series of GFP variants that can rapidly fold and mature even when fused to poorly folded peptides.

[0070] Yellow fluorescent protein (YFP) is a genetic variant of green fluorescent protein derived from Aequorea victoria. Its excitation peak is at 514 nm and its emission peak is at 527 nm.

[0071] mTFP1 is a constitutively fluorescent cyan fluorescent protein. In certain embodiments, the sequence encoding mTFP1 is shown in GenBank:ABG77397 or Sequence ID No. 100. Exemplary functional molecules include ion transporters, cell transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, microRNAs, or designer receptors (DREADDs) that are activated only by designer drugs, each possessing a specific function.

[0072] Ion transporters are transmembrane proteins that transport ions across the cell membrane. Ion transporters are found in most cells and are crucial 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 biological processes important to living cells involve the transport of calcium ions (Ca) through ion channels. 2+ ), potassium ions (K + ), sodium ions (Na + The transport of cations such as ) is involved. In certain embodiments, ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0073] Exemplary enzymes, transcription factors, receptors, membrane proteins, cell transport proteins, signaling molecules, and neurotransmitters include enzymes such as lactase, lipase, helicase, α-glucosidase, aromatic L-amino acid decarboxylase (AADC), and amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAAT / enhancer-binding protein), and Oct-1; and transforming growth factor receptor β1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, and intercellular proteins. Examples include receptors such as the leukin-8 receptor α; membrane proteins and cell transport proteins such as clathrin, dynamin, caveolin, Rab4A, and Rab-11A; signaling molecules such as nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPase, and HRas; and neurotransmitters such as cocaine-amphetamine-regulated transcripts, substance P, oxytocin, and somatostatin.

[0074] In certain embodiments, functional molecules include reporters that indicate cellular function and state, such as calcium reporters. Intracellular calcium concentration is an important predictor of numerous cellular activities, including neuronal activation, muscle cell contraction, and second messenger signaling. A highly sensitive and convenient technique for monitoring intracellular calcium concentration is the use of gene-encoded calcium indicators (GECIs). Among GECIs, calcium sensors using green fluorescent protein (GFP), known as GCaMPs, are highly efficient and widely used. GCaMPs are formed by fusing M13 and calmodulin protein to the N-terminus and C-terminus of circulating GFP. Several types of GCaMPs exhibit characteristic fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051): 1888-1891). Examples of GECIs exhibiting green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, jGCaMP7f, jGCaMP8s, jGCaMP8m, and jGCaMP8f. Furthermore, examples of GECIs exhibiting red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, from Vigene Biosciences, there are AAV8-CAG-GCaMP3 (catalog No. BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog No. BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog No. BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog No. BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog No. BS12-NXSAAV9), and AAV9-CAG-FLEX AAV products such as -jGCaMP7f-WPRE (catalog No. BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (catalog No. BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (catalog No. BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (catalog No. BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (catalog No. BS7-NXBAAV8) are available.

[0075] In certain embodiments, calcium reporters include NTnC, a gene-encoded calcium indicator (GECI); a chimeric fusion of myosin light chain kinase, GFP, and calmodulin; TN-XXL, a calcium indicator; a BRET-based auto-luminescent calcium indicator; and / or OeNL(Ca2+)-18μ, a calcium indicator protein.

[0076] In certain embodiments, functional molecules include modulators of neuronal activity-like channelrhodopsins (e.g., channelrhodopsin 1, channelrhodopsin 2, and their variants). Channelrhodopsins are a subfamily of retinilidene proteins (rhodopsins) that function as ion channels that open in response to light. 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 using site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6): 631-3) describe VChR1, a channelrhodopsin variant shifted to red light. VChR1 is less photosensitive and exhibits reduced membrane transport and expression. Other known channelrhodopsin variants include the ChR2 variant described in Nagel, et al., Proc Natl Acad Sci USA, 2003, 100(24): 13940-5, ChR2 / H134R (Nagel, G., et al., Curr Biol, 2005, 15(24): 2279-84), and ChD / ChEF / ChIEF (Lin, JY, et al., Biophys J, 2009, 96(5): 1803-14), all of which are activated by blue light (470 nm) but are not sensitive to orange / red light. Other variants are described in Lin, Experimental Physiology, 2010, 96.1: 19-25; Knopfel et al., The Journal of Neuroscience, 2010, 30(45): 14998-15004; and Mardinly et al., Nat Neurosci. 2018, 21(6):881-893.

[0077] In certain embodiments, functional molecules include DNA and RNA editing tools such as CRISPR / Cas (e.g., guide RNA and nucleases such as Cas, Cas9, cpf1, etc.). Furthermore, functional molecules include recombinant Cpf1 as described in U.S. Patent Publication 2018 / 0030425, U.S. Patent Publication 2016 / 0208243, WO / 2017 / 184768 and Zetsche et al. (2015) Cell 163: 759-771; single-chain gRNA (e.g., see Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563), editases, guide RNA molecules, microRNAs, or homologous recombinant donor cassettes.

[0078] In certain embodiments, a localization cassette is used as a functional molecule. In certain embodiments, the localization cassette is used to localize a molecule (e.g., a vector, protein, sensor) to a specific compartment below the cellular level, such as the cell body, axon, or dendrites of a neuron. In certain embodiments, the localization cassette may include a cell body tag for localization to the cell body (e.g., cell body (EE-RR)); an axon tag (e.g., derived from GAP43) or synaptophysin (sy) for localization to the axon; a hydrophobic tail for localization to the cell membrane; and a hydrophobic or alkyl chain for localization to the endoplasmic reticulum. In certain embodiments, the localization cassette is fused to a sensor molecule such as GECI. In certain embodiments, fusion proteins of the localization cassette and GECI may include cell body-jGCaMP8s, axon-jRGECO1a, syGCaMP5G, and cell body-jGCaMP7s.

[0079] In certain embodiments, tag cassettes are cited as functional molecules. Tag cassettes include His tag (HHHHHH; SEQ ID NO: 215), Flag tag (DYKDDDDK; SEQ ID NO: 216), Xpress tag (DLYDDDDK; SEQ ID NO: 217), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 218), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 219), Polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 220), Myc tag (EQKLISEEDL; SEQ ID NO: 221), Strep tag (referring to the original STREP® tag) (WRHPQFGG; SEQ ID NO: 222), STREP tag II (WSHPQFEK; SEQ ID NO: 223; (Institut fur Bioanalytik (IBA) GmbH, Germany); see, for example, U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 224), Softag Examples include 3 (TQDPSRVG; SEQ ID NO: 225) and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 226). In certain embodiments, the tag cassette may be a fused tag cassette such as 3XFLAG. In certain embodiments, the 3XFLAG may be the sequence shown in SEQ ID NO: 108.

[0080] The sequences of the aforementioned functional molecules have been published. For example, lactase (e.g., GenBank:EAX11622.1), lipase (e.g., GenBank:AAA60129.1), helicase (e.g., GenBank:AMD82207.1), amylase (e.g., GenBank:AAA51724.1), α-glucosidase (e.g., GenBank:ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot:P08047.3), transcription factor AP-1 (e.g., NP_002219.1), heat shock factor protein 1 (e.g., UniProtK B / Swiss-Prot:Q00613.1), CCAAT / enhancer-binding protein (C / EBP)β isoform a (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot:P14859.2), TGFβ (e.g., GenBank:CAF02096.2), glial cell line-derived neurotrophic factor (GDNF) (e.g., NP_001177397.1), platelet-derived growth factor receptor (e.g., GenBank:AAA60049.1), epidermal growth factor receptor (e.g., GenBank:CAA25) 240.1), vascular endothelial growth factor receptor (e.g., GenBank: AAC16449.2), interleukin-8 receptor α (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B Isoform a (e.g., NP_001825.1), Ras-related protein Rab-4A isoform 1 (e.g., NP_004569.2), Ras-related protein Rab-11A (e.g., UniProtKB / Swiss-Prot:P62491.3), platelet-derived growth factor (e.g., GenBank:AAA60552.1), transforming growth factor β3 (e.g., GenBank:AAA61161.1), nerve growth factor (e.g., GenBank:CAA37703.1), EGF (e.g., GenBank:CAA34902.2) Cocaine-amphetamine regulatory transcript (chain A) (e.g., PDB:1HY9_A), protachykinin-1 (e.g., UniProtKB-P20366), oxytocin neurophysin-1 (e.g., UniProtKB-P01178), somatostatin (e.g., GenBank:AAH32625.1), genetically encoded green calcium indicator NTnC (chain A) [synthetic construct] (e.g., PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g., GenBank:ACF93133.1), BRET-based auto-luminescent calcium indicator [synthetic construct] (e.g., GenBank: ADF42668.1), calcium indicator protein OeNL(Ca2+)-18μ [synthetic construct] (e.g., GenBank: BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (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 endonuclease Cpf1 (e.g., UniProtKB / Swiss-Prot: U2UMQ6.1), ribonuclide Examples include rease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease IIβ (e.g., GenBank:AAF76893.1), sodium channel protein type 1 subunit α (e.g., UniProtKB-P35498), member 2 of the voltage-gated potassium channel subfamily KQT (e.g., UniProtKB-O43526), ​​and voltage-gated L-type calcium channel subunit α-1C (e.g., UniProtKB-Q13936).

[0081] Further effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to Cre with improved codons. dgCre is a GFP / Cre recombinase fusion gene enhanced by the fusion of the first 159 amino acids of the dihydrofolate reductase gene (DHFR or folA) on the E. coli K12 strain chromosome to the N-terminus, possessing the G67S mutation and, through modification, the destabilizing domain mutation R12Y / Y100I. FlpO is a codon-optimized form of FLPe, significantly improving protein expression and FRT recombination efficiency in mouse cells. The FLP / FRT system, like the Cre / LoxP system, is widely used for gene expression (the creation of conditional knockout mice using the FLP / FRT system is also widespread). tTA2 refers to tetracycline transactivator.

[0082] Examples of expressible elements include expression products that do not contain effector elements, such as non-functional or defective proteins. In certain embodiments, such expressible elements can be used to carry out methods for testing the effects of their corresponding functional molecules. In certain embodiments, the expressible elements are those that have lost their functionality or become defective due to recombinant mutations that invalidate their function. In these embodiments, the non-expressible elements are as structurally similar as possible to their corresponding functional molecules.

[0083] An exemplary self-cleaving peptide is the 2A peptide, which produces two proteins from a single mRNA molecule. 2A sequences are short sequences (e.g., 20 amino acids long) and are often used in size-constrained 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 can initiate ribosome translation from a second internal site on the mRNA molecule, enabling the production of two proteins from a single mRNA molecule.

[0084] The artificial expression construct may encode nuclear localization proteins such as histone H1, histone H2A, histone H2B, histone H3, histone H4, histone-like proteins HPhA, and H2B*.

[0085] The coding sequences that encode molecules (e.g., RNA and proteins) described herein can be obtained from publicly available databases and publications. The coding sequences may further contain various sequence polymorphisms, mutations, and / or sequence variants, such changes not affecting the function of the encoded molecule. The term "coding" refers to the property of nucleic acid sequences, such as vectors, plasmids, genes, cDNA, and mRNA, to function as templates for the synthesis of other molecules, such as proteins.

[0086] The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or post-transcriptional regulatory elements (e.g., terminal regions). Furthermore, the term may include any introns and other DNA sequences spliced ​​from mRNA transcripts, as well as variants arising from alternative splice sites. These sequences may further include degenerate codons of sequences or reference sequences that may be introduced to confer codon selectivity in certain types of organisms or cells.

[0087] Promoters include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can also be described as strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters. Inducible promoters induce expression in response to specific conditions, signals, or cellular events. For example, a promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to induce transcription from the promoter. Specific examples of promoters include minBglobin (also called minBGprom), CMV, minCMV, minCMV* (minCMV* is minCMV with the SacI restriction site removed), minRho, minRho* (minRho* is minRho with the SacI restriction site removed), SV40 early promoter, Hsp68 minimal promoter (proHSP68), and Roussarcoma virus (RSV) long-chain terminal repeat (LTR) promoter. Minimal promoters do not have the activity to induce gene expression on their own, but when linked to a nearby enhancer element, they can be activated and induce gene expression.

[0088] In certain embodiments, the expression construct is provided incorporated into 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 nucleic acid to be transferred is typically ligated to the nucleic acid molecule of the vector, for example, by insertion into the nucleic acid molecule of the vector. The vector may contain sequences that induce cell self-replication, or sequences that enable integration into the DNA of a host cell. Useful vectors include, for example, plasmids (e.g., DNA plasmids and RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0089] The term "viral vector" is broadly used to refer to nucleic acid molecules containing virus-derived components that facilitate the transfer and expression of non-native nucleic acid molecules in cells. The term "adeno-associated virus vector" refers to a viral vector or plasmid containing structural elements and functional gene elements or parts thereof, primarily derived from AAV. The term "retroviral vector" refers to a viral vector or plasmid containing structural elements and functional gene elements or parts thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid containing structural elements and functional gene elements or parts thereof, primarily derived from lentiviruses, etc. The term "hybrid vector" refers to a vector containing structural elements and / or functional gene elements derived from two or more viruses.

[0090] An "adenovirus vector" refers to a construct that (a) is sufficient to facilitate the packaging of an artificial expression construct, and (b) contains sufficient adenovirus sequences to express a coded sequence cloned in the sense or antisense direction. Recombinant adenovirus vectors include genetically modified forms of adenoviruses. Because the genetic makeup of adenoviruses is a 36kb linear double-stranded DNA virus, a large portion of the adenovirus DNA can be replaced by a foreign sequence of up to 7kb. Unlike retroviruses, adenovirus DNA can replicate using episomes without causing genotoxicity, and therefore, even if adenoviruses infect host cells, they are not incorporated into the chromosomes. Furthermore, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

[0091] Adenoviruses are particularly well-suited for use as gene transfer vectors due to their moderate genome size, ease of handling, high titer, broad target cell range, and high infectivity. The adenovirus genome contains 100–200 base pair-long reverse repeat sequences (ITRs) at both ends; these ITRs are cis-elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the adenovirus genome contain various transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the transcription of the adenovirus genome and several cellular genes. Expression of the E2 region (E2A and E2B) synthesizes proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and the suppression (shut-off) of host cell protein biosynthesis. The late gene product, containing most of the adenovirus capsid protein, is expressed only after significant processing of a single primary transcript induced by the major late promoter (MLP). MLP is particularly efficient in the later stages of infection, as the mRNAs induced by this promoter all possess a 3-part 5'-leader (TPL) sequence, which is preferentially selected over mRNA for translation.

[0092] Other than the requirement that the adenovirus vector is replication-deficient or at least conditionally replication-deficient, the characteristics of the adenovirus vector are considered to be of little importance for reliably carrying out the specific embodiments disclosed herein. The adenovirus may be any of the 42 known serotypes or subgenera A to F. Adenovirus type 5 is a human adenovirus for which much biochemical and genetic information is known and has been historically used in most constructions using adenovirus as a vector. Therefore, in certain embodiments, serotype 5 adenovirus of subgenera C is preferred as a starting material to obtain a conditionally replication-deficient adenovirus vector for use in a particular embodiment.

[0093] As described herein, typical vectors are replication-deficient and lack the E1 region of the adenovirus. Therefore, the simplest method is to introduce the polynucleotide encoding the gene of interest into the position where the coding sequence of the E1 region has been removed. However, the insertion site of the construct within the adenovirus sequence is not critical. The polynucleotide encoding the gene of interest may be inserted into the deleted E3 region of the E3 substitution vector, or into the E4 region, and the deletion of the E4 region may be compensated for by a helper cell line or helper virus.

[0094] Adeno-associated virus (AAV) is a parvovirus found as a contaminant in adenovirus stocks. AAV is a ubiquitous virus (85% of the US population has anti-AAV antibodies) and does not cause disease. Furthermore, because AAV replication depends on the presence of a helper virus (e.g., adenovirus), AAV is also classified as a dependent virus. Various serotypes have been isolated, of which AAV-2 has been the most thoroughly characterized. AAV has single-stranded linear DNA, which is encapsulated by capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20–24 nm.

[0095] The length of AAV DNA is 4.7 kilobases. AAV DNA contains two open reading frames, flanked by two ITRs. The AAV genome contains two main types of genes: rep and cap. The rep gene codes for the protein responsible for AAV virus replication, while the cap gene codes for capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeat sequences are the only cis-components of AAV required for chromosomal integration. Therefore, AAV can be used as a vector from which the entire viral coding sequence can be removed and replaced with a gene cassette for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40, respectively, based on their map locations. Transcription from p5 and p19 results in the production of rep proteins, while transcription from p40 produces capsid proteins.

[0096] AAVs are outstanding for use in this disclosure due to their superior safety profile and the ability to be expressed in target cell populations by modifying their capsid and genome. scAAV refers to self-complementary AAVs. pAAV refers to plasmid adeno-associated viruses. rAAV refers to recombinant adeno-associated viruses.

[0097] Other viral vectors may be used. For example, vectors derived from viruses such as vaccinia virus, poliovirus, and herpesvirus may be used. These vectors offer beneficial properties for various mammalian cells.

[0098] Retroviruses are commonly used as tools for gene delivery. Retroviruses are RNA viruses whose genomic RNA is reverse-transcribed to produce a double-stranded linear DNA copy, which is then incorporated into the host genome via covalent bonds. The retrovirus incorporated into the host genome 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 for the production of new viral particles.

[0099] Examples of 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), Rous sarcoma virus (RSV), and lentivirus.

[0100] "Lentivirus" refers to a group of complex retroviruses (or the genus Complex Retrovirus). Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV types 1 and HIV 2); Visna-Maedivirus (VMV); Caprine arthritis-encephalomyelitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Monkey immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector skeleton (i.e., the cis-acting sequence element of HIV) can be used.

[0101] When using vectors, safety can be improved in some cases by substituting the U3 region of the 5'LTR, which induces transcription of the viral genome in viral particle production, with a heterologous promoter. Examples of heterologous promoters that can be used for this purpose include, for example, the Simian virus 40 (SV40) (e.g., early or late) promoter, the cytomegalovirus (CMV) (e.g., very early) promoter, the Moloney mouse leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, and the herpes simplex virus (HSV) (thymidine kinase) promoter. Conventional promoters can induce high levels of transcription independently of Tat. Substitution of the U3 region with a heterologous promoter reduces the likelihood of recombination occurring, which produces a replicable virus, because the complete U3 sequence is deleted from the virus production system. In certain embodiments, heterologous promoters have another advantage: they allow control over how the viral genome is transcribed. For example, a heterologous promoter may be an inducible promoter, such that the entire or partial viral genome is transcribed only in the presence of an inducer. Inducing factors include one or more compounds or physiological conditions, such as the culture temperature or culture pH of the host cells.

[0102] In certain embodiments, the viral vector includes a TAR element. "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 in which the U3 region of the 5'LTR is replaced with a heterologous promoter.

[0103] The "R region" refers to the region within the retrovirus's LTR from the start of the cap site (i.e., the transcription start site) to just before the start of the poly(A) strand. The R region is also defined as the region sandwiched between the U3 and U5 regions. The R region plays a role in moving nascent DNA from one end of the genome to the other during reverse transcription.

[0104] In certain embodiments, the expression of heterologous sequences in a viral vector can be increased by incorporating post-transcriptional regulatory elements and efficient polyadenylation sites into the viral vector, and a transcription termination signal may also be incorporated into the viral vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids. Examples of post-transcriptional regulatory elements include the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element of hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and other post-transcriptional regulatory elements (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector contains post-transcriptional regulatory elements such as WPRE and HPRE. In certain embodiments, the vector is deleting or does not contain post-transcriptional regulatory elements such as WPRE and HPRE.

[0105] The expression of heterologous genes can be increased by elements that can induce efficient transcription termination and polyadenylation of heterologous nucleic acid transcripts. Transcription termination signals are typically found downstream of polyadenylation signals. In certain embodiments, the vector includes a polyadenylation signal at the 3' end of the polynucleotide encoding the molecule to be expressed (e.g., a protein). A "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that induces both transcription termination and polyadenylation of a nascent RNA transcript transcribed by RNA polymerase II. The polyadenylation sequence can improve mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thereby contributing to improved translation efficiency. In certain embodiments, BGHpA, hGHpA, or SV40pA may be used. In certain embodiments, a preferred embodiment of the expression construct includes a terminator element. The terminator element can increase transcription levels and minimize read-through transcription from the construct to another plasmid sequence.

[0106] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may protect sequences expressed from the viral vector from integration site effects, such as effector elements or expressible elements. Integration site effects occur via cis-acting elements in genomic DNA, meaning that the imported sequence may or may not be expressed (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). In certain embodiments, the viral import vector comprises one or more insulator elements in the 3'LTR, and when the provirus is incorporated into the host genome, this insulator is incorporated into both the 5'LTR and 3'LTR during replication of the 3'LTR. Suitable insulators for use in specific embodiments include chicken β-globin insulators (see 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 (SP10 or SP10ins; 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).

[0107] In addition to those described above, various other types of suitable expression vectors are known to those skilled in the art. These known expression vectors include commercially available expression vectors designed for general recombination operations, such as plasmids containing one or more reporter genes and regulatory elements required to express the reporter genes in cells. Numerous vectors are commercially available from companies such as Invitrogen, Stratagene, and Klontech, and are described in various accompanying guidebooks. In specific embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct capable of expressing the encoded gene in mammalian cells, such as the pUC plasmid system and the Bluescript plasmid system.

[0108] Specific embodiments of the vectors disclosed herein include those listed in the following table. [Table 1] JPEG2026509974000003.jpg231168JPEG2026509974000004.jpg151167

[0109] Subcomponent sequences within large vector sequences can be readily identified by those skilled in the art based on the description herein (see Figure 17). The nucleotides between the identifiable subcomponents listed in the table above are restriction enzyme recognition sites used in construct assembly (cloning) and, if applicable, additional nucleotides that do not have identifiable function. These segments of the complete vector sequence can be modified using various cloning techniques and / or various vectors. Typically, short palindromic sequences of 6 bases represent artifacts during vector construction that are not important to the function of the vector.

[0110] In certain embodiments, a vector (e.g., AAV) having a capsid that crosses the blood-spinal barrier (BSCB) is selected. In certain embodiments, the vector is modified to include a capsid that crosses the blood-spinal barrier. Examples of AAVs having a viral capsid that crosses the blood-spinal barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014; 7:81), AAV-PHP.S (Chan et al., Nat Neurosci. 2017; 20(8): 1172), AAV-9P31, and PHP.eB. In certain embodiments, the capsid of PHP.eB differs from that of AAV9 in that, compared to AAV9 by reference, the amino acid residue S-AQ-A (SEQ ID NO: 227), starting from position 586, is changed to S-DGTLAVPFK-A (SEQ ID NO: 228). In certain embodiments, PHP.eB refers to the sequence of SEQ ID NO: 124.

[0111] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the blood-spinal barrier (BSCB) via intravenous injection. Because AAV9 transduces a wide area of ​​the central nervous system (CNS), it allows for minimally invasive treatment (Naso et al., BioDrugs. 2017; 31(4): 317). Such cases have been reported, for example, in connection with clinical trials by AveXis for the treatment of spinal muscular atrophy (SMA) syndrome (AVXS-101, NCT03505099) and for the treatment of CLN3 gene-associated neuronal ceroid lipofuscinosis (NCT03770572).

[0112] AAV-PHP.S (Addgene, Watertown, Massachusetts) is a variant of AAV9 created using the CREATE method. It encodes the 7-mer sequence QAVRTSL (sequence number 229) and transduces neurons in the enteric nervous system, strongly transducing peripheral sensory afferent nerves that project to the spinal cord and brainstem.

[0113] AAV-9P31 is a variant of AAV9. In certain embodiments, the capsid of PHP.eB differs from AAV9 in that the amino acid residue S-AQ-A (SEQ ID NO: 227), starting from position 586, is changed to S-AQWPTSYDA-A (SEQ ID NO: 230) when compared to AAV9 by reference.

[0114] (ii) Composition for administration The artificial expression constructs and vectors of this disclosure (hereinafter referred to as bioactive components) can be formulated using carriers suitable for administration to cells, tissue sections, animals (e.g., mice and non-human primates), or humans. The bioactive components contained in the compositions described herein can be prepared as neutral forms, as free bases, or as pharmacokinetically acceptable salts.

[0115] Examples of pharmaceutically acceptable salts include acid addition salts (formed from free amino groups of proteins), which are formed using, for example, inorganic acids such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed from free carboxyl groups can be derived from, for example, inorganic bases such as sodium, potassium, ammonium, calcium, and iron hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0116] Examples of carriers for bioactive ingredients include solvents, dispersions, vehicles, coatings, diluents, isotonic agents, absorption retarders, buffers, solutions, suspensions, and colloids. The use of such carriers for bioactive ingredients is well known in the art. Conventional media or agents can be used in combination with the compositions described herein, except when the conventional media or agent is incompatible with the bioactive ingredient of this disclosure.

[0117] The term "pharmaceutically acceptable carrier" refers to a carrier that, when administered to humans, in certain embodiments, does not cause allergic or similar adverse reactions when administered intravenously (e.g., into the posterior orbital plexus).

[0118] In certain embodiments, the compositions of the present disclosure can be formulated for intravenous, intraparenchymal, intraocular, intravitreous, parenteral, subcutaneous, intraventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral, or nasal inhalation, or for direct injection or direct administration into one or more cells, tissues, or organs.

[0119] The compositions of this disclosure may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres and / or nanoparticles.

[0120] The formation and use of liposomes are widely known to those skilled in the art. Liposomes have been developed to improve stability in serum and half-life in blood (see, for example, U.S. Patent No. 5,741,516). Furthermore, various methods have been reported for using liposomes and liposome-like preparations as candidate drug carriers (see, for example, U.S. Patents No. 5,567,434; U.S. Patents No. 5,552,157; ​​U.S. Patents No. 5,565,213; U.S. Patents No. 5,738,868; and U.S. Patent No. 5,795,587).

[0121] This disclosure also provides pharmaceutically acceptable nanocapsule formulations of the bioactive ingredients of this disclosure. Generally, nanocapsule formulations can encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al., Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119, 1998; Douglas et al., Crit Rev Ther Drug Carrier Syst 3(3):233-261, 1987). To avoid side effects caused by large amounts of macromolecules being taken up into cells, such ultrafine particles can be designed using polymers that can be degraded in vivo. It is also envisioned that biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements may be used in this disclosure. Such microparticles can be easily fabricated, as described, for example, in Couvreur et al., J Pharm Sci 69(2):199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlen et al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998; and U.S. Patent No. 5,145,684.

[0122] Examples of injectable compositions include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468). Injectable compositions delivered by injection are in the form of a sterile fluid that can be delivered using a syringe. In certain embodiments, the injectable composition may contain one or more preservative compounds to ensure stability during the manufacturing process and storage, and to prevent contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, which may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof, as well as / or vegetable oils. To maintain proper fluidity, coating agents such as lecithin may be used, or, in the case of dispersions, particle size may be maintained at the required size, and / or surfactants may be used. To prevent microbial action, various antibacterial and / or antifungal agents may be used, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In various embodiments, the injectable composition includes, for example, sugars and isotonic agents such as sodium chloride. Sustained absorption of the injectable composition can be achieved by incorporating absorption-delaying agents into the injectable composition, such as, for example, aluminum monostearate or gelatin. If necessary, an appropriate buffer may be added to the injectable composition, and the diluted liquid is first isotonicized with sufficient saline or glucose.

[0123] Dispersions may be prepared using glycerol, liquid polyethylene glycol, or a mixture thereof, or an oil. As described herein, under normal storage and use conditions, such preparations may contain preservatives to prevent microbial growth.

[0124] Sterile compositions can be prepared by mixing a physiologically active component with other optional components (e.g., the aforementioned components) in an appropriate amount of solvent and then sterilizing and filtering the mixture. Dispersions are typically prepared by dispersing various sterile physiologically active components in a sterile solvent containing a basic dispersion medium and other necessary raw materials (e.g., the aforementioned raw materials). In the case of sterile powders for preparing sterile injection solutions, it is preferable to pre-sterilizing and filtering a solution containing the physiologically active component and other desired raw materials, and then vacuum-drying or freeze-drying this solution to prepare a powder containing the physiologically active component and other desired raw materials.

[0125] Oral compositions may be in liquid form, such as solvents, syrups, or suspensions, and may be provided as pharmaceutical products that are reconstituted with water or other suitable solvents before use. Such liquid formulations may be prepared by conventional methods using pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, ester oils, or fractionated vegetable oils); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, or sorbic acid). The compositions of this disclosure may be prepared, for example, in the form of tablets or capsules by conventional methods using pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized 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); and wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods known in the art.

[0126] The inhalation composition can be delivered in the form of an aerosol spray dispensed 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 dosage unit may be determined by providing a valve for delivering a fixed amount. It may also be formulated into capsules or cartridges (e.g., gelatin capsules or gelatin cartridges) used in inhalers or nebulizers, and such capsules and cartridges may contain a mixed powder consisting of the composition described herein and a suitable powder base such as lactose or starch.

[0127] Furthermore, the compositions of this disclosure include 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 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0128] Furthermore, auxiliary active ingredients may also be included in the composition of this disclosure.

[0129] Typically, the compositions of this disclosure may contain at least 0.1% of a bioactive ingredient, but needless to say, the proportion of the bioactive ingredient can be varied and, for convenience, may be in the range of 1% or 2% to 70% or 80% or more, or in the range of 0.5% to 99% of the total weight or total volume of the composition of this disclosure. Naturally, the amount of the bioactive ingredient in each physiologically beneficial composition may be adjusted so that an appropriate dose is obtained from a predetermined 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 will be considered by those skilled in the art who are responsible for preparing such pharmaceutical formulations, and therefore, a variety of compositions and doses may be desirable.

[0130] In certain embodiments, when administered to humans, the compositions of this disclosure must meet sterility, pyrogenicity, general safety, and purity standards in accordance with the requirements of the U.S. Food and Drug Administration (FDA) or other relevant regulatory authorities in other countries.

[0131] (iii) Cell lines containing artificial expression constructs This disclosure includes cells containing the artificial expression constructs described herein. Cells transformed with the artificial expression constructs can be used for a variety of purposes, such as neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to assess the regulatory properties of enhancers.

[0132] Various host cell lines can be used, but in certain embodiments, the host cell is a mammalian cell. In certain embodiments, the artificial expression constructs are eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, eHGT_387m, eHGT_385m, eHGT_386m, eHGT_361h, eHGT_400h, eHGT_403h, e HGT_409h, eHGT_410m, eHGT_641m, eHGT_743m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_1185m, eHGT_1159 m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHG T_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHG T_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, 3xcore2_ eHGT_743m, 3xCore2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_113 8m, hI56i(core), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h,3xcore3_eHGT_450h and eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core), as well as / or AiP1425, CN2724, CN1390, AiP1365, CN3038, CN2102 , CN2951, CN3323, CN2237, CN2514, CN3018, CN3044, CN2229, CN2787, CN1528, CN2609, CN2360, C N2847, CN1457, CN3317, CN3318, CN3184, CN4388, CN4262, CN4263, CN4264, CN4265, CN2043, HCT 1, HCT 2, HCT 3, HCT 4, HCT 5, HCT 6, HCT 7, HCT 8, HCT 9, HCT 10, HCT 11, HCT 12, HCT 13, HCT 14, HCT 15, HCT 16, HCT 17, HCT 18, HCT The enhancer and / or vector sequence is selected from 19, CN3098, CN2122, CN2088, CN2162, CN2499, CN3062, CN2109, CN2845, CN2979, HCT32, HCT33, HCT34, HCT39, HCT40, HCT41, HCT42, HCT43, HCT44, HCT45, HCT46, CN3406, CN2253, CN2416, AiP1427, CN2786, CN2251, CN2913, CN2631, HCT47, HCT48, HCT49, HCT50, HCT69 and CN1389, and the host cell line is human cells, primate cells or mouse cells. Furthermore, cell lines that can be used for gene transfer in this disclosure include primary cell lines derived from living tissues such as rat or mouse spinal cord, and organ-type cell cultures such as spinal cord sections derived from animals, such as rat, mouse, non-human primate, or human neurosurgical tissue.

[0133] WO91 / 13150 describes various cell lines, including neuron cell lines, and methods for producing them. Similarly, WO97 / 39117 also describes neuron cell lines and methods for producing such cell lines. The neuron cell lines disclosed in these patent applications are applicable to the use of this disclosure.

[0134] In certain embodiments, the term “neuron cell” is used to describe any neuron cell, anything related to a neuron cell, or anything containing a neuron cell. A neuron cell is defined by the characteristic of having an axon and dendrites. The term “neuron-specific” refers to something found in neuron cells or cells derived therefrom, but not, or substantially not, found in cells that do not originate from neurons or in non-neuron cells (e.g., glial cells such as astrocytes and oligodendrocytes); or activity that occurs in neuron cells or cells derived therefrom, but not, or substantially not, in cells that do not originate from neurons or in non-neuron cells (e.g., glial cells such as astrocytes and oligodendrocytes).

[0135] In certain embodiments, non-neuronal cell lines, such as mouse embryonic stem cells, may be used. Cultured mouse embryonic stem cells can be transiently transfected with a plasmid construct, and the expression of the gene construct can be analyzed. Mouse embryonic stem cells are pluripotent, undifferentiated cells. Mouse embryonic stem cells can maintain their undifferentiated state with leukemia suppressor (LIF). Differentiation of mouse embryonic stem cells can be induced by removing LIF. Mouse embryonic stem cells form various types of differentiated cells in culture. Differentiation of mouse embryonic stem cells occurs through the expression of tissue-specific transcription factors, which allows for the evaluation of the function of enhancer sequences (see, for example, Fiskerstrand et al., FEBS Lett 458: 171-174, 1999).

[0136] A method for differentiating stem cells into neuronal cells involves replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenite), laminin, and polyornithine. A method for producing myelin-forming oligodendrocytes from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. Bibel, et al., 2007, Nat. Protoc. 2:1034-43 describes a protocol for producing glutamatergic neurons from stem cells, and Chatzi, et al., 2009, Exp. Neurol. 217:407-16 describes a procedure for producing GABAergic neurons. This procedure involves exposing stem cells to all-trans retinoic acid for three days. Next, by culturing in serum-free neuronal cell induction media such as neuronal basal medium supplemented with B27, bFGF, and EGF, GABAergic neurons, which account for 95% of the total cells, can be obtained.

[0137] U.S. Patent Publication 2012 / 0329714 describes the use of prolactin to increase the number of neural stem cells, and U.S. Patent Publication 2012 / 0308530 describes a culture surface having amino groups that promotes neuronal differentiation into neurons, astrocytes, and oligodendrocytes. Thus, the fate of neural stem cells can be controlled by various extracellular factors. Commonly used extracellular factors include brain-derived growth factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425); fibroblast growth factor (bFGF; U.S. Patent No. 5,766,948; FGF-1, FGF-2); neurotrophin 3 (nt-3) and neurotrophin 4 (nt-4) (Caldwell, et al., 2001, Nat. Biotechnol. Examples include 1;19:475-9); ciliary neurotrophic factor (CNTF); BMP-2 (US Patent Nos. 5,948,428 and 6,001,654); isobutyl-3-methylxanthine; leukemia growth inhibitor (LIF; US Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophin (e.g., cyclic adenosine monophosphate); epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); forskolin; ligands for GDNF family receptors; potassium; retinoic acid (US Patent No. 6,395,546); tetanus toxin; and transforming growth factor α and TGF-β (US Patent Nos. 5,851,832 and 5,753,506).

[0138] In certain embodiments, a yeast one-hybrid system may be used to identify compounds that suppress specific protein-DNA interactions, such as eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, eHGT_387m, eHGT_385m, eHG T_386m, eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, eHGT_641m, eHGT_743m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eH GT_1184m, eHGT_1185m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_ 1142m, eHGT_1049m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eH GT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, 3xcore2 _eHGT_743m, 3xCore2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, hI56i( core), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, 3xcore3_eHGT_450h,Alternatively, the transcription factor eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core) may be mentioned.

[0139] Transgenic animals are discussed below. Cell lines may be derived from such transgenic animals. For example, cell lines in which artificial expression constructs are incorporated into the genome can be obtained from primary tissue cultures derived from transgenic mice (for example, as described later) (see, for example, MacKenzie & Quinn, Proc Natl Acad Sci USA 96: 15251-15255, 1999).

[0140] (iv) Transgenic animals Another aspect of this disclosure is an operable linkage of heterogeneous code sequences, eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, eHGT_387m, eHGT_385m, eHGT_386m, eHGT_361h, eHGT_400h, eHGT_403h, eH GT_409h, eHGT_410m, eHGT_641m, eHGT_743m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_1185m, eHGT_1159m, eH GT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052 m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eH GT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, 3xcore2_eHGT_743m, 3x Core2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, hI56i(core), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, 3xcore3_eHGT_450h,and / or transgenic animals whose genome contains an artificial expression construct including eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core). In a particular embodiment, the genome of the transgenic animal is AiP1425, CN2724, CN1390, AiP1365, CN3038, CN2102, CN2951, CN3323, CN2237, CN2514, CN3018, CN3044, CN2229, CN2787, CN1528, CN2609, CN2360, CN2847, CN1457, CN3317, CN3318, CN3184, CN4388, CN4262, CN4263, CN4264, CN4265, CN2043, HCT 1, HCT 2, HCT 3, HCT 4, HCT 5, HCT 6, HCT 7, HCT 8, HCT 9, HCT 10, HCT 11, HCT Includes 12, HCT 13, HCT 14, HCT 15, HCT 16, HCT 17, HCT 18, HCT 19, CN3098, CN2122, CN2088, CN2162, CN2499, CN3062, CN2109, CN2845, CN2979, HCT32, HCT33, HCT34, HCT39, HCT40, HCT41, HCT42, HCT43, HCT44, HCT45, HCT46, CN3406, CN2253, CN2416, AiP1427, CN2786, CN2251, CN2913, CN2631, HCT47, HCT48, HCT49, HCT50, HCT69, and / or CN1389. In certain embodiments, when using non-embedded vectors, the transgenic animals are eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, eHGT_387m, eHGT_385m, eHGT_386m, eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, eHGT_641m, eHGT_743m,eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_1185m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eH GT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, 3xcore2_eHGT_743m, 3xCore2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xc ore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, hI56i(core), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, 3xcore3_eHGT_450h and / or eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(co re)-eHGT_390m(core2)-hI56i(core), and / or AiP1425, CN2724, CN1390, AiP1365, CN3038, CN2102, CN2951, CN3323, CN2237, CN2514, CN3018, CN3044, CN2229, CN2787, CN1528, CN2609, CN2360, CN2847, CN1457, CN3317, CN3318, CN3184, CN4388, CN4262, CN4263, CN4264, CN4265, CN2043, HCT 1, HCT 2, HCT 3, HCT 4, HCT 5, HCT 6,HCT 7, HCT 8, HCT 9, HCT 10, HCT 11, HCT 12, HCT 13, HCT 14, HCT 15, HCT 16, HCT 17, HCT 18, HCT 19. Contains one or more cells containing an artificial expression construct including CN3098, CN2122, CN2088, CN2162, CN2499, CN3062, CN2109, CN2845, CN2979, HCT32, HCT33, HCT34, HCT39, HCT40, HCT41, HCT42, HCT43, HCT44, HCT45, HCT46, CN3406, CN2253, CN2416, AiP1427, CN2786, CN2251, CN2913, CN2631, HCT47, HCT48, HCT49, HCT50, HCT69 and / or CN1389.

[0141] A detailed description of the method for producing transgenic animals is found in U.S. Patent No. 4,736,866. Transgenic animals may be any non-human species, but are preferably non-human primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens; or rodents such as guinea pigs, hamsters, gerbils, rats, mice, ferrets, etc.

[0142] In certain embodiments, the creation of transgenic animals yields organisms in which the recombinant construct is introduced at the same genomic integration site in every cell. Therefore, cell lines derived from such transgenic animals exhibit consistent characteristics in that they all have the recombinant construct at the same genomic integration site, and thus all these cells undergo the same variegated position effect. In contrast, introducing a gene into a cell line or primary cell culture results in heterologous expression of the construct. This method has the drawback that the expression of the introduced DNA may be influenced by the specific genetic background of the host animal.

[0143] As previously mentioned in relation to cell lines, the artificial expression constructs of this disclosure can be used for genetic modification of mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression constructs are introduced into cultured mouse embryonic stem cells. The transformed ES cells are then injected into blastocysts derived from a host mother, and the host embryo is re-implanted into the host mother. This procedure yields a chimeric mouse having tissue composed of cells derived from both embryonic stem cells present in the cultured cell line and embryonic stem cells present in the host embryo. Typically, mice with a different coat color are selected to isolate the cultured ES cells used for gene transfer from the host mouse into which the transformed cells are injected. Thus, the chimeric mouse has a mixed coat color. If at least a portion of the germline tissue is derived from the genetically modified cells, the chimeric mouse can then be crossed with an appropriate strain to obtain offspring carrying the transgene.

[0144] In addition to the delivery methods described above, other methods for delivering artificial expression constructs to target cells or target tissues or organs of animals, specifically cells, organs, or tissues of mammalian vertebrates, include sonophoresis (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 5,783,208); feedback-controlled delivery (U.S. Patent No. 5,697,899); and other available delivery methods and / or other delivery methods separately described herein.

[0145] (v) How to use In certain embodiments, compositions containing the bioactive ingredients described herein are administered to a subject to produce a physiological effect.

[0146] In certain embodiments, the Disclosure includes the use of the artificial expression constructs described herein to modulate the expression of a heterologous gene that is partially or entirely encoded downstream of a recombinant sequence enhancer. Accordingly, the Disclosure provides methods for using the artificial expression constructs described herein in the investigation, research, and future development of pharmaceuticals for the prevention, treatment, or mitigation of symptoms of disease, dysfunction, or disorder.

[0147] A particular embodiment is a method for inducing gene expression in a target type of cell by administering an artificial expression construct to the target, wherein the artificial expression construct is one of the eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, eHGT_387m, eH GT_385m, eHGT_386m, eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, eHGT_641m, eHGT_743m, eHGT_1158m, eHGT_1181m, eHGT_1182m, e HGT_1183m, eHGT_1184m, eHGT_1185m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHG T_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E13 6, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eH GT_453m, 3xcore2_eHGT_743m, 3xCore2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_ eHGT_1138m, hI56i(core), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h,3xcore3_eHGT_450h and / or eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core), and / or AiP1425, CN2724, CN1390, AiP1365, CN3038, CN2102, CN2951, CN3323, CN2237, CN2514, CN3018, CN3044, CN2229, CN2787, CN1528, CN2609, CN2360, CN2847, CN1457, CN3317, CN3318, CN3184, CN4388, CN4262, CN4263, CN4264, CN4265, CN2043, HCT 1, HCT 2, HCT 3, HCT 4, HCT 5, HCT 6, HCT 7, HCT 8, HCT 9, HCT 10, HCT 11, HCT 12, HCT 13, HCT 14, HCT 15, HCT 16, HCT 17, HCT 18, HCT 19, CN3098, CN2122, CN2088, CN2162, CN2499, CN3062, CN2109, CN2845, CN2979, HCT32, HCT33, HCT34, HCT39, HCT40, HCT41, HCT42, HCT43, HCT44, HCT45, HCT46, CN3406, CN2253, CN2416, AiP1427, CN2786, CN2251, CN2913, CN2631, HCT47, HCT48, HCT49, HCT50, HCT69 and / or CN1389, characterized by including a method. The subject may be an isolated cell, a network of cells, a tissue section, an experimental animal, a veterinary animal or a human.,

[0148] As is well known in the medical field, the dosage administered to a subject depends on various factors such as the size, surface area and age of the subject's body, the specific compound administered, sex, administration period and route, general health status, and other drugs during concomitant administration. The dosage of the compounds of the present disclosure varies variously, but in certain embodiments, the dosage of the artificial expression construct of the present disclosure is 10 5 ~10 100It may be a copy. In certain embodiments, for patients receiving intravenous, intraparenchymal, intrathecal, posterior orbital, or intrathecal administration, 10 6 ~10 22 It is possible to inject a copy of the artificial expression construct.

[0149] An "effective dose" is the amount of a composition required to produce a desired physiological change in a subject. Effective doses are often administered for research purposes. The effective doses disclosed herein are amounts that can produce a statistically significant effect in animal models, human studies, in vivo assays, or in vitro assays.

[0150] The amount of expression construct and the duration of administration of such composition will be determined by those skilled in the art who enjoy the benefits of the teachings of the present invention. However, it is conceivable that an effective dose of the composition of this disclosure may be administered by a single dose, for example, by a single injection of a sufficient number of infectious particles to impart an effect to the subject. Alternatively, depending on the circumstances, it may be desirable to administer the artificial expression construct composition or other gene construct multiple times or consecutively over a relatively short or relatively long period, and the decision of whether to administer such a dose may be determined by the person monitoring the administration of such composition. For example, the number of infectious particles administered to a mammal may be 10,0 7 pieces / ml, 10 8 pieces / ml, 10 9 pieces / ml, 10 10 pieces / ml, 10 11 pieces / ml, 10 12 pieces / ml, 10 13 The dose may be a single dose of ions / ml or more, or two or more divided doses. In certain embodiments, it may actually be desirable to administer two or more expression constructs in combination to obtain the desired effect.

[0151] In certain circumstances, it is desirable that the artificial expression construct be delivered in the form of a composition appropriately formulated herein, by pipetting, or by post-orbital injection, subcutaneous administration, intraocular administration, intravitreous administration, parenteral administration, subcutaneous administration, intravenous administration, intraparenchymal administration, intraventricular administration, intramuscular administration, intrascalar administration, intrathecal administration, intraperitoneal administration, oral administration, nasal inhalation, or direct administration or direct injection into one or more cells, tissues or organs. The method of administration may include the methods described in U.S. Patent Nos. 5,543,158; U.S. Patent Nos. 5,641,515 and U.S. Patent Nos. 5,399,363.

[0152] (vi) Kits and commercial packaging The kit and commercial packaging include the artificial expression construct described herein. This artificial expression construct can be isolated. In certain embodiments, the components of the expression product can be separated from each other. In certain embodiments, the expression product is found in a vector, a viral vector, intracellularly, in a tissue section or tissue sample, and / or in a transgenic animal. Such a kit may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or means for delivering the compositions of the Disclosure (e.g., syringes and injectables).

[0153] Embodiments of the kit or commercial package further include instructions relating to the use of the components contained in the kit or commercial package in, for example, basic research, electrophysiological research, neuroanatomical research, and / or research on and / or treatment of disorders, diseases or pathologies.

[0154] The following exemplary embodiments are provided to illustrate specific embodiments of the Disclosure. Those skilled in the art who have referred to the Disclosure will understand that various modifications can be made to the specific embodiments disclosed herein, and that such modifications will not depart from the essence and scope of the Disclosure and will result in similar or comparable outcomes.

[0155] (vii) Exemplary Embodiments 1. An artificial enhancer that includes the core region of eHGT_390m enhancer, eHGT_410m enhancer, eHGT_1139m enhancer, eHGT_1140m enhancer, eHGT_1137m enhancer, eHGT_1138m enhancer, hI56i enhancer, eHGT_367h enhancer, eHGT_453m enhancer, eHGT_779m enhancer, eHGT_743m enhancer, eHGT_140h enhancer, eHGT_121h enhancer, or eHGT_450h enhancer. 2. An artificial enhancer according to Embodiment 1, wherein the eHGT_390m enhancer, the eHGT_410m enhancer, the eHGT_1139m enhancer, the eHGT_1140m enhancer, the eHGT_1137m enhancer, the eHGT_1138m enhancer, the hI56i enhancer, the eHGT_367h enhancer, the eHGT_453m enhancer, the eHGT_779m enhancer, the eHGT_743m enhancer, the eHGT_140h enhancer, the eHGT_121h enhancer, or the eHGT_450h enhancer is a human or mouse enhancer. 3. An artificial enhancer according to Embodiment 1 or 2, comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, or comprising a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. 4. An artificial enhancer according to any one of Embodiments 1 to 3, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the eHGT_390m enhancer, the eHGT_410m enhancer, the eHGT_1139m enhancer, the eHGT_1140m enhancer, the eHGT_1137m enhancer, the eHGT_1138m enhancer, the hI56i enhancer, the eHGT_367h enhancer, the eHGT_453m enhancer, the eHGT_779m enhancer, the eHGT_743m enhancer, the eHGT_140h enhancer, the eHGT_121h enhancer, and / or the eHGT_450h enhancer. 5. An artificial enhancer according to Embodiment 4, comprising two, three, four, five, six, seven, eight, nine, or ten copies of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, or comprising two, three, four, five, six, seven, eight, nine, or ten copies of a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. 6. An artificial enhancer according to any one of Embodiments 3 to 5, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 1. 7. An artificial enhancer according to any one of embodiments 3 to 6, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 3. 8. An artificial enhancer according to any one of Embodiments 3 to 7, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 6. 9. An artificial enhancer according to any one of Embodiments 3 to 8, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 8. 10. An artificial enhancer according to any one of embodiments 3 to 9, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 11. 11. An artificial enhancer according to any one of Embodiments 3 to 10, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 13. 12. An artificial enhancer according to any one of Embodiments 3 to 11, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 15. 13. An artificial enhancer according to any one of embodiments 3 to 12, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 17. 14. An artificial enhancer according to any one of Embodiments 3 to 13, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 20. 15. An artificial enhancer according to any one of Embodiments 3 to 14, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 22. 16. An artificial enhancer according to any one of Embodiments 3 to 15, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 24. 17. An artificial enhancer according to any one of Embodiments 3 to 16, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 26. 18. An artificial enhancer according to any one of Embodiments 3 to 17, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of Sequence ID No. 28. 19. The artificial enhancer according to Embodiment 6, comprising three copies of Sequence ID No. 1. 20. The artificial enhancer according to Embodiment 7, comprising three copies of Sequence ID No. 3. 21. The artificial enhancer according to Embodiment 8, comprising three copies of Sequence ID No. 6. 22. The artificial enhancer according to Embodiment 9, comprising three copies of Sequence ID No. 8. 23. An artificial enhancer according to Embodiment 10, comprising three copies of Sequence ID No. 11. 24. The artificial enhancer according to Embodiment 11, comprising three copies of Sequence ID No. 13. 25. The artificial enhancer according to Embodiment 12, comprising three copies of Sequence ID No. 15. 26. The artificial enhancer according to Embodiment 13, comprising three copies of Sequence ID No. 17. 27. The artificial enhancer according to Embodiment 14, comprising three copies of Sequence ID No. 20. 28. The artificial enhancer according to Embodiment 15, comprising three copies of Sequence ID No. 22. 29. The artificial enhancer according to Embodiment 16, comprising three copies of Sequence ID No. 24. 30. The artificial enhancer according to Embodiment 17, comprising three copies of Sequence ID No. 26. 31. The artificial enhancer according to Embodiment 18, comprising three copies of Sequence ID No. 28. 32. An artificial enhancer according to any one of Embodiments 1 to 5, comprising one copy of Sequence ID No. 10. 33. An artificial enhancer according to any one of Embodiments 1 to 20, comprising three copies of Sequence ID No. 1 and three copies of Sequence ID No. 3. 34. The artificial enhancer according to Embodiment 19, comprising the sequence shown in Sequence ID No. 2, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 2. 35. An artificial enhancer according to Embodiment 20, comprising the sequence shown in Sequence ID No. 4, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 4. 36. An artificial enhancer according to Embodiment 21, comprising the sequence shown in Sequence ID No. 7, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 7. 37. The artificial enhancer according to Embodiment 22, comprising the sequence shown in Sequence ID No. 9, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 9. 38. An artificial enhancer according to Embodiment 23, comprising the sequence shown in Sequence ID No. 12, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 12. 39. An artificial enhancer according to Embodiment 24, comprising the sequence shown in Sequence ID No. 14, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 14. 40. An artificial enhancer according to Embodiment 25, comprising the sequence shown in Sequence ID No. 16, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 16. 41. An artificial enhancer according to Embodiment 26, comprising the sequence shown in Sequence ID No. 19, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 19. 42. An artificial enhancer according to Embodiment 27, comprising the sequence shown in Sequence ID No. 21, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 21. 43. An artificial enhancer according to Embodiment 28, comprising the sequence shown in Sequence ID No. 23, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 23. 44. An artificial enhancer according to Embodiment 29, comprising the sequence shown in Sequence ID No. 25, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 25. 45. An artificial enhancer according to Embodiment 30, comprising the sequence shown in Sequence ID No. 27, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 27. 46. ​​An artificial enhancer according to Embodiment 31, comprising the sequence shown in Sequence ID No. 29, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 29. 47. An artificial enhancer according to Embodiment 32, comprising the sequence shown in Sequence ID No. 5, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No. 5. 48. An artificial expression construct, (i)eHGT_1131h、eHGT_1132h、eHGT_1133h、eHGT_1134h、eHGT_1135h、eHGT_1137m、eHGT_1138m、eHGT_1145m、eHGT_1048m、eHGT_1050m、eHGT_1139m、eHGT_1140m、eHGT_380h、eHGT_387m、eHGT_385m、eHGT_386m、eHGT_361h、eHGT_400h、eHGT_403h、eHGT_409h、eHGT_410m、eHGT_641m、eHGT_743m、eHGT_1158m、eHGT_1181m、eHGT_1182m、eHGT_1183m、eHGT_1184m、eHGT_1185m、eHGT_1159m、eHGT_1160m、eHGT_1186m、eHGT_1187m、eHGT_1188m、eHGT_1136m、eHGT_1143m、eHGT_1144m、eHGT_1141m、eHGT_1142m、eHGT_1049m、eHGT_1052m、eHGT_1051m、eHGT_1053m、eHGT_1054m、eHGT_1055m、eHGT_1056m、MGT_E132、eHGT_638m、MGT_E136、eHGT_452h、eHGT_441h、eHGT_082h、eHGT_779m、eHGT_519h、eHGT_647m、eHGT_078h、eHGT_356h、eHGT_888m、eHGT_458m、eHGT_577h、MGT_E135、eHGT_453m、3xcore2_eHGT_743m、3xCore2_eHGT_390m、3xCore-eHGT_410m、3Xcore_eHGT_1139m、3Xcore-eHGT_1140m、3Xcore_eHGT_1137m、3Xcore_eHGT_1138m、hI56i(core)、3xhI56i(core)、core2_eHGT_367h、3xcore2_eHGT_453m、3xcore2_eHGT_779m、3xCore_eHGT_140h、3xCore_eHGT_121h、3xcore3_eHGT_450h、and an enhancer selected from eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core), (ii) Promoter and (iii) heterogeneous code sequences and Artificial expression constructs, including those mentioned above. 49. The artificial expression construct according to Embodiment 48, wherein the heterogeneous code sequence encodes an effector element or an expressible element. 50. The artificial expression construct according to Embodiment 49, wherein the effector element comprises a reporter protein or a functional molecule. 51. The artificial expression construct according to Embodiment 50, wherein the reporter protein comprises a fluorescent protein. 52. The artificial expression construct according to Embodiment 50, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug. 53. The artificial expression construct according to Embodiment 49, wherein the expressible element includes a non-functional molecule. 54. The artificial expression construct according to Embodiment 53, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated solely by a designer drug. 55. An artificial expression construct according to any one of embodiments 48 to 54, which associates with a capsid that crosses the blood-spinal barrier. 56. The artificial expression construct according to Embodiment 55, wherein the capsid comprises PHP.eB, AAV-PHP.S, or AAV-9p31. 57. An artificial expression construct according to any one of embodiments 48 to 56, which includes or codes for a skipping element. 58. The artificial expression construct according to Embodiment 57, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 59. The artificial expression construct according to Embodiment 58, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 60. eHGT_1131h、eHGT_1132h、eHGT_1133h、eHGT_1134h、eHGT_1135h、eHGT_1137m、eHGT_1138m、eHGT_1145m、eHGT_1048m、eHGT_1050m、eHGT_1139m、eHGT_1140m、eHGT_380h、eHGT_387m、eHGT_385m、eHGT_386m、eHGT_361h、eHGT_400h、eHGT_403h、eHGT_409h、eHGT_410m、eHGT_641m、eHGT_743m、eHGT_1158m、eHGT_1181m、eHGT_1182m、eHGT_1183m、eHGT_1184m、eHGT_1185m、eHGT_1159m、eHGT_1160m、eHGT_1186m、eHGT_1187m、eHGT_1188m、eHGT_1136m、eHGT_1143m、eHGT_1144m、eHGT_1141m、eHGT_1142m、eHGT_1049m、eHGT_1052m、eHGT_1051m、eHGT_1053m、eHGT_1054m、eHGT_1055m、eHGT_1056m、MGT_E132、eHGT_638m、MGT_E136、eHGT_452h、eHGT_441h、eHGT_082h、eHGT_779m、eHGT_519h、eHGT_647m、eHGT_078h、eHGT_356h、eHGT_888m、eHGT_458m、eHGT_577h、MGT_E135、eHGT_453m、3xcore2_eHGT_743m、3xCore2_eHGT_390m、3xCore-eHGT_410m、3Xcore_eHGT_1139m、3Xcore-eHGT_1140m、3Xcore_eHGT_1137m、3Xcore_eHGT_1138m、hI56i(core)、3xhI56i(core)、core2_eHGT_367h、3xcore2_eHGT_453m、3xcore2_eHGT_779m、3xCore_eHGT_140h、3xCore_eHGT_121h、3xcore3_eHGT_450h、eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)、An artificial expression construct according to any one of Embodiments 48-59, comprising or encoding a set of features selected from AAV, scAAV, rAAV, pAAV, minBglobin, CMV, minCMV, minCMV*, minRho, minRho*, fluorescent protein, hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10, tag cassette, 10aa, nuclear localization protein, self-cleaving peptide, WPRE, WPRE3, hGHpA and / or BGHpA. 61. MGT_E132-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_638m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xhI56i(core)-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; MGT_E136-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xcore2_eHGT_743m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_387m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3xCore-eHGT_410m-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; 390m(core2)-hI56i(core)-390m(core2)-hI56i(core)-390m(core2)-hI56i(core)-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; eHGT_452h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; core2_eHGT_367h-minRho*-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xSP10ins-core2_eHGT_367h-minRho*-[different code sequence]-[post-transcription regulatory element]; 3xcore2_eHGT_453m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3xcore2_eHGT_779m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_441h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xCore_eHGT_140h_minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_082h-minRho-[heterogeneous code sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_082h-minRho-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_779m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_519h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_647m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_078h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xCore2_eHGT_390m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_641m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1131h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1132h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1133h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1134h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1135h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_356h-minRho*-[different code sequence]-[post-transcription regulatory element]; 3xSP10ins-eHGT_356h-minRho*-[different code sequence]-[post-transcription regulatory element]; eHGT_1137m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1138m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1139m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1140m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1136m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1141m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1142m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1143m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1144m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1145m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1048m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1049m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1050m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1051m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1052m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1053m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1054m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1055m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1056m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_380h-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_385m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_386m-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_400h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_403h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_409h-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_410m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_361h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1158m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1159m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1160m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1181m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1182m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1183m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1184m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1185m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1186m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1187m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1188m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_888m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_458m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_577h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; MGT_E135-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_3xCore_eHGT_121h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_453m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xcore3_eHGT_450h-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_743m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3Xcore_eHGT_1137m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3Xcore_eHGT_1138m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3Xcore_eHGT_1139m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3Xcore-eHGT_1140m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; hI56i(core)-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; MGT_E132-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_638m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; 3xhI56i(core)-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; MGT_E136-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3xcore2_eHGT_743m-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; eHGT_387m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; 3xCore-eHGT_410m-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; 390m(core2)-hI56i(core)-390m(core2)-hI56i(core)-390m(core2)-hI56i(core)-minBglobin-[different code sequence]-WPRE3-BGHpA; eHGT_452h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; core2_eHGT_367h-minRho*-[heterogeneous code array]-WPRE3-BGHpA; 3xSP10ins-core2_eHGT_367h-minRho*-[different code array]-WPRE3-BGHpA; 3xcore2_eHGT_453m-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; 3xcore2_eHGT_779m-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; eHGT_441h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; 3xCore_eHGT_140h_minBglobin-[heterogeneous code array]-WPRE3-BGHpA; eHGT_082h-minRho-[different code sequence]-WPRE3-BGHpA; hsA2-eHGT_082h-minRho-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_779m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_519h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_647m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_078h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; 3xCore2_eHGT_390m-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; eHGT_641m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1131h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1132h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1133h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1134h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1135h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_356h-minRho*-[different code sequence]-WPRE3-BGHpA; 3xSP10ins-eHGT_356h-minRho*-[different code sequence]-WPRE3-BGHpA; eHGT_1137m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1138m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1139m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1140m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1136m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1141m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1142m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1143m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1144m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1145m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1048m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1049m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1050m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1051m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1052m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1053m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1054m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1055m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1056m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_380h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_385m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_386m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_400h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_403h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_409h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_410m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_361h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1158m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1159m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1160m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1181m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1182m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1183m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1184m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1185m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1186m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1187m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1188m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_888m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_458m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_577h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; MGT_E135-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_3xCore_eHGT_121h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_453m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; 3xcore3_eHGT_450h-minBglobin-[heterogeneous code array]-WPRE3-BGHpA; eHGT_743m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; 3Xcore_eHGT_1137m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3Xcore_eHGT_1138m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3Xcore_eHGT_1139m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3Xcore-eHGT_1140m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; and hI56i(core)-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA An artificial expression construct according to any one of embodiments 48 to 60, comprising or encoding a set of features selected from these. 62. A vector comprising the artificial expression construct described in any one of embodiments 48 to 61. 63. The vector according to embodiment 62, which is a viral vector. 64. The vector according to Embodiment 63, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector. 65. Adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1137m, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_380h, eHGT_387m, eHGT_385m, eHGT_386m, eHGT_361h, eHGT_ 400h, eHGT_403h, eHGT_409h, eHGT_410m, eHGT_641m, eHGT_743m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_1185m , eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049 m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT _082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, 3xcore2_eHGT_74 3m, 3xCore2_eHGT_390m, 3xCore-eHGT_410m, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, hI56i(core ), 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h, 3xcore3_eHGT_450h,AAV vectors under transcriptional control of enhancers and promoters selected from eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core). 66. The AAV vector according to Embodiment 65, wherein the heterogeneous code sequence encodes an effector element or an expressible element. 67. The AAV vector according to Embodiment 66, wherein the effector element comprises a reporter protein or a functional molecule. 68. The AAV vector according to Embodiment 67, wherein the reporter protein comprises a fluorescent protein. 69. The AAV vector according to Embodiment 67, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombinant donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug. 70. The AAV vector according to Embodiment 66, wherein the expressible element includes a non-functional molecule. 71. The AAV vector according to Embodiment 70, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated only by a designer drug. 72. Transgenic cells comprising an artificial expression construct or vector as described in any one of the prior embodiments. 73. Transgenic cells according to Embodiment 72, which are spinal motor neurons, alpha motor neurons, gamma motor neurons, spinal excitatory neurons, spinal inhibitory neurons, whole spinal neurons, cerebrospinal fluid contact neurons (CSF-cNs), or non-neuronal cells of the spinal cord. 74. The transgenic cell according to Embodiment 73, wherein the spinal motor neurons include Spp1 spinal motor neurons, Parg spinal motor neurons, Ogdh1 spinal motor neurons, or ChAT spinal motor neurons. 75. The transgenic cell according to embodiment 73, wherein the α motor neuron includes a Chold spinal motor neuron. 76. The transgenic cell according to Embodiment 73, wherein the spinal cord excitatory neurons include Mafa excitatory neurons, Esrrg, Trhr excitatory neurons, or Slc17a6 spinal cord excitatory neurons. 77. The transgenic cell according to Embodiment 73, wherein the spinal cord inhibitory neuron includes a Slc6a5 spinal cord inhibitory neuron. 78. The transgenic cell according to embodiment 73, wherein the entire spinal cord neuron includes an Esrrg spinal motor neuron. 79. The transgenic cell according to Embodiment 73, wherein the nonneuronal cells of the spinal cord include astrocytes or oligodendrocytes. 80. Transgenic cells according to any one of embodiments 72 to 79, which are mouse cells, human cells, or non-human primate cells. 81. A non-human transgenic animal comprising an artificial expression construct, vector, and / or transgenic cell as described in any one of the prior embodiments. 82. A non-human transgenic animal according to Embodiment 81, which is a mouse or a non-human primate. 83. An administerable composition comprising an artificial expression construct, vector, and / or transgenic cells as described in any one of the prior embodiments. 84. A kit comprising an artificial expression construct, vector, transgenic cells and / or a non-human transgenic animal as described in any one of the prior embodiments. 85. A method for expressing a gene in vivo or in vitro within a population of cells in the spinal cord or a population of cells derived from the spinal cord, comprising the step of expressing the gene within the cell population by providing a sample or subject containing a population of cells in the spinal cord or a population of cells derived from the spinal cord with an administerable composition according to Embodiment 83 in a sufficient dose and for a sufficient period of time. 86. The method according to Embodiment 85, wherein the gene encodes an effector element or an expressible element. 87. The method according to Embodiment 86, wherein the effector element comprises a reporter protein or a functional molecule. 88. The method according to Embodiment 87, wherein the reporter protein comprises a fluorescent protein. 89. The method according to Embodiment 87, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug. 90. The method according to Embodiment 86, wherein the expressible element includes a non-functional molecule. 91. The method according to Embodiment 90, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated only by a designer drug. 92. The method according to any one of embodiments 85 to 91, wherein the serving step includes pipetting. 93. The method according to embodiment 92, wherein the pipetting is performed on a spinal cord section. 94. The method according to Embodiment 93, wherein the spinal cord section includes spinal motor neurons, alpha motor neurons, gamma motor neurons, spinal excitatory neurons, spinal inhibitory neurons, whole spinal neurons, cerebrospinal fluid contact neurons (CSF-cNs), or non-neuronal cells of the spinal cord. 95. The method according to Embodiment 94, wherein the spinal motor neurons include Spp1 spinal motor neurons, Parg spinal motor neurons, Ogdh1 spinal motor neurons, or ChAT spinal motor neurons. 96. The method according to embodiment 94, wherein the α motor neuron includes a Chold spinal motor neuron. 97. The method according to embodiment 94, wherein the spinal cord excitatory neurons include Mafa excitatory neurons, Esrrg, Trhr excitatory neurons, or Slc17a6 spinal cord excitatory neurons. 98. The method according to embodiment 94, wherein the spinal cord inhibitory neuron includes a Slc6a5 spinal cord inhibitory neuron. 99. The method of embodiment 94, wherein the entire spinal neuron includes an Esrrg spinal motor neuron. 100. The method according to Embodiment 94, wherein the nonneuronal cells of the spinal cord include astrocytes or oligodendrocytes. 101. The method according to any one of embodiments 93 to 100, wherein the spinal cord slice is a spinal cord slice of a mouse, a human or a non-human primate. 102. The method according to any one of embodiments 93 to 101, wherein the providing step includes administration to a living subject. 103. The method according to embodiment 102, wherein the living subject is a human, a non-human primate or a mouse. 104. The method according to embodiment 102 or 103, wherein the administration to the living subject is performed by injection. 105. The method according to embodiment 104, wherein the injection includes intravenous injection, intrasubstance injection into spinal cord tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection or intrathecal injection. 106. An artificial expression construct, wherein SEQ ID NOs: 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, SEQ ID NOs: 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, It contains the sequence shown in SEQ ID NOs: 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 or 18, or SEQ ID NOs: 135, 136, 137, 138, 139, 140, 141, 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173,An artificial expression construct comprising a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 18.,

[0156] (viii) Conclusion The sequences disclosed herein and variants of the cited sequences are also included in the present application. Indicators for determining which amino acid residues can be substituted, inserted or deleted without loss of biological activity are computer programs well known in the art, such as DNASTAR TM software (Madison, Wisconsin, USA). The amino acid changes of the protein variants disclosed herein are preferably conservative amino acid changes, that is, substitutions between amino acids of similar charge or substitutions between non-charged amino acids. Conservative amino acid changes include substitutions by members of amino acid families whose side chains are related.

[0157] Appropriate conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such substitutions can usually be made without altering the biological activity of the resulting molecule. Those skilled in the art will know that substituting a single amino acid in a non-essential region of a polypeptide usually 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). Natural amino acids are typically classified into conserved substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar negative-charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar positive-charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6: (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu Groups 10 (nonpolar small aliphatic residues or slightly polar small aliphatic residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0158] When making such changes, the hydrophobicity index of amino acids may be taken into consideration. The importance of the hydrophobicity index of amino acids in conferring biological functions that interact with each other on proteins is widely understood in this art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). The hydrophobicity index for each amino acid is 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); Glutamic acid (-3.5); Gln (-3.5); Aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0159] It is well known in the art that substituting a specific amino acid with another amino acid having a similar hydrophobic index or degree of hydrophobicity can yield a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such a change, substitutions between amino acids with hydrophobic indexes within ±2 are preferred, substitutions between amino acids with hydrophobic indexes within ±1 are particularly preferred, and substitutions between amino acids with hydrophobic indexes within ±0.5 are even more particularly preferred. Furthermore, it is well known in the art that substitutions between similar amino acids can be effectively carried out based on their hydrophilicity.

[0160] As detailed in U.S. Patent No. 4,554,101, each amino acid residue is assigned a hydrophilicity value, which is as follows: Arg (+3.0); Lys (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is well known that specific amino acids can be substituted with other amino acids that have a similar hydrophilicity value, and that such substitutions can yield biologically equivalent proteins, and in particular, immunologically equivalent proteins. When making such changes, substitutions between amino acids with hydrophilicity values ​​within ±2 are preferred, substitutions between amino acids with hydrophilicity values ​​within ±1 are particularly preferred, and substitutions between amino acids with hydrophilicity values ​​within ±0.5 are even more preferred.

[0161] As outlined earlier, amino acid substitutions may be performed based on the relative similarity of substituents on the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, and size.

[0162] As otherwise described herein, gene sequence variants include codon-optimization variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0163] Variants of proteins, nucleic acids, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the proteins, nucleic acids, or gene sequences disclosed herein.

[0164] "Sequence identity (%)" refers to the relationship between two or more sequences measured by comparing them. In this technology, "identity" also means the degree of association between protein sequences, nucleic acid sequences, or gene sequences, measured by matching between protein sequence chains, nucleic acid sequence chains, or gene sequence chains. "Identity" (often called "similarity") can be easily calculated using known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods for measuring identity are preferably designed to obtain the best match between the sequences being tested. Methods for measuring identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Madison, Wisconsin), which is included in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989), using default parameters (gap penalty = 10, gap length penalty = 10)). Related programs include the GCG program suite (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, Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the program's default values. In this specification, "default values" means a set of numerical values ​​or parameters pre-registered in the software during software initialization.

[0165] The variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequences. Exemplary stringent hybridization conditions include incubation overnight at 42°C in a solution containing 50% formamide, 5×SSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and fragment-treated 20 μg / ml denatured salmon sperm DNA, followed by washing the filter at 50°C with 0.1×SSC. Modifications to the stringency of hybridization and signal detection are primarily achieved by adjusting the formamide concentration (lower formamide percentages result in lower stringency), salt conditions, or temperature. For example, moderately high stringency conditions include incubation at 37°C overnight 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 blocking salmon sperm DNA, followed by washing at 50°C with 1×SSPE and 0.1% SDS. Further lower stringency can be achieved by washing after stringent hybridization with a higher salt concentration (e.g., 5×SSC). The aforementioned conditions can be varied in various ways by adding and / or substituting other blocking reagents used to reduce the background of the hybridization experiment. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. When adding specific blocking reagents, it may be necessary to modify some of the aforementioned hybridization conditions due to compatibility issues.

[0166] The term "concatemerize" is used in a broad sense to mean linking together in a chain or in a continuous sequence. This term is used when referring to linking multiple nucleotide sequences to obtain a single nucleotide sequence, or linking multiple amino acid sequences to obtain a single amino acid sequence. Furthermore, "concatemerize" is interpreted as referring to "concatemerization."

[0167] As those skilled in the art will understand, each embodiment disclosed herein includes, substantially consists of, or comprises the specific components, processes, materials, or ingredients described herein. Therefore, the terms “includes” or “contains” should be interpreted as “includes, substantially consists of, or comprises.” The transitional phrase “includes” means, but is not limited to, the inclusion of components, processes, materials, or ingredients not described herein, even if in large quantities. The transitional phrase “consists of” excludes all components, processes, materials, or ingredients not described herein. The transitional phrase “substantially consists of” limits the scope of the embodiment to the described components, processes, materials, or ingredients, and components, or components, that do not materially affect the embodiment. A significant impact is the statistically significant decrease in target expression using a specific enhancer-target cell population combination, as measured by scRNA-Seq. Examples of these enhancer-target cell population combinations include eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, and eHGT_1137m / spinal motor neurons; eHGT_1141m and eHGT_1142m / Spp1 spinal motor neurons; eHGT_1049m and eHGT_1052m / Parg spinal motor neurons; eHGT_1051m / Ogdh1 spinal motor neurons; eHGT_1137m, eHGT_1138m, eHGT_1145m, e HGT_1048m and eHGT_1050m / ChAT spinal motor neurons; eHGT_1056m / Poln spinal motor neurons; 3Xcore_eHGT_1137m and 3Xcore_eHGT_1138m / entire spinal motor neurons; eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_1185m, 3Xcore_eHGT_1139m and 3Xcore-eHGT_1140m / α motor neurons; eHGT_1139m and eHGT_1140m / Chodl spinal motor neurons; eHGT_1186m, eHGT_1187m and eHGT_1188m / γ motor neurons; eHGT_1158m / Mafa excitatory neurons;eHGT_1136m / Esrrg, Trhr excitatory neurons; eHGT_1053m and eHGT_1054m / Slc17a6 spinal cord excitatory neurons; 3Xcore2_eHGT_743m / Tac2 excitatory neurons; MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eHGT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHG T_453m, eHGT_743m, 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h and 3xcore3_eHGT_450h / spinal cord excitatory neurons; eHGT_1055m / Slc6a5 spinal cord inhibitory neurons; MGT_E132, eHGT_638m, MGT_E136, eHGT_452h, eHGT_441h, eHGT_082h, eH GT_779m, eHGT_519h, eHGT_647m, eHGT_078h, eHGT_356h, eHGT_888m, eHGT_458m, eHGT_577h, MGT_E135, eHGT_453m, eHGT_743m, 3xhI56i(core), core2_eHGT_367h, 3xcore2_eHGT_453m, 3xcore2_eHGT_779m, 3xCore_eHGT_140h, 3xCore_eHGT_121h and 3xcore3_eHGT_450h / spinal cord inhibitory neuron hI56i(core) and eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core) / GABAergic neurons; eHGT_1143m and eHGT_1144m / Esrrg spinal motor neurons; eHGT_1159m / entire spinal neurons; eHGT_1160m / entire type of neuron found in the spinal cord; eHGT_1144m / cerebrospinal fluid contact neurons (CSF-cN);Examples include eHGT_380h, eHGT_387m, eHGT_385m, eHGT_386m, 3xCore2_eHGT_390m, and eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core) / astrocytes; as well as eHGT_361h, eHGT_400h, eHGT_403h, eHGT_409h, eHGT_410m, eHGT_641m, and 3xCore-eHGT_410m / oligodendrocytes.

[0168] In certain embodiments, "artificial" means not a natural product.

[0169] Unless otherwise stated, in this specification and in the claims, all numerical values ​​representing the quantity or properties of materials, such as molecular weight and reaction conditions, are construed in all cases as being modified with the term "approximately." Therefore, unless otherwise stated, the numerical parameters described herein and in the appended claims are approximations that will vary depending on the desired properties to be obtained by the present invention. Without limiting the scope of the claim range and equivalence principle, each numerical parameter should be interpreted at least in light of the reported significant figures and with normal rounding. To be more precise, the term “approximately” when used with a given number or range has a meaning that can be reasonably interpreted by a person skilled in the art, namely, a range of ±20% of the given number; a range of ±19% of the given number; a range of ±18% of the given number; a range of ±17% of the given number; a range of ±16% of the given number; a range of ±15% of the given number; a range of ±14% of the given number; a range of ±13% of the given number; a range of ±12% of the given number; a range of ±11% of the given number; a range of ±10% of the given number; a range of ±9% of the given number; a range of ±8% of the given number; a range of ±7% of the given number; a range of ±6% of the given number; a range of ±5% of the given number; a range of ±4% of the given number; a range of ±3% of the given number; a range of ±2% of the given number; or a range of ±1% of the given number, indicating that the number or range is somewhat greater or less than the given number or range.

[0170] While the numerical ranges and parameters representing the broad scope of this invention are approximations and approximate ranges, the numerical values ​​described in the specific examples are reported as accurately as possible. However, all numerical values ​​inherently contain certain errors that inevitably arise due to the standard deviation associated with each test measurement.

[0171] In the description of this invention (particularly in the description of the following claims), “a,” “an,” “the,” and similar demonstrative pronouns are to be interpreted as encompassing both singular and plural unless otherwise stated or the context explicitly indicates otherwise. The numerical ranges described herein are intended to be a simplified way of referring individually to each numerical value within that range. Unless otherwise stated, each numerical value is described herein as if it were described individually. Unless otherwise stated or the context explicitly indicates otherwise, any method described herein may be carried out in any suitable order. Any use of any examples provided herein, or any language indicating examples (e.g., “etc.”), is for the sole purpose of illustrating the invention in detail and does not limit the scope of the invention as described in the claims. Terms described herein should not be interpreted as referring to non-claimed components essential for carrying out the invention.

[0172] The grouping of other components of the present invention disclosed herein or the grouping of various embodiments of the present invention should not be construed as limiting the present invention. Members of each group may be described individually in this specification or in the claims, or they may be described in this specification or in the claims in combination with other members of the groups described herein or other components. For convenience and / or patentability reasons, it is anticipated that one or more members of one group may be added to another group, or one or more members may be removed from a group. In the event of such additions or deletions, this specification includes groups configured to satisfy the description of all Markush groups described in the appendix claims.

[0173] Specific embodiments of the present invention are described herein, including embodiments that the inventors consider to be the best mode for carrying out the invention. Naturally, those skilled in the art will readily understand, by carefully reading the above detailed description, that the embodiments described herein can be modified in various ways. The inventors anticipate that those skilled in the art may appropriately adopt such modifications, and intend that the present invention may be carried out in ways other than those specifically described herein. Accordingly, the present invention includes, to the extent possible within the scope of applicable law, all modifications from the subject matter of the present invention as described in the appended claims and all equivalents of the subject matter of the present invention. Furthermore, unless otherwise stated or unless the context expresses otherwise, all combinations of the aforementioned components in any modification are also included in the present invention.

[0174] Furthermore, this specification draws reference to various patents, publications, journal articles, and other documents (references herein). Each reference cited herein constitutes a part of this specification, and the teachings cited therein are incorporated herein by reference.

[0175] Finally, embodiments of the present invention disclosed herein are to be interpreted as illustrating the principles of the present invention. Other modifications may be adopted within the scope of the present invention. Therefore, as an example, other configurations of the present invention may be used in accordance with the teachings herein, but are not limited thereto. Accordingly, the present invention is not strictly limited to what is expressed and described herein.

[0176] The details described in this specification are for example only, and are presented only for the purpose of exemplifying the preferred embodiments of the present invention, to provide what is considered to be the most useful, and to enable an easy understanding of the principles and conceptual aspects of the various embodiments of the present invention. In this regard, the details of the structure of the present invention are not described in more detail than the information necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by carefully reading the description of the present invention while referring to the drawings and / or examples.

[0177] The definitions and explanations used in this disclosure are intended to control future interpretations, unless there are clear and explicit changes in the following examples, or unless the meaning of the terms becomes meaningless or substantially meaningless due to the meaning of the terms. If the definition of a term does not make sense or is substantially meaningless from the interpretation of the term, it is desired to quote the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. An artificial expression construct comprising (i) the eHGT_1137m enhancer, (ii) a promoter, and (iii) a heterologous coding sequence.

2. An artificial enhancer that includes the core region of the eHGT_1137m enhancer, the core region of the eHGT_1139m enhancer, the core region of the eHGT_1140m enhancer, the core region of the eHGT_1138m enhancer, or the core region of the eHGT_140h enhancer.

3. The artificial enhancer according to claim 2, wherein the eHGT_1137m enhancer, the eHGT_1139m enhancer, the eHGT_1140m enhancer, the eHGT_1138m enhancer, or the eHGT_140h enhancer is a human or mouse enhancer.

4. The artificial enhancer according to claim 2, comprising SEQ ID NO: 28, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26, or comprising a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 28, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO:

26.

5. The artificial enhancer according to claim 2, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of eHGT_1137m, eHGT_1139m, eHGT_1140m, eHGT_1138m and / or eHGT_140h.

6. The artificial enhancer according to claim 5, comprising two, three, four, five, six, seven, eight, nine, or ten copies of sequence number 28, sequence number 15, sequence number 22, sequence number 24, or sequence number 26, or comprising two, three, four, five, six, seven, eight, nine, or ten copies of a sequence having at least 90% sequence identity with the sequence shown in sequence number 28, sequence number 15, sequence number 22, sequence number 24, or sequence number 26.

7. The artificial enhancer according to claim 4, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 28.

8. The artificial enhancer according to claim 4, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 15.

9. The artificial enhancer according to claim 4, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 22.

10. The artificial enhancer according to claim 3, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 24.

11. The artificial enhancer according to claim 4, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 26.

12. The artificial enhancer according to claim 7, comprising three copies of sequence number 28.

13. The artificial enhancer according to claim 8, comprising three copies of sequence number 15.

14. The artificial enhancer according to claim 9, comprising three copies of sequence number 22.

15. The artificial enhancer according to claim 10, comprising three copies of sequence number 24.

16. The artificial enhancer according to claim 11, comprising three copies of sequence number 26.

17. The artificial enhancer according to claim 12, comprising the sequence shown in Sequence ID No. 29, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No.

29.

18. The artificial enhancer according to claim 13, comprising the sequence shown in Sequence ID No. 16, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No.

16.

19. The artificial enhancer according to claim 14, comprising the sequence shown in Sequence ID No. 23, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No.

23.

20. The artificial enhancer according to claim 15, comprising the sequence shown in Sequence ID No. 25, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No.

25.

21. The artificial enhancer according to claim 16, comprising the sequence shown in Sequence ID No. 27, or a sequence having at least 90% sequence identity with the sequence shown in Sequence ID No.

27.

22. It is an artificial expression construct, (i) eHGT_1137m, eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_113 5h, eHGT_1138m, eHGT_1145m, eHGT_1048m, eHGT_1050m, eHGT_1139m, eHGT_114 0m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_1184m, eHGT_118 5m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136 Enhancers selected from m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, MGT_E135, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, and 3xCore_eHGT_140h, (ii) Promoter and, (iii) Heterogeneous code sequences and Artificial expression constructs, including those mentioned above.

23. The artificial expression construct according to claim 22, wherein the heterogeneous code sequence codes for an effector element or an expressible element.

24. The artificial expression construct according to claim 23, wherein the effector element comprises a reporter protein or a functional molecule.

25. The artificial expression construct according to claim 24, wherein the reporter protein comprises a fluorescent protein.

26. The artificial expression construct according to claim 24, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombinant donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug.

27. The artificial expression construct according to claim 23, wherein the expressible element includes a non-functional molecule.

28. The artificial expression construct according to claim 27, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated solely by a designer drug.

29. The artificial expression construct according to claim 22, which associates with a capsid that crosses the blood-spinal barrier.

30. The artificial expression construct according to claim 29, wherein the capsid comprises PHP.eB, AAV-PHP.S, or AAV-9p31.

31. The artificial expression construct according to claim 22, comprising or encoding a skipping element.

32. The artificial expression construct according to claim 31, wherein the skipping element comprises a 2A peptide or an internal ribosome entry site (IRES).

33. The artificial expression construct according to claim 32, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.

34. eHGT_1137m, eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1138m, eHGT_1145m, eHGT _1048m, eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_1183m, eHGT_118 4m, eHGT_1185m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_1188m, eHGT_1136m, eHGT_1143m, e HGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_ 1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, MGT_E135, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3X core_eHGT_1137m, 3Xcore_eHGT_1138m, 3xCore_eHGT_140h, AAV, scAAV, rAAV, pAAV, minBglobin, CMV, minCMV The artificial expression construct according to claim 22, comprising or encoding a set of features selected from minCMV*, minRho, minRho*, fluorescent protein, hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10, tag cassette, 10aa, nuclear localization protein, self-cleaving peptide, WPRE, WPRE3, hGHpA and / or BGHpA.

35. eHGT_1137m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; MGT_E132-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_638m-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; MGT_E136-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3xCore_eHGT_140h_minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_1131h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1132h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1133h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1134h-minBglobin-[heterogeneous coded sequence]-[post-transcriptional regulatory element]; eHGT_1135h-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1138m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1139m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1140m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_1136m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1141m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1142m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1143m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1144m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1145m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1048m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1049m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1050m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_1051m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1052m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1053m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1054m-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; eHGT_1055m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1056m-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; eHGT_1158m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1159m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1160m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1181m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_1182m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1183m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1184m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1185m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1186m-minBglobin-[different coding sequence]-[post-transcriptional regulatory element]; eHGT_1187m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; eHGT_1188m-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; MGT_E135-minBglobin-[heterogeneous code sequence]-[post-transcriptional regulatory element]; 3Xcore_eHGT_1137m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3Xcore_eHGT_1138m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3Xcore_eHGT_1139m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; 3Xcore-eHGT_1140m-minBglobin-[heterogeneous coding sequence]-[post-transcriptional regulatory element]; eHGT_1137m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; MGT_E132-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_638m-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; MGT_E136-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3xCore_eHGT_140h_minBglobin-[heterogeneous code array]-WPRE3-BGHpA; eHGT_1131h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1132h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1133h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1134h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1135h-minBglobin-[heterogeneous code sequence]-WPRE3-BGHpA; eHGT_1138m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1139m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1140m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1136m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1141m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1142m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1143m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1144m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1145m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1048m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1049m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1050m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1051m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1052m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1053m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1054m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1055m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1056m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1158m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1159m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1160m-minBglobin-[different code sequence]-WPRE3-bGHpA; eHGT_1181m-minBglobin-[different code sequence]-WPRE3-bGHpA; eHGT_1182m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1183m-minBglobin-[different code sequence]-WPRE3-bGHpA; eHGT_1184m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1185m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1186m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1187m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; eHGT_1188m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; MGT_E135-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3Xcore_eHGT_1137m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3Xcore_eHGT_1138m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; 3Xcore_eHGT_1139m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA; and 3Xcore-eHGT_1140m-minBglobin-[heterogeneous code sequence]-WPRE3-bGHpA The artificial expression construct according to claim 22, comprising or encoding a set of features selected from these.

36. A vector comprising the artificial expression construct according to claim 22.

37. The vector according to claim 36, which is a viral vector.

38. The vector according to claim 37, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector.

39. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is eHGT_1137m, eHGT_1131h, eHGT_1132h, eHGT_1133h, eHGT_1134h, eHGT_1135h, eHGT_1138m, eHGT_1145m, eHGT_1048m , eHGT_1050m, eHGT_1139m, eHGT_1140m, eHGT_1158m, eHGT_1181m, eHGT_1182m, eHGT_118 3m, eHGT_1184m, eHGT_1185m, eHGT_1159m, eHGT_1160m, eHGT_1186m, eHGT_1187m, eHGT_11 AAV vectors under transcriptional control of enhancers and promoters selected from 88m, eHGT_1136m, eHGT_1143m, eHGT_1144m, eHGT_1141m, eHGT_1142m, eHGT_1049m, eHGT_1052m, eHGT_1051m, eHGT_1053m, eHGT_1054m, eHGT_1055m, eHGT_1056m, MGT_E132, eHGT_638m, MGT_E136, MGT_E135, 3Xcore_eHGT_1139m, 3Xcore-eHGT_1140m, 3Xcore_eHGT_1137m, 3Xcore_eHGT_1138m, and 3xCore_eHGT_140h.

40. The AAV vector according to claim 39, wherein the heterogeneous code sequence codes for an effector element or an expressible element.

41. The AAV vector according to claim 40, wherein the effector element comprises a reporter protein or a functional molecule.

42. The AAV vector according to claim 41, wherein the reporter protein comprises a fluorescent protein.

43. The AAV vector according to claim 41, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombinant donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug.

44. The AAV vector according to claim 40, wherein the expressible element includes a non-functional molecule.

45. The AAV vector according to claim 44, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombinant donor cassette, or a non-functional designer receptor (DREADD) that is activated only by a designer drug.

46. Transgenic cells comprising the artificial expression construct according to claim 22 and / or the vector according to claim 36.

47. The transgenic cell according to claim 46, which is a spinal motor neuron, an alpha motor neuron, a gamma motor neuron, a spinal excitatory neuron, a spinal inhibitory neuron, an entire spinal neuron, a cerebrospinal fluid contact neuron (CSF-cN), or a non-neuronal cell of the spinal cord.

48. The transgenic cell according to claim 47, wherein the spinal motor neurons include Spp1 spinal motor neurons, Parg spinal motor neurons, Ogdh1 spinal motor neurons, or ChAT spinal motor neurons.

49. The transgenic cell according to claim 47, wherein the alpha motor neuron includes a Chold spinal motor neuron.

50. The transgenic cell according to claim 47, wherein the spinal cord excitatory neurons include Mafa excitatory neurons, Esrrg, Trhr excitatory neurons, or Slc17a6 spinal cord excitatory neurons.

51. The transgenic cell according to claim 47, wherein the spinal cord inhibitory neuron includes a Slc6a5 spinal cord inhibitory neuron.

52. The transgenic cell according to claim 47, wherein the entire spinal neuron comprises an Esrrg spinal motor neuron.

53. The transgenic cell according to claim 47, wherein the non-neuronal cells of the spinal cord include astrocytes or oligodendrocytes.

54. The transgenic cell according to claim 46, which is a mouse cell, a human cell, or a non-human primate cell.

55. A non-human transgenic animal comprising the artificial expression construct according to claim 22, the vector according to claim 36, and / or the transgenic cell according to claim 46.

56. A non-human transgenic animal according to claim 55, which is a mouse or a non-human primate.

57. An administerable composition comprising the artificial expression construct according to claim 22, the vector according to claim 36, and / or the transgenic cells according to claim 46.

58. A kit comprising the artificial expression construct according to claim 22, the vector according to claim 36, the transgenic cells according to claim 46, and / or the non-human transgenic animal according to claim 55.

59. A method for expressing a gene in vivo or in vitro within a population of cells in the spinal cord or a population of cells derived from the spinal cord, comprising the step of providing a sample or subject containing a population of cells in the spinal cord or a population of cells derived from the spinal cord with the administerable composition according to claim 57 in a sufficient dose and for a sufficient period of time, thereby expressing the gene within the cell population.

60. The method according to claim 59, wherein the gene encodes an effector element or an expressible element.

61. The method according to claim 60, wherein the effector element comprises a reporter protein or a functional molecule.

62. The method according to claim 61, wherein the reporter protein comprises a fluorescent protein.

63. The method according to claim 61, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombinant donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug.

64. The method according to claim 60, wherein the expressible element includes a non-functional molecule.

65. The method according to claim 64, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated only by a designer drug.

66. The method according to claim 59, wherein the serving step includes pipetting.

67. The method according to claim 66, wherein the pipetting is performed on a spinal cord section.

68. The method according to claim 67, wherein the spinal cord section includes spinal motor neurons, alpha motor neurons, gamma motor neurons, spinal excitatory neurons, spinal inhibitory neurons, whole spinal neurons, cerebrospinal fluid contact neurons (CSF-cNs), or non-neuronal cells of the spinal cord.

69. The method according to claim 68, wherein the spinal motor neurons include Spp1 spinal motor neurons, Parg spinal motor neurons, Ogdh1 spinal motor neurons, or ChAT spinal motor neurons.

70. The method according to claim 68, wherein the alpha motor neuron includes a Chold spinal motor neuron.

71. The method according to claim 68, wherein the spinal cord excitatory neurons include Mafa excitatory neurons, Esrrg, Trhr excitatory neurons, or Slc17a6 spinal cord excitatory neurons.

72. The method according to claim 68, wherein the spinal cord inhibitory neuron includes a Slc6a5 spinal cord inhibitory neuron.

73. The method according to claim 68, wherein the entire spinal neuron includes Esrrg spinal motor neurons.

74. The method according to claim 68, wherein the non-neuronal cells of the spinal cord include astrocytes or oligodendrocytes.

75. The method according to claim 67, wherein the spinal cord section is a spinal cord section of a mouse, human, or non-human primate.

76. The method according to claim 59, wherein the provisioning step includes administration to a living subject.

77. The method according to claim 76, wherein the living subject is a human, a non-human primate, or a mouse.

78. The method according to claim 76, wherein the administration to the living subject is performed by injection.

79. The method according to claim 78, wherein the injection includes intravenous injection, intraparenchymal injection into spinal cord tissue, intraventricular (ICV) injection, intracisional (ICM) injection, or intrathecal injection.

80. An artificial expression construct, containing SEQ ID NOs: 163, 135, 136, 137, 138, 139, 140, 141, 143, 144, 145, 146, 147, 148, 150, 152, 154, 155, 156, 157, 158, 159, 160, 161, 164, 165, 166, 167, 167 68, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 205, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213 or SEQ ID NO: 214 The sequence consists of the sequence shown, or substantially consists of any of these sequences, or sequence numbers 163, 135, 136, 137, 138, 139, 140, 141, 143, 144, 145, 146, 147, 148, 150, 152, 154, 155, 156, 157, 158, 159, 160, 161, 164, 165, sequence Number 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 205, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212,An artificial expression construct comprising, or substantially comprising, a sequence having at least 90% sequence identity with, the sequence shown in SEQ ID NO: 213 or SEQ ID NO: 214.

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