Artificial expression constructs for regulating gene expression in the basal ganglia of the brain

Abstract

This disclosure describes artificial expression constructs for regulating gene expression in target types of central nervous system cells. The artificial expression constructs of this disclosure can be used for the expression or regulation of gene expression in basal ganglia neurons, including striatal neurons, globus pallidus neurons, and substantia nigra neurons.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 582,759, filed on 14 September 2023, which is incorporated herein by reference in its entirety as if it were described herein in its entirety.

[0002] Statement on research and development funded by the federal government This invention was made with government support under grant number MH128339, awarded by the National Institutes of Health. The U.S. government reserves certain rights with respect to this invention.

[0003] This disclosure provides artificial expression constructs for regulating gene expression in target types of central nervous system cells. The artificial expression constructs of this disclosure can be used for the expression or regulation of synthetic genes in the basal ganglia.

[0004] 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 3AR7472.XML. This file is 135,137 bytes in size, was created on September 12, 2024, and was submitted electronically via the Patent Center. [Background technology]

[0005] To fully understand brain biology, it is necessary to distinguish and define various types of cells, and to investigate them further, it is necessary to identify artificial expression constructs that can label and perturb these cells. In mice, success has been achieved in labeling cell populations that share marker gene expression using driver strains that express recombinases. However, creating, maintaining, and using such strains capable of labeling specific types of cells with high specificity is costly, often requires the crossbreeding of transgenic animals between three species, and yields the desired experimental animals infrequently. 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 artificial expression constructs for inducing gene expression in target central nervous system cell populations. Target central nervous system cell populations include striatal cholinergic neurons, striatal somatostatin (SST) chondrolectin (Chodl) neurons, dopamine D1 receptor expressing (D1R+) granule cells in the islands of Calleja (ICj), SST LIM homeobox 6 (LHX6+) striatal interneurons, globus pallidus external segment (GPe) interneurons, and substantia nigra pars compacta (SNc) dopaminergic neurons.

[0007] In certain embodiments of the artificial expression constructs of this disclosure, the following enhancers are used to induce gene expression in a target population of central nervous system cells. The combinations of enhancers and target cell populations used in this disclosure are shown below in the order of enhancer / target cell population. Striatal neurons: eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, core2_eHGT_873m, 3xcore2_eHGT_873m and eHGT_1246rh / striatal cholinergic neurons; eHGT_127m and eHGT_769m / striatal SST Chodl neurons; eHGT_1223rh and eHGT_1226rh / D1R+ICj granule cells; and eHGT_1255rh and eHGT_1256rh / SST LHX6+ striatal interneurons; globus pallidus neurons: eHGT_1239rh / GPe interneurons; and Substantia nigra neurons: eHGT_1279rh and eHGT_1280rh / SNc dopaminergic neurons.

[0008] In certain embodiments, these artificial enhancer elements include a concatemerized enhancer core. Examples include the eHGT_873m core or a concatemerized eHGT_873m core. Using such artificial enhancer elements allows for more rapid expression of the transgene and higher expression levels than when using the full-length original (natural) enhancer alone.

[0009] In certain embodiments, the core of the enhancer contains the sequence shown in Sequence ID No. 89. In certain embodiments, the core of this enhancer is concatenated and has 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, a concatemer consisting of 3 copies of the eHGT_873m enhancer core contains the sequence shown in Sequence ID No. 26.

[0010] Certain embodiments provide an artificial expression construct that includes the characteristics of the vectors described herein, including CN3362, CN4621, CN4622, CN1389, CN3445, CN4496, CN4695, CN2826, CN2602, CN4609, CN4728, CN4626, CN4731, CN4629, CN4661, and CN4662. [Brief explanation of the drawing]

[0011] [Figure 1]The sequences supporting this disclosure are listed below: eHGT_868m (SEQ ID NO: 21), eHGT_1249rh (SEQ ID NO: 22), eHGT_1250rh (SEQ ID NO: 23), hI56i(core) (SEQ ID NO: 24), eHGT_873m (SEQ ID NO: 25), core2_eHGT_873m (SEQ ID NO: 89), 3xcore2_eHGT_873m (SEQ ID NO: 26), eHGT_1246rh (SEQ ID NO: 27), eHGT_127m (SEQ ID NO: 28), eHGT_769m (SEQ ID NO: 29), eHGT_1223rh (SEQ ID NO: 30), eHGT_1226rh( SEQ ID NO: 31), eHGT_1255rh (SEQ ID NO: 32), eHGT_1256rh (SEQ ID NO: 33), eHGT_1239rh (SEQ ID NO: 34), eHGT_1279rh (SEQ ID NO: 35), eHGT_1280rh (SEQ ID NO: 36), β-globin minimal promoter (pBGmin / minBGlobin / minBGprom) (SEQ ID NO: 37), minCMV promoter (SEQ ID NO: 38), mutant minCMV promoter (SacI restriction enzyme site removed) (SEQ ID NO: 39), minRho promoter - (SEQ ID NO: 40), minRho* promoter (SEQ ID NO: 41), Hsp68 minimal promoter (proHsp68) (SEQ ID NO: 42), YFP2 (SEQ ID NO: 43), EGFP (SEQ ID NO: 44), optimized Flp recombinase (FlpO) (SEQ ID NO: 45), improved Cre recombinase (iCre) (SEQ ID NO: 46), SP10 insulator (SP10ins) (SEQ ID NO: 47), 3xSP10ins (SEQ ID NO: 48), 3XFLAG (SEQ ID NO: 49), 10aa (SEQ ID NO: 50), H2B (SEQ ID NO: 5 1) H2B* (SEQ ID NO: 90), WPRE3 (SEQ ID NO: 52), WPRE (SEQ ID NO: 53), BGHpA (SEQ ID NO: 54), hGHpA (SEQ ID NO: 55), P2A (SEQ ID NO: 56), T2A (SEQ ID NO: 57), E2A (SEQ ID NO: 58), F2A (SEQ ID NO: 59), Exemplary plasmid skeleton 1 - left ITR (SEQ ID NO: 60), Exemplary plasmid skeleton 1 - right ITR (SEQ ID NO: 61), Exemplary plasmid skeleton 2 - left ITR (SEQ ID NO: 62), Exemplary plasmid skeleton 2 - right ITR (SEQ ID NO: 63), PHP.eB capsid (SEQ ID NO: 64), AAV9 VP1 capsid protein (SEQ ID NO: 65), tet-transactivator version 2 (tTA2) (SEQ ID NO: 66), GTPase HRas (SEQ ID NO: 67), Substance P (58-68 of Protachykinin-1) (SEQ ID NO: 68), Oxytocin (20-28 of Neurophysin-1) (SEQ ID NO: 69), GCaMP6m (SEQ ID NO: 70), GCaMP6s (SEQ ID NO: 71), GCaMP6f (SEQ ID NO: 72), CN3362 (SEQ ID NO: 73), CN4621 (SEQ ID NO: 74), CN4622 (SEQ ID NO: 75), CN13 89 (SEQ ID NO: 76), CN3445 (SEQ ID NO: 77), CN4496 (SEQ ID NO: 78), CN4695 (SEQ ID NO: 79), CN2826 (SEQ ID NO: 80), CN2602 (SEQ ID NO: 81), CN4609 (SEQ ID NO: 82), CN4728 (SEQ ID NO: 83), CN4626 (SEQ ID NO: 84), CN4731 (SEQ ID NO: 85), CN4629 (SEQ ID NO: 86), CN4661 (SEQ ID NO: 87), and CN4662 (SEQ ID NO: 88). [Modes for carrying out the invention]

[0012] To fully understand brain biology, it is necessary to distinguish and define various types of cells, and to investigate them further, it is necessary 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.

[0013] This disclosure provides artificial expression constructs for inducing gene expression in target central nervous system cell populations. Target central nervous system cell populations include striatal cholinergic neurons, striatal somatostatin (SST) chondrolectin (Chodl) neurons, dopamine D1 receptor expressing (D1R+) granule cells in the islets of Kaleha (ICj), SST LIM homeobox 6 (LHX6+) striatal interneurons, globus pallidus external segment (GPe) interneurons, and substantia nigra pars compacta (SNc) dopaminergic neurons.

[0014] In certain embodiments of the artificial expression constructs of the present disclosure, the following enhancers are utilized to induce gene expression in a target central nervous system cell population. The combinations of enhancers and target cell populations used in the present disclosure are shown below in the order of enhancer / target cell population. Striatal neurons: eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, core2_eHGT_873m and eHGT_1246rh / striatal cholinergic neurons; eHGT_127m, eHGT_769m / striatal SST Chodl neurons; eHGT_1223rh, eHGT_1226rh / D1R+ ICj granule cells; and eHGT_1255rh and eHGT_1256rh / SST LHX6+ striatal interneurons; Pallidal neurons: eHGT_1239rh / GPe interneurons; and Substantia nigra neurons: eHGT_1279rh and eHGT_1280rh / SNc dopaminergic neurons.

[0015] In certain embodiments, these artificial enhancer elements comprise a concatenated enhancer core. One example thereof is the core of eHGT_873m or the concatenated core of eHGT_873m. By using such artificial enhancer elements, the transgene can be rapidly expressed to achieve high expression, compared to the case where the full-length original (natural) enhancer is used alone.

[0016] In certain embodiments, the core of the enhancer contains the sequence shown in SEQ ID NO: 89. In certain embodiments, the core of this enhancer is concatemerized and has 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, a concatemer consisting of 3 copies of SEQ ID NO: 89 contains the sequence shown in SEQ ID NO: 26.

[0017] Certain embodiments provide an artificial expression construct that includes the characteristics of the vectors described herein, including CN3362, CN4621, CN4622, CN1389, CN3445, CN4496, CN4695, CN2826, CN2602, CN4609, CN4728, CN4626, CN4731, CN4629, CN4661, and CN4662.

[0018] 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 gene expression 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.

[0019] (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 a target type of central nervous system cell; (ii) the coding sequence to be expressed; and (iii) a promoter. The artificial expression constructs of this disclosure may further include other regulatory elements as they are necessary or beneficial. In certain embodiments, the “enhancer” or “enhancer element” is a cis-acting sequence that increases the transcription amount associated with the promoter and can function in either the forward or reverse direction with respect to the promoter and the coding sequence to be transcribed, and can be positioned upstream or downstream of the promoter or the coding sequence to be transcribed. 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_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, core2_eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, and eHGT_1280rh.

[0020] 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 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 cell types, and is therefore also a target CNS enhancer with cell type-specific transcriptional activity.

[0021] When a coding sequence (or heterologous coding sequence) operably linked to an enhancer disclosed herein is expressed in a target cell type, the administered coding sequence is expressed in the intended cell type.

[0022] When a coding sequence is selectively expressed in selected cells, the administered 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 expressed” in other cell types means that the expression in the reference cell type is less than 50% compared to the target cell type; less than 40% compared to the target cell type; less than 30% compared to the target cell type; less than 20% compared to the target cell type; or less than 10% compared to the target cell type. In certain embodiments, “reference cell type” refers 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 area. In certain embodiments, the reference cell type is located within an anatomical structure adjacent to the anatomical structure containing the target cell type. In certain embodiments, the reference cell type is a non-target cell having a different gene expression profile than the target cells.

[0023] In certain embodiments, the coding sequence product may be expressed at low levels in unselected cell types, for example, at less than 1% of the expression level of the product in selected cells, or at 1%, 2%, 3%, 5%, 10%, 15%, or 20%. In certain embodiments, the target type of central nervous system cell 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 cell type.

[0024] In certain embodiments, 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.

[0025] Classification of target basal ganglia cells:

[0026] The basal ganglia include a group of subcortical nuclei, including the striatum, globus pallidus, substantia nigra, and subthalamic nucleus, and these subcortical nuclei are located in the basal forebrain of vertebrates. The basal ganglia are also strongly interconnected with several other brain regions, such as the cortex, thalamus, and brainstem. Numerous studies have shown that the basal ganglia are involved in a variety of brain functions, including voluntary movement control, procedural learning, habitual behavior, eye movements, cognition, and emotion.

[0027] Classification of target striatal cells:

[0028] The striatum (Str) is important for converting cortical activity into voluntary movement. In humans / primates, the structures corresponding to the striatum are called the putamen, caudate nucleus, and ventral striatum, with the ventral striatum containing the nucleus accumbens. The striatum of rodents includes the dorsal striatum, which contains the nucleus accumbens. Therefore, the combined putamen and caudate nucleus of humans / primates correspond to the dorsal striatum of rodents.

[0029] Classification and subclassification of striatal cells: • Medium spiny neurons (overall): These make up 95% of striatal neurons and are known to express the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2, as well as Ppp1r1b / PPP1R1B. • Medium spiny neurons, direct pathway projection type: These account for nearly 50% of striatal neurons and are rich in Drd1 / DRD1, Pdyn / PDYN, and Slc35d3 / SLC35D3. The main axonal projection destinations from direct pathway medium spiny neurons are the substantia nigra pars reticularis (SNr) or the globus pallidus internal segment (GPi). • Medium spiny neurons, indirect pathway projection type: These account for nearly 50% of striatal neurons and are rich in Drd2 / DRD2, Adora2a / ADORA2A, Gpr6 / GPR6, and Penk / PENK. The main axonal projection destination from indirect pathway medium spiny neurons is the globus pallidus external segment (GPe). • Granule cells of the Isle of Kaleja (ICj): The Isle of Kaleja (ICj) is located in the ventral striatum and contains dense clusters of granule cells. ICj is part of the limbic system, and ICj granule cells are responsible for dopamine signaling and dopamine regulation. In this specification, ICj granule cells that express the D1 dopamine receptor are referred to as "D1R+ICj granule cells". • Striatal cholinergic neurons: Striatal cholinergic neurons are small groups of cells in the striatum that release acetylcholine, which is involved in regulating sensory functions and other processes. • SST neurons: SST neurons highly express the neurotransmitter somatostatin (SST) and frequently block dendritic input to postsynaptic neurons. This subcategory includes sleep-active SST Chodl neurons (which further express Nos1 and Tacr1), which differ significantly from other SST neurons but express several shared marker genes, including SST. SST neurons expressing CHODL are a rare type of neuron corresponding to long-range projecting interneurons (Tomioka, et al. Eur. J. Neurosci. 2005; 21:1587-1600). In humans, SST gene expression is often detected in subtypes of layer 1 LAMP5+ GABAergic neurons. In this specification, SST interneurons expressing LHX6 are referred to as "SST LHX6+ interneurons".

[0030] Classification of target pallidolar cells: The globus pallidus includes the internal segment (GPi) and the external segment (GPe). GPi is responsible for transmitting information to the thalamus. GPe is responsible for receiving information from striatal neurons expressing inhibitory D2 receptors and transmitting information to the substantia nigra (SN), the output nuclei of the basal ganglia including the internal segment (GPi), and the subthalamic nucleus (STH). GPe mainly consists of GABAergic neurons and interneurons, with cholinergic neurons making up a small proportion.

[0031] Classification of target substantia nigra cells: The substantia nigra is responsible for dopamine production. The substantia nigra mainly contains dopaminergic neurons and GABAergic neurons. Dopaminergic neurons secrete dopamine. Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system. Loss of dopaminergic neurons is associated with Parkinson's disease, one of the best-known human neurological diseases. Research on developmental pathways involved in the development of dopaminergic neurons in the brain has identified several specific transcription factors, such as Nurr1, Lmx1b, and Pitx3, all of which have been shown to be important in the development of the midbrain dopaminergic system.

[0032] 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 cell marker SNAP25. Astrocytes may have a characteristic star-shaped morphology and are involved in supporting the metabolism of other cells in the brain. 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 subcategory primarily responsible for myelin formation in nerve cells. • VLMCs: Vascular leptomeningeal cells (VLMCs) are part of the meninges that surround the outer layer of the cortex and express Lum and Col1a1 as marker genes. • Pericytes: These are vascular-related cells that express the marker genes Kcnj8 and Abcc9. Pericytes surround endothelial cells and are important for regulating capillary blood flow, and are involved in crossing the blood-brain barrier. • Smooth muscle cells (SMCs): These are specialized smooth muscle cells that express the Acta2 marker gene and are associated with blood vessels. Smooth muscle cells cover arterioles in the brain and are involved in blood-brain barrier crossing. • Endothelial cells: These are cells that line the blood vessels in the brain. Endothelial cells express Tek markers and PDGF-B markers. Microglia are immune cells derived from hematopoietic cells, macrophages localized in brain tissue, which can migrate from the blood and become associated with brain tissue, and are perivascular macrophages (PVMs) that can be found as a byproduct of brain surgery. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

[0033] In certain embodiments, the coding sequence is a heterogeneous coding sequence that codes for an effector element. 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.

[0034] 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); 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.

[0035] 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.

[0036] 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.

[0037] Examples of 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 solely by designer drugs, each possessing its own unique functionality.

[0038] 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).

[0039] 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 (CCAA-T / 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, Rab-4A, 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.

[0040] 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 widely used and highly efficient tools. GCaMPs are formed by fusing M13 and calmodulin proteins 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.

[0041] 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.

[0042] In certain embodiments, functional molecules include modulators of neuronal activity, such as channelrhodopsins (e.g., channelrhodopsin 1, channelrhodopsin 2, and their variants). Channelrhodopsins are a subfamily of 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.

[0043] 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.

[0044] In certain embodiments, localization cassettes are used as functional molecules. In certain embodiments, localization cassettes are used to localize molecules (e.g., vectors, proteins, sensors) to specific compartments below the cellular level, such as the cell body, axon, and dendrites of neurons. In certain embodiments, localization cassettes include cell body tags for localization to the cell body (e.g., cell body (EE-RR)); axon tags for localization to the axon (e.g., those derived from GAP43) or synaptophysin (sy); hydrophobic tails for localization to the cell membrane; and hydrophobic or alkyl chains for localization to the endoplasmic reticulum. In certain embodiments, localization cassettes are fused to sensor molecules such as GECI. In certain embodiments, fusion proteins of localization cassettes and GECI include cell body-jGCaMP8s, axon-jRGECO1a, syGCaMP5G, and cell body-jGCaMP7s.

[0045] In certain embodiments, tag cassettes are used as functional molecules. Examples of tag cassettes include His tag (HHHHHH; SEQ ID NO: 1), Flag tag (DYKDDDDK; SEQ ID NO: 2), Xpress tag (DLYDDDDK; SEQ ID NO: 3), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 4), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 5), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 6), Myc tag (EQKLISEEDL; SEQ ID NO: 7), Strep tag (referring to the original STREP® tag) (WRHPQFGG; SEQ ID NO: 8), STREP tag II (WSHPQFEK; SEQ ID NO: 9 (Institut fur Bioanalytik (IBA) GmbH, Germany); see, for example, US Patent No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 10), Softag Examples include 3 (TQDPSRVG; Sequence ID 11) and V5 tag (GKPIPNPLLGLDST; Sequence ID 12). 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 Sequence ID 49.

[0046] 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., UniProtKB / 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:CAA25240.1), 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), gene-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), ribonuclease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease IIβ (e.g., GenBank:AAF76893.1) Examples include 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).

[0047] 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.

[0048] 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.

[0049] 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 frequently 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.

[0050] 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*. The artificial expression construct may include a sequence encoding a linker consisting of 10 amino acids (10aa). In a particular embodiment, the 10aa linker is encoded by the sequence shown in Sequence ID No. 50.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 self-replication within a cell, 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.

[0055] 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.

[0056] 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.

[0057] 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, and all mRNAs induced by this promoter have a 5' tripertite leader (TPL) sequence, which is therefore preferentially translated.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] "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.

[0067] When using certain types of vectors, safety can be improved by inducing transcription of the viral genome by substituting the U3 region of the 5'LTR with a promoter during viral particle production. Examples of promoters usable 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 by a promoter reduces the likelihood of recombination occurring, which would produce a replicable virus, because the complete U3 sequence is deleted from the virus production system. In certain embodiments, promoters have another advantage: they can control how the viral genome is transcribed. For example, the promoter may be an inducible promoter, such that the entire or partial viral genome is transcribed only if an inducer is present. Inducing factors include one or more compounds or physiological conditions, such as the culture temperature or culture pH of the host cells.

[0068] 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 promoter.

[0069] 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.

[0070] In certain embodiments, sequence expression 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 nucleic acid expression. 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.

[0071] Gene expression can be increased by elements that can induce efficient transcription termination and polyadenylation of 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 the 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 utilized. 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.

[0072] 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 (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).

[0073] 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.

[0074] Specific embodiments of the vectors disclosed herein include those listed in the following table. [Table 1]

[0075] Subcomponent sequences within large vector sequences can be readily identified by those skilled in the art based on the description herein (see Figure 2). 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.

[0076] In certain embodiments, a vector (e.g., AAV) having a capsid that can cross the blood-brain barrier (BBB) ​​is selected. In certain embodiments, the vector is modified to include a capsid that can cross the blood-brain barrier. Examples of AAVs with viral capsids that can cross the blood-brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014; 7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014; 22(7): 1299-1309), AAV1R6, AAV1R7 (Albright et al., Mol Ther. 2018; 26(2): 510), rAAVrh.8 (Yang et al., op. cit.), AAV-BR1 (Marchio et al., EMBO Mol Med. 2016; 8(6): 592), AAV-PHP.S (Chan et al., Nat Neurosci. 2017; 20(8): 1172), and AAV-PHP.B (Deverman et al., Nat Biotechnol. Examples include AAV-PPS (Chen et al., Nat Med. 2009; 15: 1215) and PHP.eB. In certain embodiments, the capsid of PHP.eB differs from AAV9 in that, compared to AAV9 by reference, the amino acid residue S-AQ-A (sequence number 14), starting from position 586, is changed to S-DGTLAVPFK-A (sequence number 15). In certain embodiments, PHP.eB refers to the sequence of sequence number 64.

[0077] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the blood-brain barrier (BBB) ​​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).

[0078] AAVrh.10 is the first AAV isolated from rhesus monkeys and exhibits weakly positive human serological reactivity compared to other common serotypes used for gene delivery purposes (Selot et al., Front Pharmacol. 2017; 8: 441). It has been evaluated in several clinical trials (LYS-SAF302, LYSOGENE, and NCT03612869).

[0079] AAV1R6 and AAV1R7 are two variants isolated from a library of chimeric AAV vectors (in which the capsid domain of AAVrh.10 is replaced with the capsid domain of AAV1). They retain the ability to cross the blood-brain barrier and transduce into the central nervous system, but their transduction into hepatic endothelium and vascular endothelium is significantly suppressed.

[0080] rAAVrh.8 is also an AAV isolated from rhesus monkeys, and peripheral administration exhibits widespread transduction to glial and neuronal cells in clinically significant areas, with suppressed peripheral tissue targeting compared to other vectors.

[0081] AAV-BR1 is an AAV2 variant that presents the NRGTEWD ​​epitope (SEQ ID NO: 17) and was isolated during in vivo screening of a random AAV display peptide library. AAV-BR1 exhibits high specificity by overexpressing the transgene in the brain, while minimizing off-target affinities (including affinity for the liver) (Korbelin et al., EMBO Mol Med. 2016; 8(6): 609).

[0082] 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 18) and transduces neurons in the enteric nervous system, and strongly transduces peripheral sensory afferent nerves projecting to the spinal cord and brainstem.

[0083] AAV-PHP.B (Addgene, Watertown, Massachusetts) is a variant of AAV9 created using the CREATE method, encoding the 7-mer sequence TLAVPFK (SEQ ID NO: 19). AAV-PHP.B delivers genes to the entire central nervous system more efficiently than AAV9, transducing a large portion of astrocytes and neurons in multiple central nervous system regions.

[0084] AAV-PPS is an AAV2 variant created by inserting the DSPAHPS epitope (SEQ ID NO: 20) into the AAV2 capsid, and exhibits dramatically improved brain directivity compared to AAV2.

[0085] For further information on capsids that cross the blood-brain barrier, see Chan et al., Nat. Neurosci. 2017 Aug: 20(8): 1172-1179.

[0086] (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.

[0087] 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.

[0088] 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.

[0089] The term "pharmaceutically acceptable carrier" refers to a carrier that, when administered to humans, and in certain embodiments, when administered intravenously (e.g., into the posterior orbital venous plexus), does not cause allergic reactions or similar adverse reactions.

[0090] In certain embodiments, the compositions of the present disclosure can be formulated for intravenous, intracerebral, intraocular, intravitreous, parenteral, subcutaneous, intraventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or direct injection or direct administration into one or more cells, tissues or organs.

[0091] The compositions of this disclosure may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres and / or nanoparticles.

[0092] The formation and use of liposomes are widely known to those skilled in the art. Liposomes have been developed to improve stability and circulating half-life in serum (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).

[0093] 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.

[0094] Examples of injectable compositions include sterile aqueous solutions or sterile water dispersions and sterile powders for the immediate preparation of sterile injectable solutions or sterile injectable dispersions (U.S. Patent No. 5,466,468). Injectable compositions delivered by injection are in the form of sterile fluids that can be delivered using a syringe. In certain embodiments, the injectable composition may be stable under manufacturing and storage conditions and may contain one or more preservative compounds 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, and in the case of dispersions, the particle size may be maintained to the required size, and / or surfactants may be used. To prevent microbial action, various antibacterial and / or antifungal agents may be used, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. 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.

[0095] 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.

[0096] Sterile compositions can be prepared by mixing a suitable amount of a bioactive component in a solvent with any other component (e.g., the component mentioned above) and then sterilizing and filtering the mixture. Dispersions are typically prepared by dispersing various sterilizing bioactive components in a sterilizing solvent containing a basic dispersion medium and other necessary raw materials (e.g., the raw materials mentioned above). In the case of sterilizing powders for preparing sterile injection solutions, it is preferable to pre-sterilizing and filtering a solution containing the bioactive component and the other desired raw materials, and then vacuum-drying or freeze-drying this solution to prepare the powder containing the bioactive component and the other desired raw materials.

[0097] Oral compositions may be in liquid form, such as solutions, 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.

[0098] 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.

[0099] 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).

[0100] Furthermore, auxiliary active ingredients may also be included in the composition of this disclosure.

[0101] 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.

[0102] In certain embodiments, when administered to humans, the compositions of this disclosure must meet standards for sterility, pyrogenicity, general safety, and purity in accordance with the requirements of the U.S. Food and Drug Administration (FDA) or other relevant regulatory authorities in other countries.

[0103] (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.

[0104] 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 of this disclosure are eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_12 The gene comprises 79rh or eHGT_1280rh and / or an enhancer and / or vector sequence selected from CN3362, CN4621, CN4622, CN1389, CN3445, CN4496, CN4695, CN2826, CN2602, CN4609, CN4728, CN4626, CN4731, CN4629, CN4661, or CN4662, wherein the host cell line is a human cell, primate cell, or mouse cell. 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 brains, and organelle cell cultures such as brain sections of animal-derived neurosurgical tissues from rats, mice, non-human primates, or humans. The PC12 cell line (available from the American Type Culture Collection (ATCC) in Manassas, Virginia) has been shown to express many neuronal marker proteins in response to nerve growth factor (NGF). The PC12 cell line is considered to be a neuronal cell line and is applicable to use in this disclosure. JAR cells (also available from ATCC) are platelet-derived cell lines that express several neuronal genes, including the serotonin transporter gene, and may also be used in the embodiments described herein.

[0105] 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 use in this disclosure.

[0106] In certain embodiments, the term “neuron” is used to describe any neuronal cell, anything related to a neuronal cell, or anything containing a neuronal cell. A neuronal cell is defined by the characteristic of having an axon and dendrites. The term “neuron-specific” refers to something found in neuronal cells or cells derived therefrom, but not, or substantially not, found in cells that do not originate from neuronal cells or in non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes); or to activity that occurs in neuronal cells or cells derived therefrom, but not, or substantially not, in cells that do not originate from neuronal cells or in non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes).

[0107] 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).

[0108] 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.

[0109] 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; neurotrophins (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).

[0110] In certain embodiments, a yeast one-hybrid system may be used to identify compounds that inhibit specific protein-DNA interactions, such as the transcription factors eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, or eHGT_1280rh.

[0111] 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).

[0112] (iv) Transgenic animals Another aspect of the present disclosure includes a transgenic animal whose genome contains an artificial expression construct comprising eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh and / or eHGT_1280rh, which is operably linked to a coding sequence. In certain embodiments, the transgenic animal has a genome that includes CN3362, CN4621, CN4622, CN1389, CN3445, CN4496, CN4695, CN2826, CN2602, CN4609, CN4728, CN4626, CN4731, CN4629, CN4661 and / or CN4662.

[0113] In certain embodiments, when using non-embedded vectors, the transgenic animal has one or more cells containing eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, e Includes an artificial expression construct containing HGT_1256rh, eHGT_1239rh, eHGT_1279rh and / or eHGT_1280rh, and / or CN3362, CN4621, CN4622, CN1389, CN3445, CN4496, CN4695, CN2826, CN2602, CN4609, CN4728, CN4626, CN4731, CN4629, CN4661 and / or CN4662.

[0114] 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 preferably include non-human primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens; and rodents such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.

[0115] In certain embodiments, creating transgenic animals yields organisms in which the recombinant construct is introduced at the same genomic integration site in all cells. Therefore, cell lines derived from such transgenic animals exhibit consistent characteristics in that all cells have the recombinant construct at the same genomic integration site, and thus all these cells undergo the same variegated positional effect. In contrast, introducing a gene into a cell line or primary cell culture results in construct expression. This method has the drawback that the expression of the introduced DNA may be influenced by the specific genetic background of the host animal.

[0116] As previously mentioned in relation to cell lines, the artificial expression constructs of this disclosure can be used to genetically modify mouse embryonic stem (ES) cells using techniques known in the art. Typically, the artificial expression constructs are introduced into cultured mouse embryonic stem (ES) 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, the mice used to isolate the cultured ES cells used for gene transfer are selected to have a different coat color than 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.

[0117] 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), as well as other available delivery methods and / or other delivery methods separately described herein.

[0118] (v) How to use In certain embodiments, compositions containing the bioactive ingredients described herein are administered to a subject to produce a physiological effect.

[0119] In certain embodiments, the Disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a gene that encodes part or all of a recombinant sequence at a downstream position of an 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.

[0120] Certain embodiments include methods of inducing gene expression in a target cell type by administering to a subject an artificial expression construct comprising eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, and / or eHGT_1280rh, and / or CN3362, CN4621, CN4622, CN1389, CN3445, CN4496, CN4695, CN2826, CN2602, CN4609, CN4728, CN4626, CN4731, CN4629, CN4661 and / or CN4662. The subject may be an isolated cell, a network of cells, a tissue section, a laboratory animal, a veterinary animal, or a human.

[0121] As is well known in the medical field, the dose administered to a subject depends on various factors such as the subject's body size, surface area and age, the particular compound administered, sex, administration duration and administration route, general health status, and other concomitant administered drugs. While the dose of the compound of the present disclosure varies, in certain embodiments, the dose of the artificial expression construct of the present disclosure is 10 5 ~10 100 copies. In certain embodiments, 10 6 ~10 22 copies of the artificial expression construct can be injected into a patient receiving intravenous administration, intraparenchymal cerebral administration, intraspinal administration, retro-orbital administration, or intrathecal administration.

[0122] An "effective amount" is the amount of the composition necessary to produce a desired physiological change in a subject. An effective amount is often administered for research purposes. The effective amount disclosed herein is an amount capable of causing a statistically significant effect in animal models, human studies, in vivo assays or in vitro assays.

[0123] 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 this disclosure. 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 in succession 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 as a dose that may be required to achieve the intended effect. 7 pieces / ml, 10 8 pieces / ml, 10 9 pieces / ml, 10 10 pieces / ml, 10 11 pieces / ml, 10 12 pieces / ml, 10 13 The number may be cells / ml or more, and these doses may be administered as a single dose or in 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.

[0124] In certain circumstances, the artificial expression construct is preferably delivered in the form of a suitably formulated composition of the present disclosure by pipetting, postorbital injection, subcutaneous administration, intraocular administration, intravitreous administration, parenteral administration, subcutaneous administration, intravenous administration, intracerebral administration, intramuscular administration, intrascalar administration, intrathecal or intraperitoneal administration, oral or 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 No. 5,399,363.

[0125] (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).

[0126] 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.

[0127] 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.

[0128] (vii) Exemplary Embodiments 1. Concatenated core of the eHGT_873m enhancer. 2. A concatemerized core according to Embodiment 1, comprising sequence number 26 or a sequence having at least 90% sequence identity with the sequence shown in sequence number 26. 3. A concatemerized core according to Embodiment 1, comprising two, three, four, five, six, seven, eight, nine, or ten copies of sequence number 89, or 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 89. 4. A concatemerized core according to Embodiment 2 or 3, comprising three copies of Sequence ID No. 89. 5. An artificial expression construct comprising (i) an enhancer selected from eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, core2_eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, and eHGT_1280rh; (ii) a promoter; and (iii) a coding sequence. 6. The artificial expression construct according to Embodiment 5, wherein the code sequence encodes an effector element or an expressible element. 7. The artificial expression construct according to Embodiment 6, wherein the effector element comprises a reporter protein or a functional molecule. 8. The artificial expression construct according to Embodiment 7, wherein the reporter protein comprises a fluorescent protein. 9. The artificial expression construct according to Embodiment 7 or 8, 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. 10. An artificial expression construct according to any one of embodiments 6 to 9, wherein the expressible element includes a non-functional molecule. 11. The artificial expression construct according to Embodiment 10, 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. 12. An artificial expression construct according to any one of embodiments 5 to 11, which is bound to a capsid that crosses the blood-brain barrier. 13. The artificial expression construct according to Embodiment 12, wherein the capsid comprises PHP.eB, AAV9, AAVrh.10, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 14. An artificial expression construct according to any of Embodiments 5 to 13, which includes or codes for a skipping element. 15. The artificial expression construct according to Embodiment 14, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 16. The artificial expression construct according to Embodiment 15, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 17. eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, 3xcore2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_122 3rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, eHGT_1280rh, AAV, scAAV, rAAV, pAAV, minBglobin, CMV, minC An artificial expression construct according to any of Embodiments 5 to 16, comprising, or encoding, a set of features selected from MV, minCMV*, minRho, minRho*, fluorescent proteins (e.g., EGFP, SYFP, GFP), hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), tag cassette, 10aa, nuclear localization protein, self-cleaving peptide, WPRE, WPRE3, hGHpA and / or BGHpA. 18. eHGT_868m-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_127m-minBglobin-[code array]-WPRE3-BGHpA, eHGT_769m-minBglobin-[code array]-WPRE3-BGHpA, eHGT_1249rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1250rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1223rh-minBglobin-[code sequence]-WPRE3-BGHpA, hI56i(core)-minBglobin-[code array]-WPRE3-BGHpA, eHGT_1239rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_873m-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_873m-minBglobin-[code array]-P2A-3XFLAG-10aa-H2B-WPRE3-BGHpA, core2_eHGT_873m-minBglobin-[code array]- WPRE3-BGHpA, core2_eHGT_873m-minBglobin-[code array]-P2A-3XFLAG-10aa-H2B*-WPRE3-BGHpA, 3xcore2_eHGT_873m-minBglobin-[code array]- WPRE3-BGHpA, 3xcore2_eHGT_873m-minBglobin-[code array]-P2A-3XFLAG-10aa-H2B*-WPRE3-BGHpA, eHGT_1255rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1226rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1246rh-minBglobin-[code sequence]-WPRE3-BGHpA eHGT_1256rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1279rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1280rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_868m-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_127m-[Minimum Promoter]-[Code Sequence]-[Post-Transcriptional Regulatory Element] eHGT_769m-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1249rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1250rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1223rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] hI56i(core)-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1239rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_873m-[minimum promoter]-[code sequence]-P2A-3XFLAG-10aa-H2B-]-[post-transcriptional regulatory element], core2_eHGT_873m-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] core2_eHGT_873m-[minimum promoter]-[code sequence]-P2A-3XFLAG-10aa-H2B*]-[post-transcriptional regulatory element], 3xcore2_eHGT_873m-[Minimum Promoter]-[Code Sequence]-[Post-Transfer Regulatory Element], 3xcore2_eHGT_873m-[minimum promoter]-[code sequence]-P2A-3XFLAG-10aa-H2B*]-[post-transcriptional regulatory element], eHGT_1255rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1226rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1246rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1256rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1279rh-[minimum promoter]-[coding sequence]-[post-transcriptional regulatory element], and eHGT_1280rh-[Minimum Promoter]-[Code Sequence]-[Post-Transcriptional Regulatory Element] An artificial expression construct according to any of embodiments 5 to 17, comprising or encoding a set of features selected from these. 19. A vector comprising an artificial expression construct as described in any of Embodiments 5 to 18. 20. The vector described in Embodiment 19, which is a viral vector. 21. The vector according to Embodiment 19 or 20, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector. 22. Transgenic cells comprising an artificial expression construct or vector as described in any of the prior embodiments. 23. A transgenic cell according to Embodiment 22, which is a basal ganglia neuron. 24. A transgenic cell according to Embodiment 23, which is a striatal neuron, a globus pallidus neuron, or a substantia nigra neuron. 25. Transgenic cells according to Embodiment 24, which are striatal cholinergic neurons, striatal SST Chodl neurons, D1R+ICj granule cells, SST LHX6+ striatal interneurons, globus pallidus external segment (GPe) interneurons, or substantia nigra pars compacta (SNc) dopaminergic neurons. 26. Transgenic cells according to any of embodiments 22 to 25, which are mouse cells, human cells, or non-human primate cells. 27. A non-human transgenic animal comprising an artificial expression construct, vector, and / or transgenic cells as described in any of the prior embodiments. 28. A non-human transgenic animal according to Embodiment 27, which is a mouse or a non-human primate. 29. An administerable composition comprising an artificial expression construct, vector, and / or transgenic cells as described in any of the prior embodiments. 30. A kit comprising an artificial expression construct, vector, transgenic cell, non-human transgenic animal and / or administerable composition as described in any of the prior embodiments. 31. A method for expressing a gene in vivo or in vitro within a target cell population in the striatum, globus pallidus, or substantia nigra of the basal ganglia, or within a target cell population derived from the striatum, globus pallidus, or substantia nigra of the basal ganglia, comprising the step of expressing the gene within the target cell population by providing a sample or subject containing the target cell population of the basal ganglia or the target cell population derived from the basal ganglia in a sufficient dose and for a sufficient period of time. 32. The method according to Embodiment 31, wherein the serving step includes pipetting. 33. The method according to embodiment 32, wherein the pipetting is performed on a brain section. 34. The method according to Embodiment 33, wherein the brain section includes basal ganglia neurons. 35. The method according to Embodiment 34, wherein the brain section includes striatal neurons, globus pallidus neurons, or substantia nigra neurons. 36. The method according to Embodiment 35, wherein the brain section includes striatal cholinergic neurons, striatal SST Chodl neurons, D1R+ICj granule cells, SST LHX6+ striatal interneurons, globus pallidus external segment (GPe) interneurons, or substantia nigra pars compacta (SNc) dopaminergic neurons. 37. The method according to any one of embodiments 33 to 36, wherein the brain section is a brain section of a mouse, human, or non-human primate. 38. The method according to Embodiment 31, wherein the dispensing step includes administration to a living subject. 39. The method according to Embodiment 38, wherein the living subject is a human, a non-human primate, or a mouse. 40. The method according to Embodiment 38 or 39, wherein the administration to the living subject is performed by injection. 41. The method according to Embodiment 40, wherein the injection includes intravenous injection, intraparenchymal injection into brain tissue, intraventricular (ICV) injection, intracisional (ICM) injection, or intrathecal injection. 42. An artificial expression construct comprising the sequence shown in SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88, or comprising a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88.

[0129] (viii) Conclusion The nucleic acid sequences and amino acid sequences provided herein are represented by abbreviations used for nucleotide bases and amino acid residues, as defined in U.S. Patent Law Enforcement Rule 37 CFR Rules 1.831-1.835 and also shown in WIPO Standard ST.26 (effective July 1, 2022). Although only one type of strand is shown for each nucleic acid sequence, complementary strands are also included in the embodiments where appropriate.

[0130] Variants of sequences disclosed herein and cited sequences are also included in this 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 This can be discovered using software (Madison, Wisconsin, USA). The amino acid changes of the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions between amino acids with similar charge or substitutions between uncharged amino acids. Conservative amino acid changes include substitutions of side chains by members of the related amino acid family.

[0131] 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.

[0132] 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 interacting biological functions on proteins is widely understood in the 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).

[0133] A specific amino acid may be substituted with another amino acid having a similar hydrophobicity index or degree of hydrophobicity, and it is well known in the art that such substitutions can yield proteins with similar biological activity, that is, proteins with biologically equivalent functionality. When making such changes, substitutions between amino acids with hydrophobicity indices within ±2 are preferred, substitutions between amino acids with hydrophobicity indices within ±1 are particularly preferred, and substitutions between amino acids with hydrophobicity indices 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 hydrophilicity.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] Variants of protein sequences, nucleic acid sequences, 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 any of the protein sequences, nucleic acid sequences, or gene sequences disclosed herein.

[0138] "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 understood to be based on the “default values” of the cited programs. In this specification, “default values” means a set of numerical values ​​or parameters that are pre-registered in the software when the software is first launched.

[0139] 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 overnight at 37°C in a solution containing 6×SSPE (20×SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml 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.

[0140] 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."

[0141] As those skilled in the art will understand, each embodiment disclosed herein may include, substantially consist of, or consist of, certain components, processes, materials, or ingredients described herein. Therefore, the terms “includes” or “contains” should be interpreted as “includes, substantially consists of, or consists of.” The transitional phrase “includes” means, but is not limited to, the inclusion of components, processes, materials, or ingredients not described herein, even in large quantities. The transitional phrase “consists of” excludes any 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, processes, materials, or ingredients that do not materially affect the embodiment. A materially affecting effect is an effect that, when measured by scRNA-Seq using a particular enhancer-target cell population combination, results in a statistically significant decrease in target expression in the target cell population, such as: Striatal neurons: eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, core2_eHGT_873m, 3xcore2_eHGT_873m and eHGT_1246rh / striatal cholinergic neurons; eHGT_127m, eHGT_769m / Striatal SST Chodl neurons; eHGT_1223rh, eHGT_1226rh / D1R+ICj granule cells; and eHGT_1255rh and eHGT_1256rh / SST LHX6+ striatal interneurons; globus pallidus neurons: eHGT_1239rh / GPe interneurons; and Substantia nigra neurons: eHGT_1279rh and eHGT_1280rh / SNc dopaminergic neurons These are some examples.

[0142] In certain embodiments, "artificial" means that it is not a natural product.

[0143] 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 of numbers 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 is somewhat more or somewhat less than the given number or range of numbers.

[0144] 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.

[0145] 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.

[0146] The grouping of alternative components or alternative embodiments of the present invention disclosed herein should not be construed as limiting the invention. Members of each group may be described individually in this specification or in the claims, or in combination with other members or components of the group described herein or in the claims. 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 that have been partially modified to satisfy the description of all Markush groups described in the appendix claims.

[0147] 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.

[0148] Furthermore, this specification draws reference to various patents, publications, journal articles, and other documents (references herein). Each reference cited herein is incorporated herein by reference in its entirety with respect to the teachings it cites.

[0149] 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 those shown and described herein.

[0150] The details described herein are illustrative and intended solely to illustrate preferred embodiments of the present invention, provided to offer what is considered most useful and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no further details of the structure of the present invention are described beyond the information necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how some forms of the present invention can be actually implemented by carefully reading the description of the present invention with reference to the drawings and / or examples.

[0151] The definitions and descriptions used in this disclosure are intended to control future interpretations unless they are explicitly and expressly changed in the following embodiments, or unless the interpretation becomes meaningless or substantially meaningless due to the meaning of the terms. If the interpretation of a term makes no sense or substantially meaningless the definition of a term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd edition) or the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. An artificial expression construct comprising the enhancer shown in Sequence ID No. 26, a promoter, and a coding sequence.

2. Concatenated core of the eHGT_873m enhancer.

3. The concatemerized core according to claim 2, comprising two, three, four, five, six, seven, eight, nine, or ten copies of sequence number 89, 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 89.

4. A concatemerized core according to claim 3, comprising three copies of sequence number 89.

5. The concatemerized core according to claim 2, comprising sequence number 26 or a sequence having at least 90% sequence identity with the sequence shown in sequence number 26.

6. An artificial expression construct comprising (i) an enhancer selected from 3xcore2_eHGT_873m, eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, core2_eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, and eHGT_1280rh; (ii) a promoter; and (iii) a coding sequence.

7. The artificial expression construct according to claim 6, wherein the code sequence encodes an effector element or an expressible element.

8. The artificial expression construct according to claim 7, wherein the effector element comprises a reporter protein or a functional molecule.

9. The artificial expression construct according to claim 8, wherein the reporter protein comprises a fluorescent protein.

10. The artificial expression construct according to claim 8, 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.

11. The artificial expression construct according to claim 7, wherein the expressible element includes a non-functional molecule.

12. The artificial expression construct according to claim 11, 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.

13. The artificial expression construct according to claim 6, which is bound to a capsid that crosses the blood-brain barrier.

14. The artificial expression construct according to claim 13, wherein the capsid comprises PHP.eB, AAV9, AAVrh.10, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS.

15. The artificial expression construct according to claim 6, comprising or encoding a skipping element.

16. The artificial expression construct according to claim 15, wherein the skipping element comprises a 2A peptide or an internal ribosome entry site (IRES).

17. The artificial expression construct according to claim 16, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.

18. 3xcore2_eHGT_873m, eHGT_868m, eHGT_1249rh, eHGT_1250rh, hI56i(core), eHGT_873m, eHGT_1246rh, eHGT_127m, eHGT_769 m, eHGT_1223rh, eHGT_1226rh, eHGT_1255rh, eHGT_1256rh, eHGT_1239rh, eHGT_1279rh, eHGT_1280rh, AAV, scAAV, rAAV, pAAV The artificial expression construct according to claim 6, comprising or encoding a set of features selected from 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.

19. 3xcore2_eHGT_873m-minBglobin-[code array]- WPRE3-BGHpA, 3xcore2_eHGT_873m-minBglobin-[code array]-P2A-3XFLAG-10aa-H2B*-WPRE3-BGHpA, eHGT_868m-minBglobin-[code array]-WPRE3-BGHpA, eHGT_127m-minBglobin-[code array]-WPRE3-BGHpA, eHGT_769m-minBglobin-[code array]-WPRE3-BGHpA, eHGT_1249rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1250rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1223rh-minBglobin-[code sequence]-WPRE3-BGHpA, hI56i(core)-minBglobin-[code array]-WPRE3-BGHpA, eHGT_1239rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_873m-minBglobin-[code array]-WPRE3-BGHpA, eHGT_873m-minBglobin-[code array]-P2A-3XFLAG-10aa-H2B-WPRE3-BGHpA, core2_eHGT_873m-minBglobin-[code array]- WPRE3-BGHpA, core2_eHGT_873m-minBglobin-[code array]-P2A-3XFLAG-10aa-H2B*-WPRE3-BGHpA, eHGT_1255rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1226rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1246rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1256rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1279rh-minBglobin-[code sequence]-WPRE3-BGHpA, eHGT_1280rh-minBglobin-[code sequence]-WPRE3-BGHpA, 3xcore2_eHGT_873m-[Minimum Promoter]-[Code Sequence]-[Post-Transfer Regulatory Element], 3xcore2_eHGT_873m-[Minimum Promoter]-[Code Sequence]-P2A-3XFLAG-10aa-H2B*-[Post-Transfer Regulation Element], eHGT_868m-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_127m-[Minimum Promoter]-[Code Sequence]-[Post-Transcriptional Regulatory Element] eHGT_769m-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1249rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1250rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1223rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] hI56i(core)-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1239rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_873m-[minimum promoter]-[code sequence]-P2A-3XFLAG-10aa-H2B-[post-transcriptional regulatory element] core2_eHGT_873m-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] core2_eHGT_873m-[minimum promoter]-[code sequence]-P2A-3XFLAG-10aa-H2B*]-[post-transcriptional regulatory element], eHGT_1255rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1226rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1246rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1256rh-[minimum promoter]-[code sequence]-[post-transcriptional regulatory element] eHGT_1279rh-[minimum promoter]-[coding sequence]-[post-transcriptional regulatory element], and eHGT_1280rh-[Minimum Promoter]-[Code Sequence]-[Post-Transcriptional Regulatory Element] The artificial expression construct according to claim 6, comprising or encoding a set of features selected from these.

20. A vector comprising the artificial expression construct according to claim 6.

21. The vector according to claim 20, which is a viral vector.

22. The vector according to claim 20, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector.

23. Transgenic cells comprising the artificial expression construct according to claim 6 and / or the vector according to claim 20.

24. The transgenic cell according to claim 23, which is a basal ganglia neuron.

25. The transgenic cell according to claim 24, which is a striatal neuron, a globus pallidus neuron, or a substantia nigra neuron.

26. The transgenic cell according to claim 25, which is a striatal cholinergic neuron, a striatal SST Chodl neuron, a D1R+ICj granule cell, an SST LHX6+ striatal interneuron, a globus pallidus external segment (GPe) interneuron, or a substantia nigra pars compacta (SNc) dopaminergic neuron.

27. The transgenic cell according to claim 23, which is a mouse cell, a human cell, or a non-human primate cell.

28. A non-human transgenic animal comprising the artificial expression construct according to claim 6, the vector according to claim 20, and / or the transgenic cell according to claim 23.

29. A non-human transgenic animal according to claim 28, which is a mouse or a non-human primate.

30. An administerable composition comprising the artificial expression construct according to claim 6, the vector according to claim 20, and / or the transgenic cells according to claim 23.

31. A kit comprising the artificial expression construct according to claim 6, the vector according to claim 20, the transgenic cells according to claim 23, and / or the non-human transgenic animal according to claim 28.

32. A method for expressing a gene in vivo or in vitro within a target cell population in the striatum, globus pallidus, or substantia nigra of the basal ganglia, or within a target cell population derived from the striatum, globus pallidus, or substantia nigra of the basal ganglia, comprising the step of expressing a gene within the target cell population by providing a sample or subject containing the target cell population of the basal ganglia or the target cell population derived from the basal ganglia in a sufficient dose and for a sufficient period of time.

33. The method according to claim 32, wherein the serving step includes pipetting.

34. The method according to claim 33, wherein the pipetting is performed on a brain section.

35. The method according to claim 34, wherein the brain section includes basal ganglia neurons.

36. The method according to claim 35, wherein the brain section includes striatal neurons, globus pallidus neurons, or substantia nigra neurons.

37. The method according to claim 36, wherein the brain section comprises striatal cholinergic neurons, striatal SST Chodl neurons, D1R+ICj granule cells, SST LHX6+ striatal interneurons, globus pallidus external segment (GPe) interneurons, or substantia nigra pars compacta (SNc) dopaminergic neurons.

38. The method according to claim 34, wherein the brain section is a brain section of a mouse, human, or non-human primate.

39. The method according to claim 32, wherein the provisioning step includes administration to a living subject.

40. The method according to claim 39, wherein the living subject is a human, a non-human primate, or a mouse.

41. The method according to claim 39, wherein the administration to the living subject is performed by injection.

42. The method according to claim 41, wherein the injection includes intravenous injection, intraparenchymal injection into brain tissue, intraventricular (ICV) injection, intracisional (ICM) injection, or intrathecal injection.

43. An artificial expression construct comprising, or substantially comprising any of the sequences shown in SEQ ID NO: 78, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88, or comprising, or substantially comprising, sequences having at least 90% sequence identity with the sequences shown in SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88, or substantially comprising, any of the sequences having at least 90% sequence identity with any of the sequences.

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