Artificial expression constructs for regulating gene expression in GABAergic neurons and astrocytes

Abstract

Artificial expression constructs for regulating gene expression in GABAergic neurons and astrocytes are described. The artificial expression constructs can be used to express SLC6A1 for the treatment of SLC6A1-associated disorders, among other uses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 144,743, filed February 2, 2021, the entire contents of which are incorporated by reference herein as if fully set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States Government support under Grant No. MH114126 from the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] References to sequence listing The sequence listing associated with this application has been provided in text form in lieu of a paper copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is A166-0029PCT_ST25.txt. This text file is 181 KB, was created on February 1, 2022, and has been submitted electronically via EFS-Web.

[0004] Field of Disclosure The present disclosure provides artificial expression constructs for regulating gene expression in GABAergic neurons and astrocytes. The gene expressed can include SLC6A1 for the treatment of SLC6A1-associated disorders, among other uses. [Background technology]

[0005] The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is released from GABAergic neurons. GABA does not undergo enzymatic degradation, but instead is removed from the extracellular space by the action of GABA transporters. GABA transporters are expressed in various cell types, such as inhibitory neurons and astrocytes, and belong to the solute carrier 6 (SLC6) family. There are six GABA transporters: A1 / GAT1, A13 / GAT2, A11 / GAT3, A6 / TauT, A8 / CT1, and A12 / BGT1 (Scimemi, Front Cell Neurosci. 2014;8:161).

[0006] The A1 / GAT1 protein is encoded by the solute carrier family 6 member 1 (SLC6A1) gene. Mutations in the SLC6A1 gene are characterized by mild to moderate intellectual disability, epilepsy, speech disorders, behavioral disorders (e.g., hyperactivity, attention deficit, aggression, autistic features, etc.), and neurological signs (e.g., ataxia, hypotonia, tremor, fine motor disorders, etc.) (Carvill, et al. Am J Hum Genet. 2015; 96: 808-15; and Johannesen, et al. Epilepsia. 2018; 59: 389-402). Summary of the Invention

[0007] The present disclosure provides artificial expression constructs that drive gene expression in GABAergic neurons and astrocytes that can be used to drive SLC6A1 gene expression to ameliorate disorders associated with SLC6A1 gene mutations, among other uses.

[0008] The artificial expression construct comprises an enhancer element that includes an I56i enhancer or its core to drive gene expression in GABAergic neurons, and one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, or their cores (e.g., eHGT_387m(core2) or eHGT_390m(core2)) to drive expression in astrocytes.

[0009] In certain embodiments, the artificial enhancer element comprises a linked core of enhancers. An example is the linked core of I56i. These artificial enhancer elements can provide higher levels and more rapid initiation of transgene expression compared to a single full-length original (native) enhancer.

[0010] In certain embodiments, the I56i core (or I56i(core)) comprises a sequence set forth in any one of SEQ ID NOs: 4 and 5. In certain embodiments, these cores are linked and have 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the core sequence. SEQ ID NOs: 6 and 7 provide 3 copy concatemers of selected enhancer cores.

[0011] In a particular embodiment, the artificial expression construct comprises a three copy concatemer of the hl56i core and a second enhancer selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, and eHGT_390m.

[0012] In certain embodiments, the artificial enhancer element comprises a combination linked enhancer. In certain embodiments, the combination linked enhancer comprises a core of an enhancer selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m (e.g., eHGT_387m(core2)) and eHGT_390m (e.g., eHGT_390m(core2)) linked with I56i(core). In certain embodiments, the core of eHGT_387m (eHGT_387m(core2)) comprises the sequence set forth in SEQ ID NO: 84. In certain embodiments, the core of eHGT_390m (eHGT_390m(core2)) comprises the sequence set forth in SEQ ID NO: 85.

[0013] In certain embodiments, the combinatorial linked enhancer comprises eHGT_387m(core2) and I56i(core) as set forth in SEQ ID NO: 88 (eHGT_387m(core2)-hI56i(core)-eHGT_387m(core2)-hI56i(core)-eHGT_387m(core2)-hI56i(core)). In certain embodiments, the combinatorial linked enhancer comprises eHGT_390m(core2) and I56i(core) as set forth in SEQ ID NO: 86. In certain embodiments, the combinatorial linked enhancer is linked to comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the combinatorial linked enhancer. In certain embodiments, SEQ ID NO: 89 provides a 3 copy concatemer of the eHGT_390m(core2)-l56i(core) combinatorial linked enhancer.

[0014] Some of the drawings presented herein may be better understood in color, and applicants consider the colored drawings to be a part of the application as originally filed and reserve the right to submit images of the colored drawings by proceeding as described below. [Brief description of the drawings]

[0015] [Figure 1A]Figure 1. Generating expression of two specificities on one AAV vector. Schematic diagram of the monospecific and bispecific AAVs tested. [Figure 1B] FIG. 2 is a further exemplary schematic diagram. [Diagram 2] Expression of bispecific vectors throughout the brain. Fluorescence images of sagittal sections of mouse brain transduced with the indicated viruses after intravenous delivery and PHP.eB encapsidation. White is SYFP expression. Images are montages. [Diagram 3] Expression of bispecific vectors in the visual cortex. Fluorescence images of sagittal sections of mouse visual cortex (VISp) transduced with the indicated viruses after intravenous delivery and PHP.eB encapsidation. White indicates SYFP expression. Images are montages. [Figure 4] Striatal expression of bispecific vectors. Fluorescent images of sagittal sections of mouse striatum transduced with the indicated viruses after intravenous delivery and PHP.eB encapsidation. White indicates SYFP expression. Images are montages. [Diagram 5] Cerebellar expression of bispecific vectors. Fluorescence images of sagittal sections of mouse cerebellum transduced with the indicated viruses after intravenous delivery and PHP.eB encapsidation. White indicates SYFP expression. Images are montages. [Figure 6] Quantification of SYFP+ cells transduced with CN1390 vector. Mouse visual cortex (VISp) transduced with CN1390 virus after intravenous delivery and PHP.eB encapsidation. Overlay of mRNA images by mFISH with SYFP fluorescent GABAergic cell marker (Gad1) and astrocyte marker (Fgfr3). Colocalization is indicated by circles of different colors and was quantified. Images are montages. [Figure 7]Quantification of SYFP+ cells transduced with CN2102 vector. Mouse visual cortex (VISp) transduced with CN2102 virus after intravenous delivery and PHP.eB encapsidation. Overlay of mRNA images by mFISH with SYFP fluorescent GABAergic cell marker (Gad1) and astrocyte marker (Fgfr3). Colocalization is indicated by circles of different colors and was quantified. Images are montages. [Figure 8] Quantification of SYFP+ cells transduced with CN2102+CN1390 vector. Mouse visual cortex (VISp) transduced with CN1390 and CN2102 viruses after intravenous delivery and PHP.eB encapsidation. Overlay of mRNA images by mFISH with SYFP fluorescent GABAergic cell marker (Gad1) and astrocyte marker (Fgfr3). Colocalization is indicated by different colored circles and was quantified. Images are montages. [Figure 9] Quantification of SYFP+ cells transduced with CN2721 vector. Mouse visual cortex (VISp) transduced with CN2721 virus after intravenous delivery and PHP.eB encapsidation. Overlay of mRNA images by mFISH with SYFP fluorescent GABAergic cell marker (Gad1) and astrocyte marker (Fgfr3). Colocalization is indicated by different colored circles and was quantified. Images are montages. [Figure 10A] Codon optimization of SLC6A1. Characterization of SLC6A1 expression vectors using different codon optimization strategies with and without introns. Expression of SLC6A1 is shown by Western blot analysis. [Figure 10B] Codon optimization of SLC6A1. Characterization of SLC6A1 expression vectors using different codon optimization strategies with and without introns. Input control is shown stained for tubulin. [Figure 10C]Quantification of SYFP expression normalized to loading control. In this experiment, HEK293 cells were transfected in 12-well plates with 1 μg DNA in triplicate for 96 hours. Cells were then lysed with RIPA. The coding sequence was as follows, under the control of the CMV promoter: (1) No DNA, (2) CN2972: hSLC6A1_myc_ddk_native_CN2522GeneOpt1Splice (sequence number 24), (3) CN2975: hSLC6A1_myc_ddk_native_Intron (sequence number 33), (4) CN2976: hSLC6A1_myc_ddk_native_CN2522GeneOpt1Splice_Intron (sequence number 36), (5) CN2974: hSLC6A1_myc_ddk_native_IDTCodonOptSplice1 (sequence number 30), and (6) CN2973: hSLC6A1_myc_ddk_native_IDTCodonOptSplice1_Intron (sequence number 27). [Figure 11A] Epifluorescence micrograph (inverted) showing native SYFP2 expression in mouse brain sagittal sections 22 days after retroorbital delivery of 1.0E12 viral genome copies of AAV vector #CN3323. Scale bar: 1 mm. [Figure 11B] High magnification of the thalamic region. [Figure 12A] Mice were injected with CN3213 (pAAV-eHGT_3xhI56i(core)_eHGT_387m-minBG-intronSLC6A1-myc-flag-WPRE3-BGHpA) packaged with 1E11vg of PHP.eB via the intracerebroventricular (ICV) route on postnatal day 2 and sacrificed on postnatal day 21. Sagittal sections showing whole brain expression of CN3213 expressing myc-tagged and codon-optimized human SLC6A1. [Figure 12B]Mice were injected with CN3213 (pAAV-eHGT_3xhI56i(core)_eHGT_387m-minBG-intronSLC6A1-myc-flag-WPRE3-BGHpA) packaged with 1E11vg PHP.eB via the intracerebroventricular (ICV) route on postnatal day 2 and sacrificed on postnatal day 21. Zoomed-in view of the cerebral cortex showing co-expression in astrocytes (arrowheads) and GABA-positive interneurons (arrows). Much of the SLC6A1 expressed in GABAergic cells was transported to dendrites. [Figure 12C] Mice were injected with CN3213 (pAAV-eHGT_3xhI56i(core)_eHGT_387m-minBG-intronSLC6A1-myc-flag-WPRE3-BGHpA) packaged with 1E11vg PHP.eB via the intracerebroventricular (ICV) route on postnatal day 2 and sacrificed on postnatal day 21. Zoomed-in view of the cerebral cortex showing co-expression in astrocytes (arrowheads) and GABA-positive interneurons (arrows). Much of the SLC6A1 expressed in GABAergic cells was transported to dendrites. [Figure 13A] Myc-tagged hSLC6A1 is trafficked to GABAergic dendrites and appears as puncta throughout the neuropil. 1E11vg PHP.eB serotype AAV expressing myc-tagged hSLC6A1 and an astrocyte-only enhancer (A), a GABAergic inhibitory cell-only enhancer (B), or a bispecific enhancer pair (C) were injected ICV into P2 aged neonatal animals. At postnatal day 21, brains were removed, sliced, and stained for myc-tagged hSLC6A1 (white). Magnification, exposure time, and post-acquisition image adjustments were the same for both vectors. The bottom panel shows a magnified view of dendritic staining in the neuropil. In (A) and (C), astrocytes clearly express myc-hSLC6A1 (arrowheads), and in (B), only GABAergic cell bodies are evident (arrows), whereas in (C), GABAergic cells clearly express myc-hSLC6A1 (asterisk), as dendrites are labeled throughout the neuropil as in (B). This staining is not seen in (A), where only astrocytes express myc-hSLC6A1. [Figure 13B] Myc-tagged hSLC6A1 is trafficked to GABAergic dendrites and appears as puncta throughout the neuropil. 1E11vg PHP.eB serotype AAV expressing myc-tagged hSLC6A1 and an astrocyte-only enhancer (A), a GABAergic inhibitory cell-only enhancer (B), or a bispecific enhancer pair (C) were injected ICV into P2 aged neonatal animals. At postnatal day 21, brains were removed, sliced, and stained for myc-tagged hSLC6A1 (white). Magnification, exposure time, and post-acquisition image adjustments were the same for both vectors. The bottom panel shows a magnified view of dendritic staining in the neuropil. In (A) and (C), astrocytes clearly express myc-hSLC6A1 (arrowheads), and in (B), only GABAergic cell bodies are evident (arrows), whereas in (C), GABAergic cells clearly express myc-hSLC6A1 (asterisk), as dendrites are labeled throughout the neuropil as in (B). This staining is not seen in (A), where only astrocytes express myc-hSLC6A1. [Figure 13C]Myc-tagged hSLC6A1 is trafficked to GABAergic dendrites and appears as puncta throughout the neuropil. 1E11vg PHP.eB serotype AAV expressing myc-tagged hSLC6A1 and an astrocyte-only enhancer (A), a GABAergic inhibitory cell-only enhancer (B), or a bispecific enhancer pair (C) were injected ICV into P2 aged neonatal animals. At postnatal day 21, brains were removed, sliced, and stained for myc-tagged hSLC6A1 (white). Magnification, exposure time, and post-acquisition image adjustments were the same for both vectors. The bottom panel shows a magnified view of dendritic staining in the neuropil. In (A) and (C), astrocytes clearly express myc-hSLC6A1 (arrowheads), and in (B), only GABAergic cell bodies are evident (arrows), whereas in (C), GABAergic cells clearly express myc-hSLC6A1 (asterisk), as dendrites are labeled throughout the neuropil as in (B). This staining is not seen in (A), where only astrocytes express myc-hSLC6A1. [Figure 14]Sequences supporting the present disclosure. Sequences include: hI56i-full human hI56i enhancer (SEQ ID NO:1), mouse I56i enhancer (core is the same as human) (SEQ ID NO:2), zebrafish I46i enhancer (SEQ ID NO:3), hI56icore-human hI56i enhancer core (SEQ ID NO:4), zebrafish I46i enhancer core (SEQ ID NO:5), 3xhl56i(core) (SEQ ID NO:6), 3xzebrafish I46i enhancer linked core (SEQ ID NO:7), eHGT_375h (SEQ ID NO:8), eHGT_376h (SEQ ID NO:9), eHGT _390h (SEQ ID NO: 10), eHGT_373m (SEQ ID NO: 11), eHGT_375m (SEQ ID NO: 12), eHGT_386m (SEQ ID NO: 13), eHGT_387m (SEQ ID NO: 14), eHGT_387m(core2) (SEQ ID NO: 84), eHGT_390m (SEQ ID NO: 15), eHGT_390m(core2) (SEQ ID NO: 85), combined linked enhancer (eHGT_387m(core2)-hI56i(core)) (SEQ ID NO: 95), combined linked enhancer (eHGT_390m(core2)-hI56i(core e)) (SEQ ID NO: 86), 3xhI56i(core)_eHGT_390m enhancer (SEQ ID NO: 87), 3x combination linked enhancer (eHGT_387m(core2)-hI56i(core)-eHGT_387m(core2)-hI56i(core)-eHGT_387m(core2)-hI56i(core)) (SEQ ID NO: 88), 3x combination linked enhancer (eHGT_390m(core2)-hI56i(core)-eHGT_390m(core2)-hI56i(core)-eHGT_390m( core2)-hI56i(core)) (SEQ ID NO: 89), beta-globin minimal promoter (SEQ ID NO: 16), minCMV promoter (SEQ ID NO: 17), mutated minCMV promoter (SEQ ID NO: 18), minRho promoter (SEQ ID NO: 19), minRho* promoter (SEQ ID NO: 20), Hsp68 minimal promoter (proHsp68) (SEQ ID NO: 21), SLC6A1 encoding sequence from CN2972 (SEQ ID NO: 22), Myc-DDK tag sequence in CN2972 (SEQ ID NO: 23), CN2972 (SEQ ID NO: 24),SLC6A1 encoding sequence derived from CN2973 (SEQ ID NO: 25), Myc-DDK tag sequence in CN2973 (SEQ ID NO: 26), CN2973 (SEQ ID NO: 27), SLC6A1 encoding sequence derived from CN2974 (SEQ ID NO: 28), Myc-DDK tag sequence in CN2974 (SEQ ID NO: 26), CN2974 (SEQ ID NO: 30), SLC6A1 encoding sequence derived from CN2975 (SEQ ID NO: 31), Myc-DDK tag sequence in CN2975 (SEQ ID NO: 32), CN2975 (SEQ ID NO: 33), SLC6A1 encoding sequence derived from CN2976 (SEQ ID NO: 34), Myc-DDK tag sequence in CN2976 (SEQ ID NO: 23), CN2976 (SEQ ID NO: 36), CN2478 (SEQ ID NO: 37), SLC6A1 transcription variant (SEQ ID NO: 38), SLC6A1 transcription variant 2 (SEQ ID NO: 39), SLC6A1 transcription variant X5 (SEQ ID NO: 40), SLC6A1 transcript variant X1 (SEQ ID NO: 41), SLC6A1 transcript variant X2 (SEQ ID NO: 42), SLC6A1 transcript variant X6 (SEQ ID NO: 43), SLC6A1 transcript variant X3 (SEQ ID NO: 44), SLC6A1 transcript variant X4 (XM_017007072.2) (SEQ ID NO: 45), sodium- and chloride-dependent GABA transporter 1 isoform a (SEQ ID NO: 46) , Myc-DDK tag (SEQ ID NO: 47), SYFP2 (SEQ ID NO: 48), EGFP (SEQ ID NO: 49), optimized Flp recombinase (FlpO) (SEQ ID NO: 50), improved Cre recombinase (iCre) (SEQ ID NO: 51), tet-transcription activation version 2 (tTA2) (SEQ ID NO: 52), GCaMP6m (SEQ ID NO: 53), GCaMP6s (SEQ ID NO: 54), GCaMP6f (SEQ ID NO: 55), SP10 Insulator (SP10ins) (SEQ ID NO:56), 3xSP10ins (SEQ ID NO:57), WPRE3 (SEQ ID NO:58), WPRE (SEQ ID NO:59), BGHpA (SEQ ID NO:60), HGHpA (SEQ ID NO:61), P2A (SEQ ID NO:62), T2A (SEQ ID NO:63), E2A (SEQ ID NO:64), F2A (SEQ ID NO:65), Exemplary Plasmid Backbone 1-Left ITR (SEQ ID NO:66), Exemplary Plasmid Backbone 1-Right ITR (SEQ ID NO:67), Exemplary Plasmid Backbone 2-Left ITR (SEQ ID NO:68), Exemplary Plasmid Backbone 2-Right ITR (SEQ ID NO:69),PHP.eB capsid (SEQ ID NO:70), AAV9 VP1 capsid protein (SEQ ID NO:71), CN1390 (SEQ ID NO:72), CN2102 (SEQ ID NO:73), CN2720 (SEQ ID NO:74), CN2721 (SEQ ID NO:75), CN2722 (SEQ ID NO:76), CN2732 (SEQ ID NO:77), CN3213 (SEQ ID NO:90), CN3322 (SEQ ID NO:91), CN3323 (SEQ ID NO:92), CN3887 (SEQ ID NO:93), and CN3888 (SEQ ID NO:94). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The solute carrier 6 (SLC6) family of proteins includes transporters for neurotransmitters, amino acids, osmolytes, and energy metabolites. These proteins play important roles in neurotransmission and homeostasis.

[0017] Gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter released from GABAergic neurons, is not subject to enzymatic degradation but instead is transported into the cell after release by the action of GABA transporters. GABA transporters are expressed in various cell types, including inhibitory neurons and astrocytes. There are six types of GABA transporters: A1 / GAT1, A13 / GAT2, A11 / GAT3, A6 / TauT, A8 / CT1, and A12 / BGT1 (Scimemi, Front Cell Neurosci. 2014;8:161).

[0018] A1 / GAT1 is expressed in GABAergic axon terminals and is also present in astrocytes, oligodendrocytes, and microglia (Fattorini,et al.,Glia 2017;65:514-22). By transferring sodium and chloride ions across the membrane in a fixed ratio with GABA, GAT1 generates a stoichiometric current (Lester,et al.,Annu Rev Pharmacol Toxicol 1994;34:219-49) and forces intracellular translocation of extracellular GABA within milliseconds of its release. GABA is removed very quickly, preventing activation of adjacent synapses (Isaacson,et al.,Neuron 1993;10:165-75).

[0019] The A1 / GAT1 protein (hereinafter referred to as GAT1) is encoded by the solute carrier family 6 member 1 (SLC6A1) gene. The SLC6A1 gene on human chromosome 4 is also referred to as GAT1, GABATR, and GABATHG. The SLC6A1 gene has a nucleic acid sequence comprising the sequences set forth in the accession numbers: NM_003042.4 (SEQ ID NO: 38), NM_001348250.2 (SEQ ID NO: 39), XM 011534027.3 (SEQ ID NO: 40), XM 011534025.3 (SEQ ID NO: 41), XM 005265410.5 (SEQ ID NO: 42), XM 005265411.5 (SEQ ID NO: 43), SM_0170070071.2 (SEQ ID NO: 44), and XM_017007072.2 (SEQ ID NO: 45). SLC6A1 sequences including codon optimized variants are also provided in FIG.

[0020] Genetic mutations in SLC6A1 are characterized by mild to moderate intellectual disability, epilepsy, language disorders, behavioral disorders (e.g., hyperactivity, attention deficit, aggression, and autistic traits), and neurological signs (e.g., ataxia, hypotonia, tremor, and fine motor disorders) (Carvill, et al. Am J Hum Genet. 2015;96:808-15; and Johannesen, et al. Epilepsia. 2018;59:389-402). For example, a study of individuals with SLC6A1 mutations, the majority of which result in loss of GAT1 function, found the most common clinical features to be epilepsy (92 / 101, 91.1%), developmental delay and cognitive impairment (46 / 56, 82.1%), and autistic traits (20 / 92, 22.8%) (Goodspeed, et al., Brain Communications 2020;2(2):fcaa170). Prior to this study, in 2015, pathogenic SLC6A1 mutations had been identified in 4% of individuals with previously undiagnosed early-onset epilepsy accompanied by myoclonic atonic seizures, and 3p microdeletions in SLC6A1 and SLC6A11 have been described in patients with Doze syndrome (Carvill, et al., Am J Hum Genet 2015;96:808-15). Further studies have identified autism spectrum disorder and developmental epileptic encephalopathy in patients with mutations in SLC6A1 (Rauch, et al., Lance 2012;380:1674-82; and Sanders, et al., Nature 2012;485:237-41). Furthermore, an exome-wide triosequencing study found an association between schizophrenia and de novo missense variants in SLC6A1 (Rees, et al., Nat Neurosci 2020;23:179-84).

[0021] The present disclosure provides an artificial expression construct that drives gene expression in GABAergic neurons and astrocytes. The artificial expression construct can be used to drive SLC6A1 gene expression to improve disorders associated with SLC6A1 gene mutations, among other uses described herein. Expression of the SLC6A1 gene can result in the expression of a functional GAT1 GABA transporter.

[0022] The artificial expression constructs disclosed herein include an I56i enhancer or its core to drive gene expression in GABAergic neurons. In certain embodiments, the I56i enhancer core can be derived from, for example, human, mouse, or zebrafish I56i enhancer (SEQ ID NO: 1, 2, and 3, respectively). The selected core of the I56i enhancer can include SEQ ID NO: 4 (human and mouse shared core) or SEQ ID NO: 5 (zebrafish core). In certain embodiments, the cores are concatenated. For example, SEQ ID NO: 6 provides a concatemer of three copies of the selected human / mouse I56i core, while SEQ ID NO: 7 provides a concatemer of three copies of the selected zebrafish I56i core.

[0023] Of particular interest is that the synthetic 3x human / mouse core (herein referred to as 3xhl56iCore; SEQ ID NO: 6), although a 3x concatemer, is shorter than the original full-length enhancer sequence reported by Dimidschstein et al. (Nat Neurosci 19(12):1743-1749, 2016). Thus, this concatenated core provides more room for enhancer-linked cargo genes, which is highly desirable. Moreover, the peak level of transgene expression driven by the 3xhl56iCore enhancer is much greater than simply 3-fold that of the original single full-length enhancer.

[0024] The artificial expression constructs drive gene expression in astrocytes by including one or more astrocyte-specific enhancers, such as eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores.

[0025] In certain embodiments, the artificial expression construct comprises a combinatorial linked enhancer. In certain embodiments, the combinatorial linked enhancer comprises a core of enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, and eHGT_390m linked with I56i(core). In certain embodiments, the combinatorial linked enhancer comprises eHGT_390m(core2) and I56i(core) as set forth in SEQ ID NO: 86. In certain embodiments, the combinatorial linked enhancer is linked to comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the combinatorial linked enhancer. In certain embodiments, SEQ ID NO: 89 provides a concatemer of 3 copies of the eHGT_390m(core2)-I56i(core) combinatorial linked enhancer.

[0026] Certain embodiments provide artificial expression constructs that include the features of vectors described herein, including vectors such as: CN2720, CN2721, CN2722, CN2732, CN3213, CN3322, CN3323, CN3887, CN3888, CN2972, CN2973, CN2974, CN2975, or CN2976. In certain embodiments, the heterologous coding sequence encoding SYFP2 in CN2720, CN2721, CN2722, CN2732, CN3213, CN3322, CN3323, CN3887, and CN3888 is replaced or supplemented with the SLC6A1 gene sequence. The SLC6A1 coding sequence can be codon-optimized (see, for example, Figures 10A-10C and 14). Particular embodiments are represented by vectors ID10.01, ID10.02, ID10.03, ID10.04, ID10.05, ID10.06, ID10.07, ID10.08, ID10.09, ID10.10, ID10.11, ID10.12, ID10.13, ID10.14, ID10.15, ID10.16, ID10.17, ID10.18, ID10.19, ID10.20, ID10.21, ID10.22, ID10.23, ID10.24, ID10.25, ID10.26, ID10.27, ID10.28, ID10.29, ID10.30, ID10.31, ID10.32, ID10.33, ID10.34, ID10.35, ID10.36, ID10.37, ID10.38, ID10.39, ID10.40, ID10.41, ID10.42, ID10.43, ID10.44, ID10.45, ID10.46, ID10.47, ID10.48, ID10.49, ID10.50, ID10.51, ID10.52, ID10.53, ID10.54, ID10.55, ID10.56, ID10.57, ID10.58, ID10.59, ID10.60, ID10.61, ID10.62, ID10.63, ID10.64, ID10.65, ID10.66, ID10.67, ID10.68, ID10.69, ID10.70, ID10.71, ID10.72 0.20, ID10.21, ID10.22, ID10.23, ID10.24, ID10.25, ID10.26, ID10.27, ID10.28, ID10.29, ID10.30 , ID10.31, ID10.32, ID11.01, ID11.02, ID11.03, ID11.04, ID11.05, ID11.06, ID11.07, ID11.08, ID11 .09, ID11.10, ID11.11, ID11.12, ID11.13, ID11.14, ID11.15, ID11.16, ID12.01, ID12.02, ID12.03, ID12.04, ID12.05, ID12.06, ID12.07, ID12.08, ID12.09, ID12.10, ID12.11, ID12.12, ID12.13, ID12 .14, ID12.15, ID12.16, ID13.01, ID13.02, ID13.03, ID13.04, ID13.05, ID13.06, ID13.07, ID13.08, ID13.09, ID13.10, ID13.11, ID13.12, ID13.13, ID13.14, ID13.15, ID13.16, ID14.01, ID14.02, ID14.03、ID14.04、ID14.05、ID14.06、ID14.07、ID14.08、ID14.09、ID14.10、ID14.11、ID14.12、ID14.13、ID14.14、ID14.15、ID14.16、ID15.01、ID15.02、ID15.03、ID15.04、ID15.05、ID15.06、ID15.07、ID15.08、ID15.09、ID15.10、ID15.11、ID15.12、ID15.13、ID15.14、ID15.15、ID15.16、ID16.01、ID16.02、ID16.03、ID16.04、ID16.05、ID16.06、ID16.07、ID16.08、ID16.09、ID16.10、ID16.11、ID16.12、ID16.13、ID16.14、ID16.15、ID16.16、ID17.01、ID17.02、ID17.03、ID17.04、ID17.05、ID17.06、ID17.07、ID17.08、ID17.09、ID17.10、ID17.11、ID17.12、ID17.13、ID17.14、ID17.15、ID17.16、ID18.01、ID18.02、ID18.03、ID18.04、ID18.05、ID18.06、ID18.07、ID18.08、ID18.09、ID18.10、ID18.11、ID18.12、ID18.13、ID18.14、ID18.15、ID18.16、ID19.01、ID19.02、ID19.03、ID19.04、ID19.05、ID19.06、ID19.07、ID19.08、ID19.09、ID19.10、ID19.11、ID19.12、ID19.13、ID19.14、ID19.15、ID19.16、ID20.01、ID20.02、ID20.03、ID20.04、ID20.05、ID20.06、ID20.07、ID20.08、ID20.09、ID20.10、ID20.11、ID20.12、ID20.13、ID20.14、ID20.15、ID20.16、ID21.01、ID21.02、ID21.03、ID21.04、ID21.05、ID21.06、ID21.07、ID21.08、ID21.09、ID21.10、ID21.11、ID21.12、ID21.13、ID21.14、ID21.15、ID21.16, ID22.01, ID22.02, ID22.03, ID22.04, ID22.05, ID22.06, ID22.07, ID22.08, ID22.09, ID22.10, ID22.11, ID22.12, ID 22.13, ID22.14, ID22.15, ID22.16, ID23.01, ID23.02, ID23.03, ID23.04, ID23.05, ID23.06, ID23.07, ID23.08, ID23.09 , ID23.10, ID23.11, ID23.12, ID23.13, ID23.14, ID23.15, ID23.16, ID24.01, ID24.02, ID24.03, ID24.04, ID24.05, ID24 .06, ID24.07, ID24.08, ID24.09, ID24.10, ID24.11, ID24.12, ID24.13, ID24.14, ID24.15, ID24.16, ID25.01, ID25.02, I D25.03, ID25.04, ID25.05, ID25.06, ID25.07, ID25.08, ID25.09, ID25.10, ID25.11, ID25.12, ID25.13, ID25.14, ID25.1 5, ID25.16, ID26.01, ID26.02, ID26.03, ID26.04, ID26.05, ID26.06, ID26.07, ID26.08, ID26.09, ID26.10, ID26.11, ID2 The present invention provides artificial expression constructs that include the features of the vectors described herein, including ID26.6.12, ID26.13, ID26.14, ID26.15, ID26.16, ID27.01, ID27.02, ID27.03, ID27.04, ID27.05, ID27.06, ID27.07, ID27.08, ID27.09, ID27.10, ID27.11, ID27.12, ID27.13, ID27.14, ID27.15, and ID27.16. Of note, vectors CN2972, CN2973, CN2974, CN2975, and CN2976 do not contain enhancer sequences as disclosed herein and are therefore not used to provide target gene expression in GABAergic neurons and astrocytes.

[0027] Aspects of the disclosure are now described in further options and details: (i) artificial expression constructs and vectors for targeted expression of genes in targeted cell types; (ii) compositions for administration; (iii) cell lines containing artificial expression constructs; (iv) transgenic animals; (v) methods of use; (vi) kits and commercial packages; (vii) exemplary embodiments; and (viii) concluding paragraphs. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the disclosure.

[0028] (i) Artificial expression constructs and vectors for targeted expression of genes in targeted cell types. The artificial expression constructs disclosed herein comprise (i) at least two enhancer sequences, at least one of which directs expression of a coding sequence in GABAergic neurons and at least one of which directs expression of a coding sequence in astrocytes, (ii) a coding sequence to be expressed, and (iii) a promoter. The artificial expression constructs can also comprise other regulatory elements if necessary or beneficial.

[0029] An "enhancer" or "enhancer element" increases the level of transcription in association with a promoter. In certain instances, an enhancer can function in either orientation with respect to the promoter and the coding sequence to be transcribed, and can be located upstream or downstream with respect to the promoter or the coding sequence to be transcribed. Methods and techniques for measuring the function(s) of an enhancer element exist and are recognized in the art. Specific examples of enhancer sequences for use in the artificial expression constructs disclosed herein include the I56i enhancer, or a core thereof (e.g., hI56i core or 3XhI56i core), and one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores.

[0030] An artificial expression construct comprising at least two enhancer sequences can have the two enhancer sequences adjacent to each other or not adjacent to each other. The term "adjacent" refers to a position relative to each other such that there is no intervening functional sequence (e.g., promoter, enhancer, or heterologous coding sequence) between the two reference sequence segments (e.g., enhancers). The term "non-adjacent" refers to two sequence segments (e.g., enhancers) positioned such that there is an intervening functional sequence, such as a promoter, enhancer, and / or heterologous coding sequence, between the two sequence segments. Adjacent enhancer sequences can have small binding sequences between them, e.g., residues that appear based on cloning techniques. These small binding segments are not considered to be functional within the context of the current disclosure. In certain embodiments, an artificial expression construct comprising two enhancer sequences comprises a first enhancer and a second enhancer. In certain embodiments, the first enhancer is adjacent to the second enhancer. In certain embodiments, the first enhancer is not adjacent to the second enhancer. In certain embodiments, the first enhancer is 5' to the second enhancer. In certain embodiments, the second enhancer is 5' to the first enhancer.

[0031] In certain embodiments, the targeted CNS cell type enhancer is an enhancer that is uniquely or primarily utilized by the targeted CNS cell type. The targeted CNS cell type enhancer enhances expression of a gene in the targeted CNS cell type. In certain embodiments, the targeted CNS cell type enhancer enhances expression of a gene in the targeted CNS cell type and does not substantially affect expression of a gene in other non-targeted cell types, and is therefore also a selectively targeted CNS type enhancer that has cell type specific transcriptional activity.

[0032] When a heterologous coding sequence operably linked to an enhancer disclosed herein is expressed in the targeted cell type, the administered heterologous coding sequence is expressed in the intended cell type.

[0033] When a heterologous coding sequence is selectively expressed in a selected cell, it results in the expression of the administered heterologous coding sequence in the intended cell type, as described in further detail below. In certain embodiments, it is not substantially expressed in other cell types, and the expression in the reference cell type is less than 50% compared to the target cell type; the expression in the reference cell type is less than 40% compared to the target cell type; the expression in the reference cell type is less than 30% compared to the target cell type; the expression in the reference cell type is less than 20% compared to the target cell type; or the expression in the reference cell type is less than 10% compared to the target cell type. In certain embodiments, the reference cell type refers to a non-targeted cell. The non-targeted cell is in the same anatomical structure as the targeted cell and / or occurs in a common anatomical region. In certain embodiments, the reference cell type is in an anatomical structure adjacent to the anatomical structure containing the targeted cell type. In certain embodiments, the reference cell type is a non-targeted cell that has a gene expression profile different from that of the targeted cell.

[0034] In certain embodiments, the product of the coding sequence may be expressed in non-selected cell types at low levels, for example, less than 1% or 1%, 2%, 3%, 5%, 10%, 15%, or 20% of the level of expression of the product in selected cell types. In certain embodiments, the targeted central nervous system cell type is the only cell type that expresses the appropriate combination of transcription factors that bind to the enhancers disclosed herein to drive gene expression. Thus, in certain embodiments, expression occurs only in the targeted cell type.

[0035] In certain embodiments, target cell types (e.g., neuronal and / or non-neuronal) can be identified based on transcriptional profiles, such as, for example, Tasic et al., Nature 563, 72-78 (2018) and Hodge et al., Nature, 573, 61-68 (2019). For reference, a description of cell types and identifying characteristics is provided as follows:

[0036] Neocortical GABAergic neuron subclasses: ●All: express the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2. • Lamp5, and Vip GABAergic neurons: are developmentally derived from neuronal precursors originating from the caudal ganglion luminal (CGE) or preoptic area (POA). ● Sst and Pvalb GABAergic neurons: are developmentally derived from neuronal precursors in the medial ganglionic emittance (MGE). ●Lamp5 GABAergic neurons: are found in many cortical layers, especially the upper layers (L1-L2 / 3), and mainly have glial and single plexus morphologies. ● Lamp5_Lhx6 GABAergic neurons: A subset of Lamp5 GABAergic neurons that co-express Lamp5 and Lhx6. ● Sncg GABAergic neurons: found in many cortical layers and have molecular overlap with Lamp5 and Vip cells, but express Lamp5 or Vip inconsistently and Sncg more consistently. Serpinf1 GABAergic neurons are found in many cortical layers and have molecular overlap with Sncg and Vip cells, but they express Sncg or Vip inconsistently and Serpinf1 more consistently. ●Vip GABAergic neurons: They are found in many cortical layers, but are particularly abundant in the upper layers (L1-L4) and highly express the neurotransmitter vasoactive intestinal peptide (Vip). ●Sst GABAergic neurons: found in many cortical layers, but especially frequent in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic input to postsynaptic neurons. This subclass includes sleep-active Sst Chodl neurons (which also express Nos1 and Tacr1), which are very distinct from other Sst neurons but express several shared marker genes with Sst. In humans, SST gene expression is often detected in layer 1 LAMP5+ GABAergic neuron subtypes. ●Pvalb GABAergic neurons: found in many cortical layers, but especially frequent in the lower layers (L5-L6). They highly express the calcium-binding protein parvalbumin (Pvalb), express the neuropeptide Tac1, and often attenuate the output of postsynaptic neurons. Most GABAergic neurons strongly express Pvalb. This subclass includes chandelier cells, which have a distinct chandelier-like morphology and express the markers Cpne5 and Vipr2 in mice, and NOG and UNC5B in humans. Meis2: A distinct subclass defined as the only neocortical GABAergic neuron type that expresses the Meis2 gene and does not express some other genes expressed by all other neocortical GABAergic neuron types (e.g., Thy1 and Scn2b). This type is found in L6b and subcortical white matter.

[0037] Neocortical glutamatergic neuron subclasses: All: express the glutamate transmitters Slc17a6 and / or Slc17a7. They all express Snap25 and lack expression of Gad1 / Gad2. • L2 / 3 IT glutamatergic neurons: Predominantly located in layer 2 / 3 and with predominantly intratelencephalic (cortico-cortical) projections. • L4 IT glutamatergic neurons: are found primarily in layer 4 and have primarily either local or intratelencephalic (cortico-cortical) projections. L5 IT glutamatergic neurons: located mainly in layer 5 and with mainly intratelencephalic (cortico-cortical) projections. Also called L5a. L5 PT glutamatergic neurons: Predominantly found in layer 5 and with predominantly cortico-subcortical (pyramidal or corticofugal) projections. Also referred to as L5b or L5 CF (corticofugal), or L5 ET (super telencephalic). This subclass includes cells that are corticospinal projection neurons located in the primary motor cortex and adjacent regions and associated with motor neuron / movement disorders such as ALS. This subclass includes thick tufted pyramidal neurons, including distinctive subtypes found only in specialized areas, e.g., Betz, Meynert, and von Economo cells. ●L5 NP glutamatergic neurons: reside mainly in layer 5 and have projections mainly in the vicinity. • L6 CT glutamatergic neurons: are located mainly in layer 6 and have mainly corticothalamic projections. • L6 IT glutamatergic neurons: Predominantly located in layer 6 and with predominantly intracortical (cortico-cortical) projections. This subclass includes the L6 IT Car3, which are highly similar to the intracortical projection cells of the claustrum. • L6b glutamatergic neurons: Predominantly located in the neocortical subplate (L6b), with local (close to the cell body) processes and some cortico-cortical projections from the VISp to the anterior cisternae, and cortico-subcortical projections to the thalamus. ● CR neurons: Cajal-Retzius cells, a distinct subclass defined as a single type in L1, express distinct molecular markers Lhx5 and Trp73.

[0038] Cerebellar Purkinje cells: large GABAergic neurons that are the only projection neurons and the only product of the cerebellum. Their cell bodies form a single layer called the "Purkinje cell layer" and express parvalbumin.

[0039] Deep cerebellar nucleus neurons: neurons located in the deep cerebellar nucleus structures. These include glutamatergic and GABAergic cells that express the Pvalb gene.

[0040] Non-neuronal subclasses: Astrocytes: Neuroectodermally derived glial cells that express the marker Aqp4 and often express GFAP, but not the neuronal marker SNAP25. They can have a distinct star-shaped morphology and are involved in the metabolic support of other cells in the brain. Multiple astrocytic morphologies are found in mice and humans. Oligodendrocytes: Glial cells derived from the neuroectoderm that express the marker Sox10. This category includes oligodendrocyte precursor cells (OPCs). Oligodendrocyte cells are the subclass primarily responsible for myelination of neurons. • VLMC: Vascular leptomeningeal cells (VLMC) are part of the meninges that surround the outer layer of the cortex and express the marker genes Lum and Col1a1. Pericytes: Blood vessel-associated cells that express the marker genes Kcnj8 and Abcc9. Pericytes wrap around endothelial cells and are important in regulating capillary blood flow and are involved in blood-brain barrier permeability. SMC: A specialized smooth muscle cell that is a blood vessel-associated cell that expresses the marker gene Acta2. SMCs line the arteries of the brain and are involved in blood-brain barrier permeability. ●Endothelial cells: cells that line the blood vessels in the brain. Endothelial cells express the markers Tek and PDGF-B. Microglia: Hematopoietic-derived immune cells that are brain-resident macrophages and perivascular macrophages (PVMs) that may be transiently associated with brain tissue or included as a by-product of brain dissection methods. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

[0041] In certain embodiments, the coding sequence is a heterologous coding sequence and encodes GAT1. The heterologous coding sequence encoding GAT1 may be, for example, a codon-optimized SLC6A1 variant as shown in FIG. 14 (SEQ ID NOs: 22, 25, 28, 31, 34).

[0042] In certain embodiments, the coding sequence is a heterologous coding sequence that encodes an effector element. An effector element is a sequence that is expressed to achieve, and actually achieves, an intended effect. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0043] Exemplary reporter genes / proteins include those expressed by Addgene ID numbers 83894 (pAAV-hDlx-Flex-DTomato-Fishell_7), 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), 83900 (pAAV-mDlx-GFP-Fishell-1), and 89897 (pcDNA3-FLAG-mTET2(N500)).Exemplary reporter genes include, among others, those encoding expressible fluorescent proteins, or expressible biotin: blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, Amcyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™ (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific); far-red fluorescent proteins (e.g., mPlum, and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0044] GFP consists of 238 amino acids (26.9 kDa) and was originally isolated from Aequorea victoria / Aequorea aequorea / Aequorea forskalea, a jellyfish that fluoresces green when exposed to blue light. GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, which is in the lower green part of the visible spectrum. GFP from Renilla reniformis has a single major excitation peak at 498 nm. Due to its potential for widespread use and the evolving needs of researchers, many different variants of GFP have been genetically engineered to produce them. The first major improvement was a single point mutation (S65T) reported in Nature by Roger Tsien in 1995. This mutation dramatically improved the spectral properties of GFP, resulting in increased fluorescence, photostability, and a shift in the main excitation peak to 488 nm, with peak emission maintained at 509 nm. Addition of the 37°C folding efficiency (F64L) point mutant to this scaffold resulted in enhanced GFP (EGFP). EGFP has a molecular weight of 9.13×10−21 m, also cited as 55,000 L / (mol cm). 2 / has an extinction coefficient (denoted by ε), also known as the optical cross section of the molecule. In 2006, we reported superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides.

[0045] "Yellow fluorescent protein" (YFP) is a genetic variant of the green fluorescent protein derived from the jellyfish Aequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm.

[0046] Exemplary functional molecules include functional ion transporters, cellular transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, microRNAs, or designer receptors exclusively activated by designer drugs (DREADDs).

[0047] Ion transporters are transmembrane proteins that mediate the transport of ions across cell membranes. These transporters are widespread in most cell types and are important in regulating cellular excitability and homeostasis. Ion transporters are involved in numerous cellular processes, such as action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many important biological processes in living cells involve the movement of cations, such as calcium (Ca2+), potassium (K+), and sodium (Na+) ions, through such ion channels. In certain embodiments, the ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0048] Exemplary enzymes, transcription factors, receptors, membrane proteins, cellular transport proteins, signaling molecules, and neurotransmitters include enzymes such as lactase, lipase, helicase, alpha-glucosidase, amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAA-T / enhancer binding protein), and Oct-1; receptors such as transforming growth factor receptor beta 1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, and interleukin 8 receptor alpha; membrane proteins, cellular transport proteins, such as clathrin, dynamin, caveolin, Rab-4A, and Rab-11A; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGFβ), epidermal growth factor (EGF), GTPases, and HRas; and neurotransmitters such as cocaine- and amphetamine-regulated transcription factors, substance P, oxytocin, and somatostatin.

[0049] In certain embodiments, the functional molecules include reporters of cell functions and states, such as calcium reporters. Intracellular calcium concentration is an important predictor of numerous cellular activities, such as neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels uses genetically encoded calcium indicators (GECIs). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor, called GCaMP, is an efficient and widely used tool. GCaMP is formed by fusing M13 and calmodulin proteins to the N- and C-termini of circularly permuted GFP. Some GCaMPs produce distinct fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs that exhibit green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Additionally, GECIs that exhibit red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV8-CAG-GCaMP3 (catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog number: BS12-NXSAAV9), AAV9-CAG-FLEX- The company offers AAV products including jGCaMP7f-WPRE (catalog number: BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (catalog number: BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (catalog number: BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (catalog number: BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (catalog number: BS7-NXBAAV8).

[0050] In certain embodiments, the calcium reporters include the genetically encoded calcium indicator GECI, NTnC; myosin light chain kinase, GFP, calmodulin chimera; the calcium indicator TN-XXL; a BRET-based autoluminescent calcium indicator; and / or the calcium indicator protein OeNL(Ca2+)-18u).

[0051] In certain embodiments, functional molecules include regulators of neuronal activity, such as channelrhodopsins (e.g., channelrhodopsin-1, channelrhodopsin-2, and their variants). Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels. In addition to channelrhodopsin 1 (ChR1) and channelrhodopsin 2 (ChR2), several channelrhodopsin variants have been developed. For example, Lin et al. (Biophys J, 2009, 96(5):1803-14) describe the creation of chimeras of the transmembrane domains of ChR1 and ChR2 in combination with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6):631-3) describe a red-shifted channelrhodopsin variant, VChR1. VChR1 has low light sensitivity and poor membrane trafficking and expression. Other known channelrhodopsin mutants include the ChR2 mutant described by Nagel et al., Proc Natl Acad Sci USA, 2003, 100(24):13940-5, ChR2 / H134R (Nagel, G. et al., Curr Biol, 2005, 15(24):2279-84), and ChD / ChEF / ChIEF (Lin, JY et al. Biophys J, 2009, 96(5):1803-14), which are activated by blue light (470 nm) but show no sensitivity to orange / red light. Further mutants are described in Lin, Experimental Physiology, 2010, 96.1:19-25, and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.

[0052] 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 or cpf1). Functional molecules may also include engineered Cpf1, such as those described in US2018 / 0030425, US2016 / 0208243, WO / 2017 / 184768, and Zetsche et al. (2015) Cell 163:759-771; single gRNA (see, e.g., Jinek et al. (2012) Science 337:816-821, Jinek et al. (2013) eLife 2:e00471, Segal (2013) eLife 2:e00563), or editases, guide RNA molecules, microRNAs, or homologous recombination donor cassettes.

[0053] As indicated, sequences are publicly available. Further examples include lactase (e.g., GenBank: EAX11622.1), lipase (e.g., GenBank: AAA60129.1), helicase (e.g., GenBank: AMD82207.1), amylase (e.g., GenBank: AAA51724.1), alpha-glucosidase (e.g., GenBank: ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot: P08047.3), transcription factor AP-1 (e.g., NP_002219.1), heat shock factor protein Protein 1 (e.g., UniProtKB / Swiss-Prot:Q00613.1), CCAAT / enhancer-binding protein (C / EBP) beta isoform a (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot:P14859.2), TGFβ (e.g., GenBank:CAF02096.2), platelet-derived growth factor receptor (e.g., GenBank:AAA60049.1), epidermal growth factor receptor (e.g., GenBank:CAA25240.1), vascular endothelial growth factor receptor (VAG) (e.g., GenBank:CAA25240.1), and vascular endothelial growth factor receptor (VAG) (e.g., GenBank:CAA25240.1). receptor (e.g., GenBank: AAC16449.2), interleukin 8 receptor alpha (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B isoform Ras-related protein Rab-4A isoform 1 (e.g., NP_004569.2), ras-related protein Rab-11A (e.g., UniProtKB / Swiss-Prot:P62491.3), platelet-derived growth factor (e.g., GenBank:AAA60552.1), transforming growth factor-beta 3 (e.g., GenBank:AAA61161.1), nerve growth factor (e.g., GenBank:CAA37703.1), EGF (e.g., GenBank:CAA34902.2), cocaine- and amphetamine-regulated transcription (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 autoluminescent calcium indicator [synthetic construct] (e.g., GenBank ADF42668.1), calcium indicator protein OeNL(Ca2+)-18u [synthetic construct] (e.g., GenBank BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (chain A) [synthetic construct] (e.g., PDB: 3EKJ_A), channel opsin 1 (e.g., UniProtKB-F8UVI5), channel opsin 1 (e.g., GenBank AER58 217.1), channelrhodopsin-2 (e.g., UniProtKB-B4Y105), channelrhodopsin2 [synthetic construct] (e.g., GenBank: ABO64386.1), CRISPR-associated protein (Cas) (e.g., GenBank: AKG27598.1), Cas9 [synthetic construct] (e.g., GenBank: AST09977.1), CRISPR-associated endonuclease Cpf1 (e.g., UniProtKB / Swiss-Prot: U2UMQ6.1), ribonuclease 4 or ribonuclease ase L (e.g., UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease II beta (e.g., GenBank:AAF76893.1), sodium channel protein type 1 subunit alpha (e.g., UniProtKB-P35498), voltage-gated potassium channel subfamily KQT member 2 (e.g., UniProtKB-O43526), ​​and voltage-gated L-type calcium channel subunit alpha-1C (e.g., UniProtKB-Q13936).

[0054] Further effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to codon-improved Cre. dgCre refers to an enhanced GFP / Cre recombinase fusion gene with an N-terminal fusion of the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) with a G67S mutation, modified to also contain an R12Y / Y100I instability domain mutation. FlpO refers to a codon-optimized form of FLPe that significantly increases protein expression and FRT recombination efficiency in mouse cells. Similar to the Cre / LoxP system, the FLP / FRT system is widely used for gene expression (and the generation of conditional knockout mice mediated by the FLP / FRT system). tTA2 refers to tetracycline transactivator. 4x2C is a synthetic microRNA binding site element that allows silencing of virus-mediated transgene expression in certain cell types. For example, it can be used to reduce or eliminate expression in numerous glutamatergic neuronal populations in the brain. 4x2C is described in Sayeg et al., ACS Synth. Biol. 2015, 4, 7, 788-795.

[0055] Exemplary expressible elements are expression products that do not contain effector elements, such as, for example, non-functional or defective proteins. In certain embodiments, expressible elements can provide a method for studying the effects of their functional counterparts. In certain embodiments, expressible elements are non-functional or defective based on engineered mutations that render them non-functional. In these aspects, non-expressible elements are as similar in structure as possible to their functional counterparts.

[0056] An exemplary self-cleaving peptide is the 2A peptide, which leads to the production of two proteins from one mRNA. The 2A sequence is short (e.g., 20 amino acids), allowing for more use in size-restricted constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct comprises an internal ribosome entry site (IRES) sequence. The IRES allows the ribosome to begin translation at a second internal site on the mRNA molecule, leading to the production of two proteins from one mRNA.

[0057] Coding sequences encoding the molecules (e.g., RNA, 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 modifications do not affect the function of the encoded molecule. The terms "encode" or "encoding" refer to the property of a sequence of a nucleic acid, such as a vector, plasmid, gene, cDNA, mRNA, etc., that serves as a template for the synthesis of another molecule, such as a protein.

[0058] The term "gene" may include not only the coding sequence, but also regulatory regions such as promoters, enhancers, insulators, and / or termination regions. The term may further include all introns and other DNA sequences spliced ​​from the mRNA transcript, as well as variants resulting from alternative splice sites. The sequence may also include degenerate codons of the reference sequence, or sequences that may be introduced to provide codon preferences in a particular organism or cell type.

[0059] The promoter may include a general promoter, a tissue-specific promoter, a cell-specific promoter, and / or a cytoplasm-specific promoter. The promoter may be a strong promoter, a weak promoter, a constitutive expression promoter, and / or an inducible promoter. An inducible promoter induces expression in response to a specific condition, signal, or cellular event. For example, the promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to effect transcription from the promoter. Specific examples of promoters include minBglobin (or 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 immediate early promoter, Hsp68 minimal promoter (proHSP68), and Rous sarcoma virus (RSV) long repeat (LTR) promoter. A minimal promoter has no activity to drive gene expression by itself, but can be activated to drive gene expression when linked to proximal enhancer elements.

[0060] In certain embodiments, the expression construct is provided in a vector. The term vector refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is generally linked, e.g., inserted, into a vector nucleic acid molecule. The vector may include sequences that direct autonomous replication in the cell or may include sequences that allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0061] Viral vector is widely used to refer to nucleic acid molecules that contain nucleic acid elements derived from viruses that facilitate the transfer and expression of non-natural nucleic acid molecules into cells. The term adeno-associated viral vector refers primarily to viral vectors or plasmids that contain structural and functional genetic elements, or portions thereof, derived from AAV. The term "retroviral vector" refers primarily to viral vectors or plasmids that contain structural and functional genetic elements, or portions thereof, derived from retroviruses, etc. The term "lentiviral vector" refers primarily to viral vectors or plasmids that contain structural and functional genetic elements, or portions thereof, derived from lentiviruses, etc. The term "hybrid vector" refers to vectors that contain structural and / or functional genetic elements from two or more types of viruses.

[0062] Adenoviral vector refers to a construct that contains sufficient adenoviral sequences to (a) support packaging of an artificial expression construct and (b) express coding sequences cloned therein in either sense or antisense orientation. Recombinant adenoviral vectors include genetically engineered forms of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb linear double-stranded DNA virus, allows for the replacement of large elements of adenoviral DNA with up to 7 kb of foreign sequences. In contrast to retroviruses, adenoviral infection of host cells does not result in chromosomal integration, since adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Adenoviruses are also structurally stable, and no genome rearrangements have been detected after extensive amplification.

[0063] Adenoviruses are particularly suitable for use as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in the regulation of transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) leads to the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript derived from the major late promoter (MLP). The MLP is particularly efficient at the late stages of infection, and all mRNAs derived from this promoter contain a 5'-triple leader (TPL) sequence, making this the preferred mRNA for translation.

[0064] Other than the requirement that the adenoviral vector be replication-defective, or at least conditionally defective, the nature of the adenoviral vector is not believed to be critical to the successful practice of certain embodiments disclosed herein. The adenovirus can be any of the 42 different known serotypes or subgroups A-F. In certain embodiments, adenovirus type 5 of subgroup C is the preferred starting material for obtaining a conditionally replication-defective adenoviral vector for use in certain embodiments, since adenovirus type 5 is a human adenovirus for which a great deal of biochemical and genetic information is known, and which has been used historically for most constructs using adenovirus as a vector.

[0065] As stated, typical vectors are replication-defective and do not have the adenovirus E1 region.Therefore, it will be most convenient to introduce the polynucleotide encoding the gene of interest into the position where the E1 coding sequence is deleted.However, the insertion position of the construct within the adenovirus sequence is not critical.The polynucleotide encoding the gene of interest may be inserted in place of the deleted E3 region in the E3 replacement vector or in the E4 region where the helper cell line or helper virus complements the E4 defect.

[0066] Adeno-associated virus (AAV) is a parvovirus discovered as a contamination of adenovirus strains. It is a ubiquitous virus that is not associated with any disease (antibodies are present in 85% of the human population in the United States). It is also classified as a dependent virus because its replication depends on the presence of a helper virus such as adenovirus. Various serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is packaged in the capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20 to 24 nm.

[0067] AAV DNA is 4.7 kilobases long. It contains two open reading frames flanked by two ITRs. There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins involved in viral replication, while cap codes for the capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of AAV for chromosomal integration. Thus, AAV can be used as a vector for delivery, with all viral coding sequences removed and replaced with a cassette of genes. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their map locations. Transcription from p5 and p19 leads to the production of rep proteins, while transcription from p40 produces the capsid proteins.

[0068] AAV is prominent for use within the present disclosure due to its excellent safety profile and the fact that their capsids and genomes can be tailored to allow expression in target cell populations. scAAV refers to self-complementary AAV. pAAV refers to plasmid adeno-associated virus. rAAV refers to recombinant adeno-associated virus.

[0069] Other viral vectors may be employed, such as those derived from viruses such as vaccinia virus, poliovirus, and herpes virus, which offer several attractive features for a variety of mammalian cells.

[0070] Retroviruses are common tools for gene delivery. "Retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates the genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II to induce the expression of RNA molecules that code for structural proteins and enzymes required to produce new viral particles.

[0071] Exemplary retroviruses suitable for use in certain embodiments include: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0072] "Lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include: HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2); Visnamedi virus (VMV); Capric arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) can be used.

[0073] Improved safety for using some vectors may be achieved by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used for this purpose include, for example, the virus simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters can drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent viruses, since the complete U3 sequence is not present in the virus production system. In certain embodiments, heterologous promoters have the added advantage of controlling how the viral genome is transcribed. For example, the heterologous promoter may be inducible, such that transcription of all or part of the viral genome occurs only when an inducer is present. Inducers can include one or more chemical compounds or the physiological conditions under which the host cells are cultured, such as temperature or pH.

[0074] In certain embodiments, the viral vector comprises a TAR element. The term "TAR" refers to the "transactivation response" genetic element located in the R region of lentiviral LTR. This element interacts with the lentiviral transactivator (tat) genetic element to enhance viral replication. However, this element is not necessary in the embodiment in which the U3 region of 5'LTR is replaced with a heterologous promoter.

[0075] "R region" refers to the region within a retroviral LTR that begins at the beginning of the capping group (i.e., at the beginning of transcription) and ends just before the beginning of the poly(A) tract. The R region is also defined as flanking the U3 and U5 regions. The R region serves to allow the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

[0076] In certain embodiments, expression of heterologous sequences in viral vectors is enhanced by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and, optionally, transcription termination signals into the vector. Various post-transcriptional regulatory elements can enhance expression of heterologous nucleic acids. Examples include the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE, Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in Hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and the like (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector comprises a post-transcriptional regulatory element such as a WPRE or HPRE. In certain embodiments, the vector lacks or does not comprise a post-transcriptional regulatory element such as a WPRE or HPRE.

[0077] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts can enhance heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors include a polyadenylation sequence 3' of a polynucleotide that encodes a molecule (e.g., a protein) to be expressed. The term "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that directs both the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a poly(A) tail to the 3' end of a coding sequence, thus contributing to improved translation efficiency. Certain embodiments may utilize BGHpA or SV40pA. In certain embodiments, preferred embodiments of expression constructs include terminator elements. These elements can help increase transcription levels and minimize readthrough from the construct into other plasmid sequences.

[0078] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may contribute to protecting viral vector expression sequences, such as effector elements or expressible elements, from integration site effects (i.e., position effects, see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002, and Zhan et al., Hum. Genet., 109:471, 2001), which may result in deregulated expression of the imported sequence, which may be mediated by cis-acting elements present in the genomic DNA. In certain embodiments, the viral import vector comprises one or more insulator elements in the 3'LTR, and when the provirus is integrated into the host genome, the provirus comprises one or more insulators in both the 5'LTR and the 3'LTR by replicating the 3'LTR. Suitable insulators for use in certain embodiments include the chicken β-globin insulator (Chung et al. Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al. Cell 98:387, 1999), the SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer-blocking insulators (Liu et al. Nature, Biotechnology, 33:198, 2015).

[0079] Beyond the above description, a wide range of suitable expression vector types are known to those skilled in the art. These may include commercially available expression vectors designed for general recombinant procedures, such as plasmids containing one or more reporter genes and regulatory elements necessary for the expression of the reporter gene in cells. Many vectors are commercially available, such as from Invitrogen, Stratagene, Clontech, etc., and are described in many related guides. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct that can support the expression of a coding gene in mammalian cells, such as the pUC or Bluescript plasmid series.

[0080] Particular embodiments of the vectors disclosed herein include the following: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14]

[0081] Those skilled in the art can easily identify the sequences of subcomponents within a larger vector sequence, and can easily identify them based on the present disclosure (see FIG. 14). The nucleotides between the identifiable and listed subcomponents reflect restriction enzyme recognition sites used in the assembly (cloning) of the construct, and in some cases, the additional nucleotides convey no discernable function. These segments of the complete vector sequence can be adjusted based on different cloning strategies and / or vector uses. Generally, short six nucleotide palindromic sequences reflect vector construction artifacts that are not critical to vector function.

[0082] In certain embodiments, a vector (e.g., AAV) is selected that has a capsid that crosses the blood-brain barrier (BBB). In certain embodiments, the vector is modified to include a capsid that crosses the BBB. Examples of AAVs with viral capsids that can cross the blood-brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014;7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014;22(7):1299-1309), AAV1R6, AAV1R7 (Albright et al., Mol Ther. 2018;26(2):510), rAAVrh.8 (Yang, et al., supra), AAV-BR1 (Marchio et al., EMBO Mol Med. 2016;8(6):592), AAV-PHP.S (Chan et al., Nat Neurosci. 2017;20(8):1172), and AAV-PHP.B (Deverman et al., Nat Biotechnol. 2016;34(2):204), AAV-PPS (Chen et al., Nat Med. 2009;15:1215), and PHP.eB. In certain embodiments, the PHP.eB capsid is distinct from and references AAV9, in which the amino acid sequence beginning at residue 586: S-AQ-A (SEQ ID NO:78) is changed to S-DGTLAVPFK-A (SEQ ID NO:79). In certain embodiments, PHP.eb refers to SEQ ID NO:70.

[0083] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the BBB after intravenous injection. It transforms large parts of the central nervous system (CNS), thus allowing for minimally invasive treatment, as described, for example, in connection with clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and for the treatment of CLN3 gene-associated neuronal cerebrovascular-lipofusion syndrome (NCT03770572) (Naso et al., BioDrugs. 2017;31(4):317).

[0084] AAVrh.10 was originally isolated from rhesus macaques, has shown low seropositivity in humans when compared to other common serotypes used in gene delivery applications (see Selot et al., Front Pharmacol. 2017;8:441), and is being evaluated in the LYS-SAF302, LYSOGENE, and NCT03612869 clinical trials.

[0085] Two variants isolated from a library of chimeric AAV vectors, AAV1R6 and AAV1R7 (in which the AAV1 capsid domain was exchanged with AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing a significant reduction in liver and vascular endothelial transduction.

[0086] Similarly, rAAVrh.8 isolated from rhesus macaques demonstrates global transduction of glial and neuronal cell types in clinically important areas after peripheral administration and also displays reduced peripheral tissue tropism compared to other vectors.

[0087] AAV-BR1 is an AAV2 variant displaying the NRGTEWD ​​(SEQ ID NO: 80) epitope isolated during in vivo screening of a random AAV-display peptide library, which shows high specificity with high transgene expression in the brain and has minimal off-target affinity (including the liver) (Korbelin et al., EMBO Mol Med. 2016;8(6):609).

[0088] AAV-PHP.S (Addgene, Watertown, Mass.) is a CREATE-generated variant of AAV9 that encodes the 7-mer sequence QAVRTSL (sequence number 81) and potently transduces neurons within the enteric nervous system and peripheral sensory afferents entering the spinal cord and brainstem.

[0089] AAV-PHP.B (Addgene, Watertown, Mass.) is a CREATE method-generated variant of AAV9 that encodes the 7-mer sequence TLAVPFK (SEQ ID NO:82). It transfers genes throughout the CNS more efficiently than AAV9 and transduces the majority of astrocytes and neurons across multiple CNS regions.

[0090] AAV-PPS was generated by inserting the DSPAHPS (SEQ ID NO: 83) epitope into the capsid of AAV2 and exhibits dramatically improved brain tropism compared to AAV2.

[0091] For more information regarding capsids crossing the blood-brain barrier, see Chan et al., Nat. Neurosci, 2017 Aug:20(8):1172-1179.

[0092] (ii) Compositions for Administration. The artificial expression constructs and vectors of the present disclosure (referred to herein as physiologically active components) can be formulated with a carrier suitable for administration to cells, tissue slices, animals (e.g., mice, non-human primates), or humans. The physiologically active components in the compositions described herein can be prepared in neutral form, as a free base, or as a pharmacologically acceptable salt.

[0093] Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like, and salts formed with the free carboxyl groups are derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, as well as such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0094] Carriers for physiologically active ingredients may include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active ingredients is well known in the art. Any conventional media or agent may be used with the compositions described herein, except insofar as it is incompatible with the physiologically active ingredient.

[0095] The phrase "pharmaceutical acceptable carrier" refers to a carrier that does not produce an allergic or similar untoward reaction when administered to a human, and in certain embodiments, when administered intravenously (e.g., into the retroorbital plexus).

[0096] In certain embodiments, the compositions can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or intranasal inhalation, or for direct injection or application to one or more cells, tissues, or organs.

[0097] The composition may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles.

[0098] The formation and use of liposomes is generally known to those skilled in the art. Liposomes have been developed with improved serum stability and circulation half-life (see, for example, U.S. Patent No. 5,741,516). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (see, for example, U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868; and 5,795,587).

[0099] The present disclosure also provides pharma- ceutically acceptable nanocapsule formulations of physiologically active ingredients. Nanocapsules can generally encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al. Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al. Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119, 1998; Douglas et al. Crit, Rev Ther Drug Carrier Syst 3(3):233-261, 1987). To avoid side effects due to intracellular polymer overload, such ultrafine particles can be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be readily produced as described in Couvreuret al., J Pharm Sci 69(2):199-202, 1980; Couvreuret al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlenet al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998, and U.S. Pat. No. 5,145,684.

[0100] Injectable compositions may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468). For delivery by injection, the form is sterile and fluid to the extent that it can be delivered by syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage, and optionally contains one or more preservative compounds to protect against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, to maintain the required particle size in the case of dispersions, and / or by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation includes isotonic agent(s), such as sugar(s) or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption into the composition, such as aluminum monostearate and gelatin. Injectable compositions can be suitably buffered, if necessary, and liquid diluents can be first rendered isotonic with sufficient saline or glucose, for example.

[0101] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils.As indicated, under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0102] Sterile compositions can be prepared by incorporating physiologically active ingredients in an appropriate amount of solvent containing other optional ingredients (e.g., as described above), followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilized physiologically active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients (e.g., as described above). In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method can be vacuum drying and freeze-drying technology, which obtains a powder of physiologically active ingredients and any additional desired ingredients from the solution previously sterile-filtered.

[0103] Oral compositions may be in liquid form, for example, as solution, syrup or suspension, or may be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use.Such liquid preparations can be prepared by conventional means using pharmacopoeia-acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, or fractionated vegetable oils); preservatives (for example, methyl or propyl-p-hydroxybenzoic acid or sorbic acid). The composition may take the form of, for example, a tablet or capsule prepared with pharma- ceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.

[0104] Inhalable compositions can be delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or filler can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0105] Compositions also include microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208), and feedback controlled delivery (U.S. Pat. No. 5,697,899).

[0106] Supplementary active ingredients can also be incorporated into the compositions.

[0107] Typically, the composition will contain at least 0.1% of the physiologically active ingredient, although the percentage of the physiologically active ingredient may of course vary, and may conveniently be 1 or 2% to 70% or 80% or more, or 0.5 to 99% by weight or volume of the total composition. Of course, the amount of the physiologically active ingredient in each physiologically useful composition may be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, as well as other pharmacological considerations, are contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various compositions and dosages may be desirable.

[0108] In certain embodiments, for human administration, compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by the U.S. Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.

[0109] (iii) Cell lines containing the artificial expression constructs. The present disclosure includes cells containing the artificial expression constructs described herein. Cells transformed with artificial expression constructs can be used for many purposes, including neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to evaluate the regulatory properties of enhancers.

[0110] Although various host cell lines can be used, in certain embodiments, the cells are mammalian cells. In certain embodiments, the artificial expression construct comprises an I56i enhancer, its linked core, or its linked core and one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores, and the cell line is, for example, a human, primate, or mouse cell. Cell lines that can be utilized for transformation in the present disclosure also include primary cell lines derived from living tissues such as rat or mouse brain, and organotypic cell cultures including brain slices from animals such as rats or mice. The PC12 cell line (available from the American Type Culture Collection, ATCC, Manassas, VA) has been shown to express a number of neuronal marker proteins in response to neuronal growth factor (NGF). The PC12 cell line is believed to be a neuronal cell line and may be used in the present disclosure. JAR cells (available from ATCC) are a platelet-derived cell line that expresses some neuronal genes, such as the serotonin transporter gene, and may be used with the embodiments described herein.

[0111] WO 91 / 13150 describes various cell lines, including neuronal cell lines, and methods for their production. Similarly, WO 97 / 39117 describes neuronal cell lines and methods for the production of such cell lines. The neuronal cell lines disclosed in these patent applications are applicable for use in the present disclosure.

[0112] In certain embodiments, "neuronal" describes something that is, relates to, or includes a nerve cell. A nerve cell is defined by the presence of an axon and a dendrite. The term "neuronal-specific" refers to activity that is found or occurs in nerve cells or cells derived from nerve cells, but that is not found or occurs or is substantially not found or occurs in non-neuronal cells or cells that are not derived from nerve cells, e.g., glial cells such as astrocytes or oligodendrocyte cells.

[0113] In certain embodiments, non-neuronal cell lines including mouse embryonic stem cells can be used. Cultured mouse embryonic stem cells can be used to analyze the expression of gene constructs using transient transfection with plasmid constructs. Mouse embryonic stem cells are pluripotent and undifferentiated. These cells can be maintained in this undifferentiated state by leukemia inhibitory factor (LIF). Withdrawal of LIF induces differentiation of embryonic stem cells. In culture, stem cells form various differentiated cell types. Differentiation is triggered by the expression of tissue-specific transcription factors, and the function of enhancer sequences can be evaluated. (See, for example, Fiskerstrand et al., FEBS Lett 458:171-174, 1999).

[0114] A method for differentiating stem cells into different cell types includes replacing stem cell culture medium with medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplement (e.g., transferrin, insulin, progesterone, putrescine, and selenate), laminin, and polyornithine. A process for producing myelinating oligodendrocyte cells from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. Bibel, et al., 2007, Nat. Protoc. 2:1034-43, describes a protocol for producing glutamatergic neurons from stem cells, while Chatzi, et al., 2009, Exp. Neurol. 217:407-16, describes a procedure for producing GABAergic neurons. This procedure includes exposing stem cells to all-trans RA for 3 days. After subsequent culture in serum-free neuronal induction medium containing Neurobasal medium supplemented with B27, bFGF, and EGF, up to 95% GABA neurons were obtained.

[0115] US Publication No. 2012 / 0329714 describes the use of prolactin to increase neural stem cell numbers, and US Publication No. 2012 / 0308530 describes culture surfaces with amino groups that promote neuronal differentiation into neurons, astrocytes, and oligodendrocytes. Thus, the fate of neural stem cells can be controlled by a variety of extracellular factors. Commonly used factors include brain-derived growth factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425); fibroblast growth factor (bFGF; U.S. Patent No. 5,766,948; FGF-1, FGF-2); neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4); Caldwell, et al. al., 2001, Nat. Biotechnol. 1;19:475-9); ciliary neurotrophic factor (CNTF); BMP-2 (U.S. Pat. Nos. 5,948,428 and 6,001,654); isobutyl 3-methylxanthine; leukemia inhibitory growth factor (LIF; U.S. Pat. No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (a glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Pat. No. 6,395,546); tetanus toxin; and transforming growth factor-α and TGF-β (U.S. Pat. Nos. 5,851,832 and 5,753,506).

[0116] In certain embodiments, the yeast one-hybrid system may also be used to identify compounds that inhibit specific protein / DNA interactions, such as transcription factors, of the I56i enhancer, its core, or its linked cores and one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores.

[0117] Transgenic animals are described below. Cell lines can also be derived from such transgenic animals. For example, primary tissue cultures from transgenic mice (e.g., as described below) can provide cell lines that have artificial expression constructs already integrated into their genome (see, e.g., MacKenzie and Quinn, Proc Natl Acad Sci USA 96:15251-15255, 1999).

[0118] (iv) Transgenic Animals. Another aspect of the present disclosure includes a transgenic animal, the genome of which includes an I56i enhancer, a core thereof, or a linked core thereof, operably linked to a heterologous coding sequence, and one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores. In certain embodiments, the genome of the transgenic animal is selected from the group consisting of CN2720, CN2721, CN2722, CN2732, CN3213, CN3322, CN3323, CN3887, CN3888, CN2972, CN2973, CN2974, CN2975, CN2976, ID10.01, ID10.02, ID10.03, ID10.04, ID10.05, ID10.06, ID10.07, ID10.08, ID10.09, ID10.10, ID10.11, ID10.12, ID10.13, ID10.14, ID10.15, ID10.16, ID10.17, ID10.18, ID10.19, ID10.20, ID10.21, ID10.22, ID10.23, ID10.24, ID10.25, ID10.26, ID10.27, ID10.28, ID10.29, ID10.30, ID10.31, ID10.32, ID10.33, ID10.34, ID10.35, ID10.36, ID10.37, ID10.38, ID10.39, ID10.40, ID10.41, ID10.42, ID10.43, ID10.44, ID10.45, ID10.46, ID10.47, ID10.48, ID10.49, ID10.50, ID10.51, ID10.52, ID10.53, ID10.54, ID10.55, ID10.56, ID10 D10.10, ID10.11, ID10.12, ID10.13, ID10.14, ID10.15, ID10.16, ID10.17, ID10.18, ID10.19, ID10.20, ID10.21, ID10 .22, ID10.23, ID10.24, ID10.25, ID10.26, ID10.27, ID10.28, ID10.29, ID10.30, ID10.31, ID10.32, ID11.01, ID11.02, ID11.03, ID11.04, ID11.05, ID11.06, ID11.07, ID11.08, ID11.09, ID11.10, ID11.11, ID11.12, ID11.13, ID11.14, ID1 1.15, ID11.16, ID12.01, ID12.02, ID12.03, ID12.04, ID12.05, ID12.06, ID12.07, ID12.08, ID12.09, ID12.10, ID12.1 1, ID12.12, ID12.13, ID12.14, ID12.15, ID12.16, ID13.01, ID13.02, ID13.03, ID13.04, ID13.05, ID13.06, ID13.07, I D13.08, ID13.09, ID13.10, ID13.11, ID13.12, ID13.13, ID13.14, ID13.15, ID13.16, ID14.01, ID14.02, ID14.03, ID14.04、ID14.05、ID14.06、ID14.07、ID14.08、ID14.09、ID14.10、ID14.11、ID14.12、ID14.13、ID14.14、ID14.15、ID14.16、ID15.01、ID15.02、ID15.03、ID15.04、ID15.05、ID15.06、ID15.07、ID15.08、ID15.09、ID15.10、ID15.11、ID15.12、ID15.13、ID15.14、ID15.15、ID15.16、ID16.01、ID16.02、ID16.03、ID16.04、ID16.05、ID16.06、ID16.07、ID16.08、ID16.09、ID16.10、ID16.11、ID16.12、ID16.13、ID16.14、ID16.15、ID16.16、ID17.01、ID17.02、ID17.03、ID17.04、ID17.05、ID17.06、ID17.07、ID17.08、ID17.09、ID17.10、ID17.11、ID17.12、ID17.13、ID17.14、ID17.15、ID17.16、ID18.01、ID18.02、ID18.03、ID18.04、ID18.05、ID18.06、ID18.07、ID18.08、ID18.09、ID18.10、ID18.11、ID18.12、ID18.13、ID18.14、ID18.15、ID18.16、ID19.01、ID19.02、ID19.03、ID19.04、ID19.05、ID19.06、ID19.07、ID19.08、ID19.09、ID19.10、ID19.11、ID19.12、ID19.13、ID19.14、ID19.15、ID19.16、ID20.01、ID20.02、ID20.03、ID20.04、ID20.05、ID20.06、ID20.07、ID20.08、ID20.09、ID20.10、ID20.11、ID20.12、ID20.13、ID20.14、ID20.15、ID20.16、ID21.01、ID21.02、ID21.03、ID21.04、ID21.05、ID21.06、ID21.07、ID21.08、ID21.09、ID21.10、ID21.11、ID21.12、ID21.13、ID21.14、ID21.15、ID21.16、ID22.01, ID22.02, ID22.03, ID22.04, ID22.05, ID22.06, ID22.07, ID22.08, ID22.09, ID22.10, ID22.11, ID22.12, ID22 .13, ID22.14, ID22.15, ID22.16, ID23.01, ID23.02, ID23.03, ID23.04, ID23.05, ID23.06, ID23.07, ID23.08, ID2 3.09, ID23.10, ID23.11, ID23.12, ID23.13, ID23.14, ID23.15, ID23.16, ID24.01, ID24.02, ID24.03, ID24.04, ID 24.05, ID24.06, ID24.07, ID24.08, ID24.09, ID24.10, ID24.11, ID24.12, ID24.13, ID24.14, ID24.15, ID24.16, ID 25.01, ID25.02, ID25.03, ID25.04, ID25.05, ID25.06, ID25.07, ID25.08, ID25.09, ID25.10, ID25.11, ID25.12, I D25.13, ID25.14, ID25.15, ID25.16, ID26.01, ID26.02, ID26.03, ID26.04, ID26.05, ID26.06, ID26.07, ID26.08, I D26.09, ID26.10, ID26.11, ID26.12, ID26.13, ID26.14, ID26.15, ID26.16, ID27.01, ID27.02, ID27.03, ID27.04, ID27.05, ID27.06, ID27.07, ID27.08, ID27.09, ID27.10, ID27.11, ID27.12, ID27.13, ID27.14, ID27.15, or ID27.In certain embodiments, when a non-integrating vector is utilized, the transgenic animal contains within one or more of its cells an I56i enhancer, its core, or linked cores thereof, as well as one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores, and / or CN2720, CN2721, CN2722, CN2732, CN3213,. CN3322、CN3323、CN3887、CN3888、CN2972、CN2973、CN2974、CN2975、CN2976、ID10.01、ID10.02、ID10.03、ID10.04、ID10.05、ID10.06、ID10.07、ID10. 08、ID10.09、ID10.10、ID10.11、ID10.12、ID10.13、ID10.14、ID10.15、ID10.16、ID10.17、ID10.18、ID10.19、ID10.20、ID10.21、ID10.22、ID10.23、ID 10.24、ID10.25、ID10.26、ID10.27、ID10.28、ID10.29、ID10.30、ID10.31、ID10.32、ID11.01、ID11.02、ID11.03、ID11.04、ID11.05、ID11.06、ID11.07、ID11.08、ID11.09、ID11.10、ID11.11、ID11.12、ID11.13、ID11.14、ID11.15、ID11.16、ID12.01、ID12.02、ID12.03、ID12.04、ID12.05、ID12.06、ID1 2.07、ID12.08、ID12.09、ID12.10、ID12.11、ID12.12、ID12.13、ID12.14、ID12.15、ID12.16、ID13.01、ID13.02、ID13.03、ID13.04、ID13.05、ID13.06 、ID13.07、ID13.08、ID13.09、ID13.10、ID13.11、ID13.12、ID13.13、ID13.14、ID13.15、ID13.16、ID14.01、ID14.02、ID14.03、ID14.04、ID14.05、ID14 .06、ID14.07、ID14.08、ID14.09、ID14.10、ID14.11、ID14.12、ID14.13、ID14.14、ID14.15、ID14.16、ID15.01、ID15.02、ID15.03、ID15.04、ID15.05、 ID15.06、ID15.07、ID15.08、ID15.09、ID15.10、ID15.11、ID15.12、ID15.13、ID15.14、ID15.15、ID15.16、ID16.01、ID16.02、ID16.03、ID16.04、ID16.05、ID16.06、ID16.07、ID16.08、ID16.09、ID16.10、ID16.11、ID16.12、ID16.13、ID16.14、ID16.15、ID16.16、ID17.01、ID17.02、ID17.03、ID17.04、ID17.05、ID17.06、ID17.07、ID17.08、ID17.09、ID17.10、ID17.11、ID17.12、ID17.13、ID17.14、ID17.15、ID17.16、ID18.01、ID18.02、ID18.03、ID18.04、ID18.05、ID18.06、ID18.07、ID18.08、ID18.09、ID18.10、ID18.11、ID18.12、ID18.13、ID18.14、ID18.15、ID18.16、ID19.01、ID19.02、ID19.03、ID19.04、ID19.05、ID19.06、ID19.07、ID19.08、ID19.09、ID19.10、ID19.11、ID19.12、ID19.13、ID19.14、ID19.15、ID19.16、ID20.01、ID20.02、ID20.03、ID20.04、ID20.05、ID20.06、ID20.07、ID20.08、ID20.09、ID20.10、ID20.11、ID20.12、ID20.13、ID20.14、ID20.15、ID20.16、ID21.01、ID21.02、ID21.03、ID21.04、ID21.05、ID21.06、ID21.07、ID21.08、ID21.09、ID21.10、ID21.11、ID21.12、ID21.13、ID21.14、ID21.15、ID21.16、ID22.01、ID22.02、ID22.03、ID22.04、ID22.05、ID22.06、ID22.07、ID22.08、ID22.09、ID22.10、ID22.11、ID22.12、ID22.13、ID22.14、ID22.15、ID22.16、ID23.01、ID23.02、ID23.03、ID23.04、ID23.05、ID23.06、ID23.07、ID23.08、ID23.09、ID23.10、ID23.11、ID23.12、ID23.13、ID23.14、ID23.15、ID23.16、ID24.01、ID24.02, ID24.03, ID24.04, ID24.05, ID24.06, ID24.07, ID24.08, ID24.09, ID24.10, ID24.11, ID24.12, ID24.13, ID24.14, ID24.15, ID24.16, ID25.01, ID25. 02, ID25.03, ID25.04, ID25.05, ID25.06, ID25.07, ID25.08, ID25.09, ID25.10, ID25.11, ID25.12, ID25.13, ID25.14, ID25.15, ID25.16, ID26.01, ID26.0 2, ID26.03, ID26.04, ID26.05, ID26.06, ID26.07, ID26.08, ID26.09, ID26.10, ID26.11, ID26.12, ID26.13, ID26.14, ID26.15, ID26.16, ID27.01, ID27.02, ID27.03, ID27.04, ID27.05, ID27.06, ID27.07, ID27.08, ID27.09, ID27.10, ID27.11, ID27.12, ID27.13, ID27.14, ID27.15, or ID27.16.

[0119] Detailed methods for producing transgenic animals are described in U.S. Patent No. 4,736, 866. The transgenic animals can be any non-human species, but preferably include non-human primates (NHPs), sheep, horses, cows, pigs, goats, dogs, cats, rabbits, chickens, and rodents, such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.

[0120] In certain embodiments, the construction of transgenic animals results in organisms with genetically engineered constructs present in all cells in the same genomic integration site.Therefore, cell lines derived from such transgenic animals will be as consistent as genetically engineered constructs in all cells in the same genomic integration site, and therefore will undergo the same position effect mutations.In contrast, introducing genes into cell lines or primary cell cultures can result in heterologous expression of constructs.The drawback of this approach is that the expression of introduced DNA can be affected by the specific genetic background of the host animal.

[0121] As indicated above in relation to cell lines, the artificial expression constructs of the present disclosure can be used to genetically modify mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression constructs are introduced into cultured mouse embryonic stem cells. The transformed ES cells are then injected into a blastocyst from a host mother, and the host embryo is reimplanted into the mother. This results in chimeric mice whose tissues are composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Usually, the mouse from which the cultured ES cells used for the transfer are derived is selected to have a coat color that is different from the host mouse whose embryo the transformed cells are injected into. The chimeric mice then have a variety of coat colors. The chimeric mice are bred with the appropriate strain to produce offspring that carry the transgene, at least in part, as long as the germline tissue is derived from the genetically modified cells.

[0122] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering artificial expression constructs to target cells or targeted tissues and organs of animals, particularly cells, organs, or tissues of vertebrates: ultrasound (e.g., ultrasound as described in U.S. Pat. No. 5,656,016); intraosseous injection (U.S. Pat. No. 5,779,708); microchip devices (U.S. Pat. No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208); feedback controlled delivery (U.S. Pat. No. 5,697,899), and other delivery methods available and / or described elsewhere in this disclosure.

[0123] (v) Methods of Use. In certain embodiments, a composition comprising a bioactive ingredient described herein is administered to a subject to achieve a physiological effect.

[0124] In certain embodiments, the disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a heterologous gene partially or completely encoded at a position downstream of an enhancer in an engineered sequence. Accordingly, methods of using the disclosed artificial expression constructs in the research, clinical trials, and potential development of pharmaceuticals to prevent, treat, or alleviate symptoms of a disease, dysfunction, or disorder are provided herein.

[0125] Certain embodiments include the I56i enhancer, its core, or linked cores described herein, as well as one or more enhancers selected from eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_387m, eHGT_390m, and their cores, and / or CN2720, CN2721, CN2722, CN2732, CN3213, CN3322, CN3323, CN3887, CN3888, CN2972, CN2973, CN2974, CN29 75, CN2976, ID10.01, ID10.02, ID10.03, ID10.04, ID10.05, ID10.06, ID10.07, ID10.08, ID10.09, ID10.10, ID10.11, ID10.12, ID10.13, ID10.14, ID1 0.15, ID10.16, ID10.17, ID10.18, ID10.19, ID10.20, ID10.21, ID10.22, ID10.23, ID10.24, ID10.25, ID10.26, ID10.27, ID10.28, ID10.29, ID10.30, ID10.31, ID10.32, ID11.01, ID11.02, ID11.03, ID11.04, ID11.05, ID11.06, ID11.07, ID11.08, ID11.09, ID11.10, ID11.11, ID11.12, ID11.13, ID11. 14, ID11.15, ID11.16, ID12.01, ID12.02, ID12.03, ID12.04, ID12.05, ID12.06, ID12.07, ID12.08, ID12.09, ID12.10, ID12.11, ID12.12, ID12.13, ID 12.14, ID12.15, ID12.16, ID13.01, ID13.02, ID13.03, ID13.04, ID13.05, ID13.06, ID13.07, ID13.08, ID13.09, ID13.10, ID13.11, ID13.12, ID13.13 , ID13.14, ID13.15, ID13.16, ID14.01, ID14.02, ID14.03, ID14.04, ID14.05, ID14.06, ID14.07, ID14.08, ID14.09, ID14.10, ID14.11, ID14.12, ID14.13、ID14.14、ID14.15、ID14.16、ID15.01、ID15.02、ID15.03、ID15.04、ID15.05、ID15.06、ID15.07、ID15.08、ID15.09、ID15.10、ID15.11、ID15.12、ID15.13、ID15.14、ID15.15、ID15.16、ID16.01、ID16.02、ID16.03、ID16.04、ID16.05、ID16.06、ID16.07、ID16.08、ID16.09、ID16.10、ID16.11、ID16.12、ID16.13、ID16.14、ID16.15、ID16.16、ID17.01、ID17.02、ID17.03、ID17.04、ID17.05、ID17.06、ID17.07、ID17.08、ID17.09、ID17.10、ID17.11、ID17.12、ID17.13、ID17.14、ID17.15、ID17.16、ID18.01、ID18.02、ID18.03、ID18.04、ID18.05、ID18.06、ID18.07、ID18.08、ID18.09、ID18.10、ID18.11、ID18.12、ID18.13、ID18.14、ID18.15、ID18.16、ID19.01、ID19.02、ID19.03、ID19.04、ID19.05、ID19.06、ID19.07、ID19.08、ID19.09、ID19.10、ID19.11、ID19.12、ID19.13、ID19.14、ID19.15、ID19.16、ID20.01、ID20.02、ID20.03、ID20.04、ID20.05、ID20.06、ID20.07、ID20.08、ID20.09、ID20.10、ID20.11、ID20.12、ID20.13、ID20.14、ID20.15、ID20.16、ID21.01、ID21.02、ID21.03、ID21.04、ID21.05、ID21.06、ID21.07、ID21.08、ID21.09、ID21.10、ID21.11、ID21.12、ID21.13、ID21.14、ID21.15、ID21.16、ID22.01、ID22.02、ID22.03、ID22.04、ID22.05、ID22.06、ID22.07、ID22.08、ID22.09、ID22.10, ID22.11, ID22.12, ID22.13, ID22.14, ID22.15, ID22.16, ID23.01, ID23.02, ID23.03, ID23.04, ID23.05, ID2 3.06, ID23.07, ID23.08, ID23.09, ID23.10, ID23.11, ID23.12, ID23.13, ID23.14, ID23.15, ID23.16, ID24.01, I D24.02, ID24.03, ID24.04, ID24.05, ID24.06, ID24.07, ID24.08, ID24.09, ID24.10, ID24.11, ID24.12, ID24.13 , ID24.14, ID24.15, ID24.16, ID25.01, ID25.02, ID25.03, ID25.04, ID25.05, ID25.06, ID25.07, ID25.08, ID25. 09, ID25.10, ID25.11, ID25.12, ID25.13, ID25.14, ID25.15, ID25.16, ID26.01, ID26.02, ID26.03, ID26.04, ID2 6.05, ID26.06, ID26.07, ID26.08, ID26.09, ID26.10, ID26.11, ID26.12, ID26.13, ID26.14, ID26.15, ID26.16, I The present invention includes a method of administering an artificial expression construct comprising D27.01, ID27.02, ID27.03, ID27.04, ID27.05, ID27.06, ID27.07, ID27.08, ID27.09, ID27.10, ID27.11, ID27.12, ID27.13, ID27.14, ID27.15, or ID27.16 to a subject to drive expression of genes in GABAergic neurons and astrocytes. The subject may be an isolated cell, a network of cells, a tissue slice, a laboratory animal, a veterinary animal, or a human.

[0126] As is well known in the medical arts, dosage for any subject will depend on many factors, including the subject's size, surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. Dosages for the compounds of the present disclosure may vary, but in certain embodiments, dosages are within the range of 10-20 mg / kg of the artificial expression construct of the present disclosure.5 ~10 100 In certain embodiments, patients receiving intravenous, intraparenchymal, intraspinal, retroorbital, or intrathecal administration receive 10 copies. 6 ~10 22 One copy of the artificial expression construct can be injected.

[0127] Treating a subject includes delivering a therapeutically effective amount, including an amount that provides effective, prophylactic and / or therapeutic treatment.

[0128] "Effective amount" refers to the amount of composition required to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amounts disclosed herein may cause statistically significant effects in animal models or in vitro assays related to the evaluation of the onset, progression, and / or resolution of SLC6A1-associated disorders.

[0129] "Prophylactic treatment" includes treatment administered to a subject who does not show signs or symptoms of an SLC6A1-related disorder or who shows only early signs or symptoms of an SLC6A1-related disorder, and the treatment is administered for the purpose of further reducing or decreasing the risk of developing an SLC6A1-related disorder. Thus, prophylactic treatment functions as a preventative treatment against an SLC6A1-related disorder. In certain embodiments, prophylactic treatment reduces, delays, or prevents the worsening of an SLC6A1-related disorder.

[0130] "Therapeutic treatment" includes treatment administered to a subject exhibiting symptoms or signs of an SLC6A1-related disorder and for the purpose of reducing or eliminating those signs or symptoms of an SLC6A1-related disorder. Therapeutic treatment can reduce, control, or eliminate the presence or activity of an SLC6A1-related disorder and / or reduce or eliminate the side effects of an SLC6A1-related disorder.

[0131] The functions of an effective amount, prophylactic or therapeutic treatment, are not mutually exclusive, and in certain embodiments, an administered dosage may achieve more than one type of treatment.

[0132] In certain embodiments, methods for determining the efficacy of treatment with the constructs disclosed herein include measurements taken before treatment, during the first year after treatment, and at other time points, In certain embodiments, the efficacy of treatment with the constructs disclosed herein is determined to be effective if the measured values ​​assessed remain at normal levels or are less severe than previous levels of impairment.

[0133] Therapeutically effective amounts can be assessed using developmental tests for cognitive and motor function. Those skilled in the art will be aware of appropriate conditions for assessing cognitive function, which may include a variety of commonly used tests. Representative tests include the Continuous Performance Test (CPT), Wisconsin Card Sorting Test, Trailmaking A+B, Mini Mental State Exam (MMSE), List Learning (verbal memory), Digit Sequencing Task (working memory), Token Motor Task (motor speed), Category Instances (semantic fluency), Controlled Oral Word Association Test (literal fluency), Tower of London Test (executive function), Symbol Coding (attention and motor speed), Affective Interference Test-Delayed Recognition Task, Stroop Test, Brief Assessment of Cognition in Schizophrenia, and the MMSE. These include neuropsychological tests such as the Assessment and Cognitive Functioning in Schizophrenia (BACS; which includes many of the tests mentioned above), the Measurement and Treatment Research to Improve Cognition in Schizophrenia battery (MATRICS), and tests included in the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog).

[0134] In certain embodiments, the method for determining the effectiveness of treatment for mild to moderate intellectual disability can be measured by observing the positive change in the clinical symptoms of the patient.The classification of intellectual disability can be determined using the American Association on Intellectual and Developmental Disabilities (AAIDD) or the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5), published by the American Psychiatric Association (Committee to Evaluate the Supplemental Security Income Disability Program for Children with Mental Disorders; Board on the Health of Select Populations; Board on Children, Youth, and Families; Institute of Medicine; Division of Behavioral and Social Sciences and Education; The National Academies of Sciences, Engineering, and Medicine; Boat TF, Wu JT, editors. Mental Disorders and Disabilities Among Low-Income Children. Washington (DC): National Academies Press (US); 2015 Oct 28.9, Clinical Characteristics of Intellectual Disabilities). In certain embodiments, the effectiveness of treatment of intellectual disability can be measured using IQ, severity based on daily living skills, severity based on the intensity of support needed, or the SSI list criteria. The SSI list does not specify severity levels, but rather describes criteria commensurate with the listed levels of severity.

[0135] In certain aspects, the method of determining the effectiveness of the treatment of epilepsy may be the therapeutic effect on the reduction or prevention of seizures. In certain embodiments, the provided methods may reduce or prevent one or more different types of seizures. Ideally, the disclosed methods prevent seizures completely. However, the disclosure also includes methods in which the incidence of seizures is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0136] In general, seizures may include convulsions, repetitive movements, abnormal sensations, and combinations thereof. Seizures can be classified as focal seizures (also called partial seizures) and generalized seizures. Focal seizures affect only one side of the brain, while generalized seizures affect both sides of the brain. Specific types of focal seizures include simple focal seizures, complex focal seizures, and secondarily generalized seizures. Simple focal seizures are restricted or concentrated to a particular lobe (e.g., the temporal, frontal, parietal, or occipital lobe). Complex focal seizures generally affect a larger portion of the brain hemisphere than simple focal seizures, but typically occur in the temporal or frontal lobe. When simple focal seizures extend from one side (hemisphere) of the brain to both sides, the seizure is called a secondarily generalized seizure. Specific types of generalized seizures include absence (also called petit mal), tonic, atonic, myoclonic, tonic-clonic (also called grand mal), and clonic seizures.

[0137] In certain embodiments, the methods described herein may reduce the frequency of seizures, reduce the severity of seizures, change the type of seizures (e.g., from a more severe type to a less severe type), or a combination thereof, in a patient after treatment compared to no treatment (e.g., before treatment) or compared to treatment with another conventional therapy.

[0138] In certain embodiments, the method of determining the effectiveness of a treatment for speech difficulties may include the use of a speech assessment. In certain embodiments, a speech pathologist evaluates the patient's speech production. Assessment tools include the Diadochokinetic (DDK) rate, which measures repetitive sounds within a set period of time; the Motor ABC test, the Beery-Buktenica Developmental Test of Visual-Motor Coordination (Beery VMI); MRI, CT, or EMG tests; the Voice Handicap Index (VHI); the Frenchay Dysarthria Assessment (FDA); the Radbound Dysarthria Assessment (RDA); oral motor tests; and other speech and language tests.

[0139] In certain embodiments, the method for determining the effectiveness of a treatment for a behavioral disorder may include improving at least one clinical symptom and / or at least one physical parameter associated with the behavioral disorder. In certain embodiments, the behavioral disorder may include Attention Deficit Disorder (ADD) or Attention Deficit Hyperactivity Disorder (ADHD). Effective treatment results in an improvement in the patient's ADHD Rating Scale-IV (ARS-IV), ADHD Self-Report Scale (ASRS), Clinical Global Impression (CGI), and / or cognitive function.

[0140] The ADHD Rating Scale IV (ARS-IV) assesses the following behaviors: 1. Trouble paying close attention to details or making careless mistakes on tasks; 2. Keeping hands and feet moving or squirming when seated; 3. Difficulty maintaining attention during a task or play; 4. Leaving seat when required to be seated; 5. Not seeming to listen when spoken to directly; 6. Excessive running or climbing inappropriately; 7. Not following directions or being unable to complete a task; 8. Difficulty playing quietly or participating in leisure activities; 9. Difficulty organizing tasks and activities; 10. "Running" or acting as if "motorized"; 11. Avoiding tasks that require sustained mental effort; 12. Talking excessively; 13. Forgetting what is needed for a task or activity; 14. Blurring answers before the question is complete; 15. Easily distracted; 16. Difficulty waiting for turns. 17. Often forgets things in daily activities. 18. Interrupts or intrudes on other people's conversations.

[0141] Kessler et al (Psychological Medicine,35:245-256,2005) report the WHO Adult ADHD Self-Report Scale (ASRS) for the general public. The ASRS Symptom Checklist is a self-report questionnaire used to aid in the diagnosis of adult ADHD.

[0142] The Clinical Global Impression-Improvement scale (CGI-I) is a 7-point scale that asks clinicians to rate the extent to which the patient's illness has improved or worsened compared with the baseline state at the start of the intervention: 1, very significantly improved; 2, significantly improved; 3, slightly improved; 4, not improved; 5, slightly worse; 6, significantly worse; or 7, very significantly worse.

[0143] In certain embodiments, the efficacy of treatment in autism spectrum disorder may be evaluated. A variety of standardized assessment schemes are available for monitoring the course, severity, and spectrum of impairment in patients with autism spectrum disorder or patients suspected of being at risk for autism spectrum disorder. Such schemes may also be used to evaluate the evolution of autism symptoms over time or response to treatment. Among these, the Autism Diagnostic Observation Schedule (ADOS-2, its latest version, described in Gotham et al. J Autism Dev Disord. 2007 Apr;37(4):613-27) has various modules with explanations related to the developmental level and age of the patient, making it reasonably useful for patients of a wide age range. It includes standardized administration of examiner-initiated interactive activities, which are designed to elicit social interaction, communication, and repetitive behaviors for the purpose of diagnosing autism spectrum disorder, and includes procedures optimized for patients from under 48 months to adulthood. Also useful in assessing communication disorders in autism spectrum disorders is the Expressive One Word Picture Vocabulary Test (EOWPVT), which assesses language expression and vocabulary and language production abilities (Chapman et al. Early Hum Dev. 2015 Jun;91(6):373-379.). Additional measures that can be used to measure improvement in ASD patients include the caregiver-administered Aberrant Behavior Checklist (ABC, see e.g., Kaat et al. J Autism Dev Disord. 2014 May;44(5):1 103-16.), and the Autism Treatment Evaluation Checklist (ATEC, see e.g., Geier et al. Mental Health Research in Intellectual Disabilities 2013;6:255-67).In addition, a modified version of the Clinical Global Impressions scale may be used to assess patient progress.

[0144] In certain embodiments, the method of determining the effectiveness of a treatment for a neurological symptom may include a procedure that improves the patient's symptoms or otherwise alleviates, relieves, or minimizes an undesirable condition. In certain embodiments, the neurological symptom may include ataxia, hypotonia, and other movement disorders. In certain embodiments, changes in muscle tone, strength, reflexes, hyperflexibility, posture, endurance, MRI, CT, EMG, or EEG scans may be assessed to measure effective treatment of hypotonia. In certain embodiments, evaluation of writing and feeding skills, eye movement, gait, balance and coordination, speech, MRI, or CT scans may be used to measure effective treatment of ataxia.

[0145] Numerous movement disorders affect infants and children, and in this context, numerous movement and developmental tests can be used to assess therapeutic effectiveness, such as the Peabody Developmental Motor Scale (PDMS-II), the Alberta Infant Motor Scale (AIMS), the Bayley Scales of Infant and Toddler Development® - Third Edition (Bayley-III), or the Comprehensive Developmental Inventory for Infants and Toddlers (CDIIT).

[0146] The PDMS-II is a performance-based measure of gross and fine motor development for infants and children from birth to 5 years of age. The tool separates motor development into gross motor skills and fine motor skills. The combination of gross and fine motor skills composite scores allows the examiner to have a reliable estimate of a child's motor abilities. It includes four gross and two fine motor subtests: Reflexes (gross motor); Steadiness (gross motor); Locomotion (gross motor); Object Manipulation (gross motor); Grasping (fine motor); and Visual-Motor Integration (fine motor).

[0147] Scoring the PDMS-II depends on raw scores, percentiles, standard scores, and age equivalents for the subtests, as well as component allocations. Raw scores are the total points a child accumulates on the subtests. Developmental ages are often used to communicate information to parents of young children. Age equivalents for the PDMS-II are referred to as "motor ages" and inform parents that their children are "passing" items that children of a particular age typically pass. Age equivalents for the PDMS-II subtests are found in Table C.1 of the PDMS-II manual or are generated by the PDMS-II software scoring and reporting system.

[0148] AIMS is a 58-item observational measure of infant motor skills used from birth to the age of independent walking (18 months). It assesses the sequential development of motor goals in terms of the progressive development and consolidation of antigravity muscle control. The test assesses infant movement in four positions: prone, supine, sitting, and standing. The AIMS total score is calculated by summing the scores of the 58 items, with a range of scores from 0 to 58. Higher scores indicate more mature motor development. Infant scores can then be converted to percentiles and compared with age-matched peers from a normative sample.

[0149] The Bayley-III provides a standardized assessment of cognitive and motor development for children aged 1-42 months. The assessment measures development in cognitive, communication, physical, social / emotional, and adaptive domains to identify children with developmental delays. The test includes five developmental scales: cognitive, language, motor, social-emotional, and adaptive behavior. It is possible to present developmental age vs. chronological age results for each subscale.

[0150] The CDIIT diagnostic test is a child development test and includes five developmental subtests for use with children aged 3 to 72 months.

[0151] The amount of expression constructs of such compositions and the time of administration will be within the purview of one of ordinary skill in the art having the benefit of the present teachings. However, it is likely that administration of an effective amount of the disclosed compositions can be achieved by a single administration, such as, for example, a single injection of a sufficient number of infectious particles to produce an effect in a subject. Alternatively, in some situations, it may be desirable to provide multiple or successive administrations of an artificial expression construct composition or other genetic construct over either a relatively short or relatively long period of time, as may be determined by the individual overseeing the administration of such compositions. For example, the number of infectious particles administered to a mammal may be as little as 10 to 20 mg / kg, given either as a single dose or divided into two or more administrations, as may be required to achieve the intended effect. 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or even higher infectious particles / ml. Indeed, in certain embodiments, it may be desirable to administer two or more different expression constructs in combination to achieve a desired effect.

[0152] In certain circumstances, it may be desirable to deliver the artificial expression construct in an appropriately formulated composition as disclosed herein, either by pipette, retro-orbital injection, subcutaneous, intraocular, intravitreal, parenteral, subcutaneous, intravenous, intraparenchymal, intraventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or by direct application or injection into one or more cells, tissues, or organs. Methods of administration may also include those forms as described in U.S. Patent No. 5,543,158, U.S. Patent No. 5,641,515, and U.S. Patent No. 5,399,363.

[0153] (vi) Kits and commercial packages. Kits and commercial packages contain the artificial expression constructs described herein. The artificial expression constructs can be sequestered. In certain embodiments, the components of the expression product can be sequestered from one another. In certain embodiments, the expression product can be in a vector, in a viral vector, in a cell, in a tissue slice or sample, and / or in a transgenic animal. Such kits can further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or a means of delivery of the composition, such as a syringe, injection, etc.

[0154] Kit or commercial package embodiments also include instructions for use of the included components, for example, in basic research, electrophysiological studies, neuroanatomical studies, and / or in the study and / or treatment of a disorder, disease or condition.

[0155] The following illustrative embodiments and experimental examples are included to demonstrate specific embodiments of the present disclosure. Those of skill in the art will appreciate in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain like or similar results without departing from the spirit and scope of the present disclosure.

[0156] (vii) Exemplary embodiments. 1. An artificial expression construct comprising: (i) a first enhancer comprising the core of the I56i enhancer; (ii) a second enhancer comprising one or more of eHGT_387m, eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_390m, or their cores; (iii) a promoter; and (iv) a heterologous coding sequence. 2. The artificial expression construct of embodiment 1, wherein said first enhancer is adjacent to said second enhancer. 3. The artificial expression construct of embodiment 1, wherein said first enhancer is not adjacent to said second enhancer. 4. An artificial expression construct according to any of embodiments 1 to 3, wherein said first enhancer is 5' to said second enhancer. 5. An artificial expression construct according to any of embodiments 1 to 3, wherein said second enhancer is 5' to said first enhancer. 6. An artificial expression construct according to any one of embodiments 1 to 5, wherein said core of said I56i enhancer is an I56i human core or an I56i zebrafish core. 7. The artificial expression construct according to embodiment 6, wherein said core comprises a sequence set forth in SEQ ID NO: 4 or 5, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4 or 5. 8. An artificial expression construct according to any one of embodiments 1 to 7, in which the cores of the I56i enhancer are linked. 9. The artificial expression construct of embodiment 8, wherein the linked core of the I56i enhancer has 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the I56i human core or the I56i zebrafish core. 10. An artificial expression construct according to embodiment 9, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 4 and / or SEQ ID NO: 5, or comprising a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4 or 5. 11. An artificial expression construct according to embodiment 9, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO:4 or a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:4. 12. An artificial expression construct according to embodiment 9, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO:5, or comprising a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:5. 13. An artificial expression construct according to embodiment 9, comprising three copies of SEQ ID NO:4 or three copies of a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:4. 14. An artificial expression construct according to embodiment 9, comprising three copies of SEQ ID NO:5 or three copies of a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:5. 15. An artificial expression construct according to embodiment 8, wherein the linkage core of the I56i enhancer comprises the sequence set forth in SEQ ID NO:6 or comprises a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:6. 16. The artificial expression construct according to embodiment 8, wherein the linkage core of the I56i enhancer comprises the sequence set forth in SEQ ID NO:7 or comprises a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:7. 17. An artificial expression construct according to embodiment 8, wherein the second enhancer comprises eHGT_387m or eHGT_390m or a sequence having at least 90% sequence identity with the sequence of eHGT_387m or eHGT_390m. 18. An artificial expression construct according to any of the preceding embodiments, wherein the second enhancer is the core of an enhancer selected from eHGT_387m, eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, or eHGT_390m. 19. An artificial expression construct according to any one of the preceding embodiments, wherein the second enhancer is a core of eHGT_387m or eHGT_390m or a sequence having at least 90% sequence identity with the sequence of the core of eHGT_387m or eHGT_390m. 20. An artificial expression construct according to embodiment 19, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 84, or comprising a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 84. 21. An artificial expression construct according to embodiment 19 or 20, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 85, or comprising a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 85. 22. An artificial expression construct according to any of the preceding embodiments, wherein said second enhancer core is linked to said core of said I56i enhancer to generate a combined linked enhancer. 23. The artificial expression construct of embodiment 22, wherein the combinatorial linked enhancer comprises a sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86. 24. The artificial expression construct of embodiment 22 or 23, wherein the combined linked enhancer has 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the core of the second enhancer and 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the core of the I56i enhancer. 25. An artificial expression construct according to any of embodiments 22 to 24, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of said combinatorially linked enhancer. 26. An artificial expression construct according to any of embodiments 22 to 25, wherein the combined linked enhancer comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of eHGT_387m(core2); 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of eHGT_390m(core2); and / or 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the core of the I56i enhancer. 27. An artificial expression construct according to any of embodiments 22 to 26, wherein the combinatorial linked enhancer comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86. 28. An artificial expression construct according to any one of embodiments 22 to 27, wherein the combinatorial linked enhancer comprises three copies of the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86. 29. An artificial expression construct according to any one of embodiments 22 to 28, wherein the combinatorial linked enhancer comprises a sequence as set forth in SEQ ID NO: 88 or SEQ ID NO: 89, or a sequence having at least 90% sequence identity with the sequence as set forth in SEQ ID NO: 88 or SEQ ID NO: 89. 30. An artificial expression construct according to any of embodiments 1 to 29, wherein the heterologous coding sequence encodes GAT1. 31. The artificial expression construct of embodiment 30, wherein the heterologous coding sequence is a codon-optimized SLC6A1 gene. 32. The artificial expression construct according to embodiment 31, wherein the heterologous coding sequence has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 22, 25, 28, 31, 34, 38, 39, 40, 41, 42, 43, 44 or 45. 33. An artificial expression construct according to embodiment 31 or 32, wherein the heterologous coding sequence has a sequence according to SEQ ID NO: 22, 25, 28, 31, 34, 38, 39, 40, 41, 42, 43, 44 or 45. 34. An artificial expression construct according to any of the preceding embodiments, wherein the heterologous coding sequence encodes an effector element or an expressible element. 35. The artificial expression construct of embodiment 34, wherein the effector element comprises a reporter protein or a functional molecule. 36. The artificial expression construct of embodiment 35, wherein the reporter protein comprises a fluorescent protein. 37. The artificial expression construct of embodiment 35, wherein the functional molecule comprises a functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signal transduction molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a microRNA, a homologous recombination donor cassette, or a designer receptor exclusively activated by a designer drug (DREADD). 38. The artificial expression construct of embodiment 34, wherein the expressible element comprises a non-functional molecule. 39. The artificial expression construct of embodiment 38, wherein the non-functional molecule comprises a non-functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signaling molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a microRNA, a homologous recombination donor cassette, or a DREADD. 40. An artificial expression construct according to any of the preceding embodiments, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier. 41. The artificial expression construct of embodiment 40, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 42. An artificial expression construct according to any of the preceding embodiments, wherein the artificial expression construct comprises or encodes a skip element. 43. The artificial expression construct of embodiment 42, wherein the skip element comprises a 2A peptide or an internal ribosome entry site (IRES). 44. The artificial expression construct of embodiment 43, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 45. The artificial expression construct comprises: I56i enhancer linked core, eHGT_387m, eHGT_375h, eHGT_376h, eHGT_390h, eHGT_373m, eHGT_375m, eHGT_386m, eHGT_390m, eHGT_387m(core2), eHGT_375h(core), eHGT_376h(core), eHGT_390h(core), eHGT_373m(core), eHGT_375m(core), eHGT_3 45. An artificial expression construct according to any of the preceding embodiments, comprising or encoding a set of features selected from: 86m(core), eHGT_390m(core2), AAV, scAAV, rAAv, minBglobin, CMV, minCMV, minRho, minRho*, fluorescent protein, codon-optimized SLC6A1, 4X2C, Cre, iCre, dgCre, FlpO, tTA2, SP10, WPRE, WPRE3, hGHpA, and / or BGHpA. 46. ​​The artificial expression construct is: CN2721, CN3213, CN2720, CN2722, CN2732, CN3322, CN3323, CN3887, CN3888, CN2972, CN2973, CN2974, CN2975, CN2976, ID10.01, ID10.02, ID10.03, ID10.04, ID10.05, ID10.06, ID10.07, ID10.08, ID10.09, ID10.10, ID10.11, ID10.12, ID10.13, ID10.14, ID10.15, ID10.16, ID10.17, I D10.18, ID10.19, ID10.20, ID10.21, ID10.22, ID10.23, ID10.24, ID10.25, ID10.26, ID10.27, ID10.28, ID10.29, ID10.30, ID10.31, ID10.32, ID11. 01, ID11.02, ID11.03, ID11.04, ID11.05, ID11.06, ID11.07, ID11.08, ID11.09, ID11.10, ID11.11, ID11.12, ID11.13, ID11.14, ID11.15, ID11.16, ID 12.01, ID12.02, ID12.03, ID12.04, ID12.05, ID12.06, ID12.07, ID12.08, ID12.09, ID12.10, ID12.11, ID12.12, ID12.13, ID12.14, ID12.15, ID12.1 6, ID13.01, ID13.02, ID13.03, ID13.04, ID13.05, ID13.06, ID13.07, ID13.08, ID13.09, ID13.10, ID13.11, ID13.12, ID13.13, ID13.14, ID13.15, ID1 3.16, ID14.01, ID14.02, ID14.03, ID14.04, ID14.05, ID14.06, ID14.07, ID14.08, ID14.09, ID14.10, ID14.11, ID14.12, ID14.13, ID14.14, ID14.15, ID14.16, ID15.01, ID15.02, ID15.03, ID15.04, ID15.05, ID15.06, ID15.07, ID15.08, ID15.09, ID15.10, ID15.11, ID15.12, ID15.13, ID15.14, ID15.15、ID15.16、ID16.01、ID16.02、ID16.03、ID16.04、ID16.05、ID16.06、ID16.07、ID16.08、ID16.09、ID16.10、ID16.11、ID16.12、ID16.13、ID16.14、ID16.15、ID16.16、ID17.01、ID17.02、ID17.03、ID17.04、ID17.05、ID17.06、ID17.07、ID17.08、ID17.09、ID17.10、ID17.11、ID17.12、ID17.13、ID17.14、ID17.15、ID17.16、ID18.01、ID18.02、ID18.03、ID18.04、ID18.05、ID18.06、ID18.07、ID18.08、ID18.09、ID18.10、ID18.11、ID18.12、ID18.13、ID18.14、ID18.15、ID18.16、ID19.01、ID19.02、ID19.03、ID19.04、ID19.05、ID19.06、ID19.07、ID19.08、ID19.09、ID19.10、ID19.11、ID19.12、ID19.13、ID19.14、ID19.15、ID19.16、ID20.01、ID20.02、ID20.03、ID20.04、ID20.05、ID20.06、ID20.07、ID20.08、ID20.09、ID20.10、ID20.11、ID20.12、ID20.13、ID20.14、ID20.15、ID20.16、ID21.01、ID21.02、ID21.03、ID21.04、ID21.05、ID21.06、ID21.07、ID21.08、ID21.09、ID21.10、ID21.11、ID21.12、ID21.13、ID21.14、ID21.15、ID21.16、ID22.01、ID22.02、ID22.03、ID22.04、ID22.05、ID22.06、ID22.07、ID22.08、ID22.09、ID22.10、ID22.11、ID22.12、ID22.13、ID22.14、ID22.15、ID22.16、ID23.01、ID23.02、ID23.03、ID23.04、ID23.05、ID23.06、ID23.07、ID23.08、ID23.09、ID23.10、ID23.11、ID23.12, ID23.13, ID23.14, ID23.15, ID23.16, ID24.01, ID24.02, ID24.03, ID24.04, ID24. 05, ID24.06, ID24.07, ID24.08, ID24.09, ID24.10, ID24.11, ID24.12, ID24.13, ID24.1 4, ID24.15, ID24.16, ID25.01, ID25.02, ID25.03, ID25.04, ID25.05, ID25.06, ID25.0 7, ID25.08, ID25.09, ID25.10, ID25.11, ID25.12, ID25.13, ID25.14, ID25.15, ID25.16 46. ​​An artificial expression construct according to any of the preceding embodiments, comprising the features of ID26.01, ID26.02, ID26.03, ID26.04, ID26.05, ID26.06, ID26.07, ID26.08, ID26.09, ID26.10, ID26.11, ID26.12, ID26.13, ID26.14, ID26.15, ID26.16, ID27.01, ID27.02, ID27.03, ID27.04, ID27.05, ID27.06, ID27.07, ID27.08, ID27.09, ID27.10, ID27.11, ID27.12, ID27.13, ID27.14, ID27.15, or ID27.16. 47. A vector comprising an artificial expression construct according to any one of embodiments 1 to 46. 48. The vector of embodiment 47, wherein the vector comprises a viral vector. 49. The vector of embodiment 48, wherein the viral vector comprises a recombinant adeno-associated viral (AAV) vector. 50. A transgenic cell comprising an artificial expression construct according to any one of embodiments 1 to 46 and / or a vector according to embodiments 47 to 49. 51. The transgenic cell of embodiment 50, wherein the transgenic cell is a GABAergic neuron or an astrocyte. 52. The transgenic cell of embodiment 50 or 51, wherein the transgenic cell is murine, human, or non-human primate. 53. A non-human transgenic animal comprising an artificial expression construct according to any of embodiments 1 to 46, and / or a vector according to embodiments 47 to 49, and / or a transgenic cell according to embodiment 51 or 52. 54. The non-human transgenic animal of embodiment 53, wherein the non-human transgenic animal is a mouse or a non-human primate. 55. An administrable composition comprising an artificial expression construct according to any of embodiments 1 to 46, and / or a vector according to embodiments 47 to 49, and / or a transgenic cell according to embodiment 51 or 52. 56. A kit comprising an artificial expression construct according to any of embodiments 1 to 46, and / or a vector according to embodiments 47 to 49, and / or a transgenic cell according to embodiment 51 or 52, and / or a non-human transgenic cell according to embodiment 53 or 54, and / or an administrable composition according to embodiment 55. 57. A method for expressing a gene in a cell population in vivo or in vitro, comprising providing an administrable composition of embodiment 55 to a sample or subject comprising a population of cells in a sufficient dosage and for a sufficient period of time, thereby expressing the gene in the cell population. 58. The method of embodiment 57, wherein the gene encodes GAT1, an effector element, or an expressible element. 59. The method of embodiment 58, wherein the gene is a codon-optimized SLC6A1 gene. 60. The method of embodiment 57 or 58, wherein the gene has at least 90% sequence identity with the sequence set forth in SEQ ID NO: 22, 25, 28, 31 or 34. 61. The method according to any of embodiments 57 to 60, wherein the gene has a sequence according to SEQ ID NO: 22, 25, 28, 31 or 34. 62. The method of embodiment 57, wherein the population of cells comprises GABAergic neurons and astrocytes. 63. The method of embodiment 57, wherein the providing comprises pipetting. 64. The method of embodiment 63, wherein the pipetting is performed on a brain slice. 65. The method of embodiment 64, wherein the brain slice comprises GABAergic neurons and astrocytes. 66. The method of embodiment 64 or 65, wherein the brain slice is from a mouse, a human, or a non-human primate. 67. The method of embodiment 57, wherein said providing comprises administering to a living subject. 68. The method of embodiment 67, wherein the living subject is a human, a non-human primate, or a mouse. 69. The method of embodiment 67, wherein the living subject has a SL6CA1-associated disorder. 70. The method of embodiment 69, wherein the SL6CA1-related disorder comprises cognitive impairment, motor impairment, mild to moderate intellectual disability, epilepsy, language disorder, attention deficit disorder, attention deficit hyperactivity disorder, or autism spectrum disorder. 71. The method according to any one of embodiments 67 to 70, wherein said administering to a living subject is by injection. 72. The method of embodiment 71, wherein the injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternomagnathus (ICM) injection, or intrathecal injection. 73. An artificial expression construct having a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:94. 74. An artificial expression construct having a sequence as set forth in SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:94.

[0157] (viii) Concluding paragraph. Variants of sequences disclosed and referenced herein are also included. Computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software, are available for guidance in determining which amino acid residues can be substituted, inserted, or deleted without disrupting biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of a family of amino acids that are related in their side chains.

[0158] Suitable conservative substitutions of amino acids in a peptide or protein are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families as follows: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified into polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified into polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, nonpolar residues): isoleucine (I Group 7 (uncharged polar): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10: (small aliphatic, non-polar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0159] In making such changes, the hydrophilicity index of amino acids may be taken into consideration. The importance of the hydrophilic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid is assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are as follows: Ile (+4.5), Val (+4.2), Leu (+3.8), Phe (+2.8), Cys (+2.5), Met (+1.9), Ala (+1.8), Gly (-0.4), Thr (-0.7), Ser (-0.8), Trp (-0.9), Tyr (-1.3), Pro (-1.6), His (-3.2), Glutamate (-3.5), Gln (-3.5), Aspartate (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5).

[0160] It is known in the art that certain amino acids may be substituted by other amino acids with similar hydrophilicity indexes or scores, resulting in proteins that still have similar biological activity, i.e., still obtain biologically functional equivalent proteins. In making such changes, substitutions of amino acids with hydrophilicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitutions of similar amino acids can be effectively made based on hydrophilicity.

[0161] As described in detail in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: Arg (+3.0), Lys (+3.0), Aspartic acid (+3.0±1), Glutamic acid (+3.0±1), Ser (+0.3), Asn (+0.2), Gln (+0.2), Gly (0), Thr (-0.4), Pro (-0.5±1), Ala (-0.5), His (-0.5), Cys (-1.0), Met (-1.3), Val (-1.5), Leu (-1.8), Ile (-1.8), Tyr (-2.3), Phe (-2.5), Trp (-3.4). It is understood that an amino acid may be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically valuable, and in particular an immunologically valuable, protein. In such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0162] As discussed above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0163] As noted elsewhere herein, variants of a gene sequence may include codon-optimized variants, sequence polymorphisms, splice variants, and / or variants that do not affect to a statistically significant extent the function of the encoded product.

[0164] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0165] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, see Computational Molecular Biology (Lesk, A. M. ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W. ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G. ed.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G. ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. ed.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignment and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Related programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin), and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, 1999). Within the context of this disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the program referenced. As used herein, "default values" refers to any set of values ​​or parameters that are initially loaded into the software when first initialized.

[0166] Variants also include nucleic acid molecules that hybridize to sequences disclosed herein under stringent hybridization conditions and provide the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 43°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20μg / ml denatured sheared salmon sperm DNA, followed by washing the filter in 0.1xSSC at 50°C. Changes in the stringency of hybridization and signal detection are primarily achieved by manipulating formamide concentration (lower percentages of formamide result in lower stringency), salt conditions, or temperature. For example, moderately high stringency conditions include overnight incubation at 37° C. in a solution containing 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm blocking DNA, followed by washing at 50° C. in 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed after stringent hybridization can be performed at higher salt concentrations (e.g., 5×SSC). Modifications in the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Exemplary blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of certain blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

[0167] The term ligation is used broadly to describe joining together into a chain or series. It is used to describe joining nucleotide or amino acid sequences into a single nucleotide or amino acid sequence, respectively. The term "concatamerize" should be construed as indicating "concatenate."

[0168] As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular described elements, steps, ingredients, or components. Thus, the term "include" or "including" should be interpreted as describing "comprises, consists, or consists essentially of." The transitional term "comprises" or "comprises" means to anticipate the inclusion of unspecified elements, steps, ingredients, or components, even if in major amounts, without limitation. The transitional phrase "consisting of" excludes any elements, steps, ingredients, or components not specified. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients, or components, and those that do not have a substantial effect on the embodiment. A substantial effect results in a statistically significant decrease in target expression in GABAergic neurons and astrocytes utilizing the artificial expression constructs disclosed herein.

[0169] In certain embodiments, artificial means means not occurring in nature.

[0170] Unless otherwise indicated, all numbers expressing properties, such as amounts of ingredients, molecular weights, reaction conditions, and the like, used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of numerical principles to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where further clarity is needed, the term "about" has the meaning reasonably assigned by a person of ordinary skill in the art to a stated value or range, i.e., ±20% of the stated value, ±19% of the stated value, ±18% of the stated value, ±17% of the stated value, ±16% of the stated value, ±15% of the stated value, ±14% of the stated value, ±13% of the stated value, ±12% of the stated value, ±11% of the stated value, ±10% of the stated value, ±9% of the stated value, ±8% of the stated value, ±7% of the stated value, ±6% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1% of the stated value.

[0171] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0172] As used in the context of describing the present invention (particularly in the context of the claims below), "a," "an," "the," and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0173] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the modified group, thereby satisfying the description of all Markush groups described as used in the appended claims.

[0174] Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to adopt such variations as necessary, and the inventors intend the invention to be practiced other than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0175] Additionally, throughout this specification, numerous references are made to patents, printed publications, journal articles, and other written texts, the materials of which are referenced herein. Each of the referenced materials is individually incorporated herein by reference in its entirety for the teachings referenced.

[0176] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not limited to that precisely as shown and described.

[0177] The details set forth herein are presented by way of example and for the sole purpose of illustrative discussion of preferred embodiments of the present invention, and to provide what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt is made to show structural details of the present invention, but rather an attempt is required to provide a basic understanding of the present invention, and to make clear to those skilled in the art how some forms of the present invention may be embodied in practice, through the description taken in conjunction with the drawings and / or examples.

[0178] The definitions and explanations used in this disclosure are meant and intended to control in any future interpretations, unless clearly and unambiguously amended in the following examples, or unless the application of the meaning makes any interpretation meaningless or essentially meaningless. If the interpretation of a term makes it meaningless or essentially meaningless, the definition should be taken from a dictionary known to those skilled in the art, such as Webster's Dictionary, 3rd Edition, or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. (i) a first enhancer having a sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5 and having the same enhancer activity as the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; (ii) a second enhancer having a sequence set forth in SEQ ID NO:14, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:84 or SEQ ID NO:85, or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:84 or SEQ ID NO:85 and having the same enhancer activity as the sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:84 or SEQ ID NO:85; (iii) a promoter; and (iv) comprises a coding sequence; An artificial expression construct, wherein the same enhancer activity comprises selective expression of a coding sequence in GABAergic neurons or in astrocytes.

2. The first enhancer comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 and having the same enhancer activity as the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; 2. The artificial expression construct of claim 1, wherein the same enhancer activity comprises selective expression of a coding sequence in GABAergic neurons.

3. The first enhancer has a sequence as set forth in SEQ ID NO:6 or SEQ ID NO:7, or a sequence having at least 95% sequence identity with the sequence as set forth in SEQ ID NO:6 or SEQ ID NO:7 and having the same enhancer activity as the sequence as set forth in SEQ ID NO:6 or SEQ ID NO:7; 2. The artificial expression construct of claim 1, wherein the same enhancer activity comprises selective expression of a coding sequence in GABAergic neurons.

4. The second enhancer comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 85, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 85 and having the same enhancer activity as the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 85; 2. The artificial expression construct of claim 1, wherein the same enhancer activity comprises selective expression of the coding sequence in astrocytes.

5. linking the second enhancer to the first enhancer to generate a combined linked enhancer; The combined linked enhancer has a sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 86, or a sequence having at least 95% sequence identity with the sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 86 and having the same enhancer activity as the sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 86; 5. The artificial expression construct of claim 4, wherein the same enhancer activity comprises selective expression of the coding sequence in astrocytes.

6. The combinatorially linked enhancer comprises: 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 84, 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 84 and having the same enhancer activity as the sequence set forth in SEQ ID NO: 84, 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 85, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 85 and having the same enhancer activity as the sequence set forth in SEQ ID NO: 85; and / or characterized in that it has 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 and having the same enhancer activity as the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, 6. The artificial expression construct of claim 5, wherein the same enhancer activity comprises selective expression of the coding sequence in GABAergic neurons and / or in astrocytes.

7. The combined linked enhancer comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86 and having the same enhancer activity as the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 86; 6. The artificial expression construct of claim 5, wherein the same enhancer activity comprises selective expression of the coding sequence in GABAergic neurons and in astrocytes.

8. The combined linked enhancer has a sequence as set forth in SEQ ID NO: 88 or SEQ ID NO: 89, or a sequence having at least 95% sequence identity with the sequence as set forth in SEQ ID NO: 88 or SEQ ID NO: 89, and has the same enhancer activity as the sequence as set forth in SEQ ID NO: 88 or SEQ ID NO: 89; 6. The artificial expression construct of claim 5, wherein the same enhancer activity comprises selective expression of the coding sequence in GABAergic neurons and in astrocytes.

9. 2. The artificial expression construct of claim 1, wherein the coding sequence is a codon-optimized SLC6A1 gene having a sequence as set forth in SEQ ID NO: 22, 25, 28, 31, 34, 38, 39, 40, 41, 42, 43, 44 or 45, or a sequence having at least 95% sequence identity with the sequence as set forth in SEQ ID NO: 22, 25, 28, 31, 34, 38, 39, 40, 41, 42, 43, 44 or 45 and having the same enhancer activity as the sequence as set forth in SEQ ID NO: 22, 25, 28, 31, 34, 38, 39, 40, 41, 42, 43, 44 or 45.

10. 2. The artificial expression construct of claim 1, wherein the coding sequence encodes a fluorescent protein or a neurotransmitter.

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

12. The artificial expression construct of claim 11, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, AAV9, AAVrh.10, AAV1R6 or AAV1R7.

13. 2. The artificial expression construct of claim 1, wherein the artificial expression construct comprises or encodes a 2A peptide or an internal ribosome entry site (IRES).

14. 2. The artificial expression construct of claim 1, wherein the artificial expression construct is in a viral vector.

15. 1. A method of expressing a coding sequence in a cell population in vivo or in vitro, comprising providing an administrable composition comprising an artificial expression construct to a sample or subject comprising said cell population, in a sufficient dose and for a sufficient period of time, thereby expressing said coding sequence in said cell population; The artificial expression construct comprises: (i) a first enhancer having a sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5 and having the same enhancer activity as the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, wherein the same enhancer activity includes selective expression of a coding sequence in GABAergic neurons; (ii) a second enhancer having a sequence set forth in SEQ ID NO:14, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:84 or SEQ ID NO:85, or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:84 or SEQ ID NO:85 and having the same enhancer activity as the sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:84 or SEQ ID NO:85, wherein the same enhancer activity comprises selective expression of the coding sequence in astrocytes; (iii) a promoter; and (iv) a coding sequence.

16. The method of claim 15, wherein the coding sequence encodes GAT1.

17. The method of claim 15, wherein the coding sequence is a codon-optimized SLC6A1 gene.

18. 16. The method of claim 15, wherein the cell population comprises GABAergic neurons and astrocytes.

19. The method of claim 15, wherein said providing comprises pipetting the brain slice.

20. The method of claim 15 , wherein said providing comprises administering to a living subject.