Artificial expression constructs for selectively modulating gene expression in mediating neurons

Abstract

To provide artificial expression constructs for selectively modulating gene expression in selected central nervous cell types, capable of selectively expressing synthetic genes or modify gene expression in GABAergic mediating neurons.SOLUTION: Provided herein is an artificial expression construct comprising: (i) an enhancer of a specific sequence; (ii) a promoter; and (iii) a heterologous encoding sequence encoding an effector element.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 742,835, filed October 8, 2018; No. 62 / 749,012, filed October 22, 2018; and No. 62 / 810,281, filed February 25, 2019, each of which is incorporated by reference in its entirety as if fully set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. RF1MH114126 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing Reference The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named A166-0006PCT_ST25.txt. The text file is 379KB, was created on October 3, 2019, and has been submitted electronically via EFS-Web.

[0004] The present disclosure provides artificial expression constructs for selectively regulating gene expression in selected central nervous system cell types. The artificial expression constructs can be used to selectively express synthetic genes or modify gene expression in GABAergic forebrain interneurons. [Background technology]

[0005] GABAergic interneurons play an important role in central nervous system processing and development. Dysfunction of these cells may also contribute to a number of neuropsychiatric disorders, such as schizophrenia and autism. GABAergic interneurons also play a role in epilepsy.

[0006] Cell type or cell class specific gene delivery using non-pathogenic recombinant adeno-associated viruses (rAAV) has shown increasing support for the treatment of a wide range of diseases. The inclusion of one or more cis-acting DNA regulatory elements, such as specific promoters or enhancers, in rAAV has been beneficial to provide specificity of expression in specific target cells, including specific cell types or cell classes in the brain.

[0007] Dimidschstein and colleagues (Nat Neurosci 19(12):1743-1749, 2016) developed a rAAV that allows highly selective gene expression in GABAergic interneurons within the telencephalon. This rAAV contains a 527 bp enhancer sequence (called mI56i or mDlx) from the intergenic interval between the distal-less homeobox 5 and 6 genes (Dlx5 / 6), which is naturally expressed by forebrain GABAergic interneurons during embryonic development. The Dimidschstein et al. construct is available on Addgene under ID number 83900 (the enhancer drives eGFP expression). Additional constructs using mouse or human I56i enhancers to drive various transgenes are available through Addgene, for example, Plasmid ID Nos. 83899 (driving GCaMP6f expression), 83898 (driving ChR2 expression), 83895 (driving synthetic eGFP expression), 89897 (driving hM3DREADD expression), 83896 (driving hM4Di expression), and 83894 (driving synthetic tdTomato expression). See also U.S. Patent Application Publication No. 2018 / 0078658.

[0008] Furthermore, the mI56i enhancer has previously been used to reliably target reporter genes in a pattern highly similar to the normal pattern of Dlx5 / 6 expression during embryonic development (Zerucha et al., J Neuroscience 20:709-721, 2000; Stuhmer et al., Cerebral Cortex 12:75-85, 2002; Stenman et al., J Neuroscience 23:167-174, 2003; Monory et al., Neuron. 51:455-455, 2006; Miyoshi et al., J Neuroscience 30:1532-1594, 2010).

[0009] One major drawback of using rAAV as a gene delivery system is the limited packaging limit of AAV, which is particularly restrictive for the inclusion of long gene control and expression elements. Furthermore, many existing interneuron-specific rAAV expression constructs can result in weak gene expression, which reduces their usefulness in research and therapeutic applications. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Patent Application Publication No. 2018 / 0078658 [Non-patent literature]

[0011] [Non-Patent Document 1] Dimidschstein et al., Nat Neurosci 19(12):1743-1749,2016 [Non-Patent Document 2] Zerucha et al., J Neuroscience 20:709-721,2000 [Non-Patent Document 3] Stuhmer et al.,Cerebral Cortex 12:75-85,2002 [Non-Patent Document 4] Stenman et al., J Neuroscience 23:167-174,2003 [Non-Patent Document 5] Monory et al.,Neuron.51:455-455,2006 [Non-Patent Document 6] Miyoshi et al.,J Neuroscience 30:1532-1594,2010 Summary of the Invention [Means for solving the problem]

[0012] The present disclosure overcomes the shortcomings of the prior art by providing an engineered enhancer element that results in rapid, strong, cell-specific expression of heterologous coding sequences in forebrain GABAergic interneurons.

[0013] In certain embodiments, the artificial enhancer elements comprise a concatemerized core of the I56i enhancer. These artificial enhancer elements result in a more rapid onset of transgene expression compared to a single full-length original (native) enhancer.

[0014] In certain embodiments, the I56i enhancer core may be derived from, for example, human, mouse or zebrafish I56i enhancer (SEQ ID NO: 1, 4 and 5, respectively). The selected core of the I56i enhancer may include SEQ ID NO: 2 (core shared by human and mouse) or SEQ ID NO: 6 (zebrafish core). In certain embodiments, the core is concatemerized. For example, SEQ ID NO: 3 provides a 3-copy concatemer of the selected human / mouse I56i core, and SEQ ID NO: 7 provides a 3-copy concatemer of the selected zebrafish I56i core.

[0015] Particularly interestingly, the synthetic 3x human / mouse core (herein referred to as 3xhl56iCore; SEQ ID NO: 3), despite being a 3x concatemer, is shorter than the original full-length enhancer sequence reported in Dimidschstein et al. (Nat Neurosci 19(12):1743-1749, 2016). Thus, this concatemerized core provides more locations for enhancer-linked cargo genes, which is highly desirable. Furthermore, the peak level of transgene expression driven by the 3xhI56iCore enhancer is much greater than a simple 3-fold increase in the level of the original single full-length original enhancer.

[0016] The engineered concatemerized I56i cores disclosed herein enable new and improved gene delivery vectors that are particularly useful for achieving selective transgene expression in forebrain GABAergic interneurons of various animal species, including humans.

[0017] Many of the drawings presented herein are better understood in color, and applicants reserve the right to consider color versions of the drawings as part of the original application and to show color images of the drawings in later prosecutions. [Brief description of the drawings]

[0018] [Figure 1]Virus CN1244 / PHP.eB. 10 genome copies delivered intravenously (IV) to adult mice. PHP.eB encodes a capsid derived from AAV9 that allows efficient AAV translocation across the mouse blood-brain barrier and allows delivery of AAV vectors in a brain-wide manner. This capsid differs from AAV9 such that the amino acid starting at residue 586: SAQA (SEQ ID NO: 98) is changed to SDGTLAVPFKA (SEQ ID NO: 33). The Gad2-T2A-nls-mCherry reporter represents nearly all inhibitory neurons in the mouse brain (here, the V1 visual cortex is represented), and the delivered CN1244 / PHP.eB virus drives specific SYFP2 reporter activity in forebrain GABAergic neurons. [Diagram 2] Figures 2A, 2B. Comparison of CN1244 vs. CN1389 vs. CN1390. (Figure 2A) Schematic diagram of the three vector constructs, CN1390, CN1389 and CN1244 (CN1203 scAAV). Key: hI56i-full-length human enhancer (black box; SEQ ID NO:1); selected hI56i core (gray box; SEQ ID NO:2) and 3x concatemer of core (gray box; SEQ ID NO:3); minBG-minimal beta-globin promoter; SYFP2-Super Yellow Fluorescent Protein 2; WPRE3-Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element 3; BGHpA-bovine growth hormone polyA sequence; L-ITR and R-ITR-Adeno-associated virus-2 (AAV2) inverted terminal repeats (ITRs). (Figure 2B) Fluorography images showing the relative expression of SYFP2 from AAV vector constructs CN1244, CN1389, and CN1390. Adult wild-type mice were injected retro-orbitally with 1E+11 genome copies of the indicated viruses. Animals were maintained for 3-4 weeks, then euthanized, brains extracted and sliced, followed by live tissue epifluorescence imaging of native fluorescence. Exposure times were matched so that transgene expression levels could be directly compared. The first three panels are 500 ms exposures for each of the constructs shown, and the fourth panel is a shorter (50 ms) exposure image of CN1390. CN1390, which has an engineered concatemerized core, showed strong and more rapid transgene expression. [Diagram 3] CN1390 retains cell type specificity for pan-GABAergic neuronal populations. Cortical / hippocampal brain slice cultures were prepared from P5-10 Gad2-IRES-Cre het / Ai75 het animals. After 1 h of culture, CN1390 virus suspension was pipetted onto the slice surface to transduce brain cell types. At 10 DIV / 10 DPI, native fluorescence was imaged in green and red channels on a Nikon inverted microscope. 10 DIV / 10 DPI. DIV: days in vitro, DPI: days post infection. [Figure 4] 4A, 4B. Comparison of CN1244 vs. CN1390 in non-human primate ex vivo brain slice cultures. (Fig. 4A) Fluorography images showing the relative expression of SYFP2 from AAV vector constructs CN1244 and CN1390. Neocortical slices were cultured from adult macaque brains and infected with nominally matched titers of the indicated viruses. Brain slice cultures were maintained in an incubator for 4 days in vitro (4DIV / 4DPI) 4 days post-infection and then used for live tissue epifluorescence imaging of native fluorescence. Exposure times were matched so that transgene expression levels could be directly compared. CN1390, which has an engineered concatemerized core, showed strong and more rapid transgene expression. (Fig. 4B) Fluorography images showing the relative expression of SYFP2 from AAV vector constructs CN1244 and CN1390. Hippocampal slices were cultured from adult macaque brains and infected with nominally matched titers of the indicated viruses. Brain slice cultures were maintained in an incubator for 6 days in vitro (6 DIV / 6 DPI) after infection and then used for live tissue epifluorescence imaging of native fluorescence. Exposure times were matched to allow direct comparison of transgene expression levels. CN1390, which has an engineered concatemerized core, showed strong and more rapid transgene expression. [Diagram 5]Figures 5A-5E. CN1390 shows rapid onset of transgene expression in human ex vivo brain slices. Human ex vivo neocortical brain slice cultures were prepared from live neurosurgical specimens as described in Ting et al., Scientific Reports 8(1):8407,2018. After 1 h in culture, CN1390 virus suspension was pipetted onto the slice surface to transduce brain cell types. At 1, 3 and 6 DIV / DPI, native SYFP2 fluorescence was imaged using matched exposure times on a Nikon microscope. Figures 5A-5D show rapid viral genetic labeling of human neocortical interneurons for targeted patch clamp recording and analysis. (Figure 5A) Time course of viral-mediated YFP expression after human brain slice transduction with CN1390 eB. (Figure 5B) Magnification of the boxed area in (Figure 5A). (Figure 5C) High magnification image of a virally labeled interneuron showing a bipolar morphology. (FIG. 5D) Examples of whole-cell recordings from four different virally labeled YFP+ human interneurons showing different firing patterns in response to suprathreshold current injections. (FIG. 5E) At various time points in culture, slices were taken for terminal patch clamp recording analysis to establish the firing properties of the labeled neurons. Functional analysis of human neocortical interneuron firing patterns and electrical properties by patch clamp recordings was feasible as early as 40 hours after CN1390 AAV-PHP.eB virus infection. [Figure 6]CN1390 maintains GABAergic cell class selectivity. At 7-34 DIV / DPI, virally transduced human organotypic slices from four unique human donors were isolated and 234 single SYFP2+ cells were FACS sorted from glia- and debris-depleted cell suspensions and profiled by single-cell RNA-seq (SMARTer V.4). These cells were mapped to the existing MTG cell type taxonomy. The bar at the bottom of the taxonomy indicates the number of SYFP+ cells that were mapped to the final leaf. The circle further up the taxonomy indicates the number of cells that could only be mapped to that branch point. Note that all major GABAergic classes of cells were labeled and no glutamatergic or glial cells were recovered. The cell types listed from top to bottom are: GABAergic; 3 Inh L1-2 PAX-6 CDH12, 4 Inh L1-2 PAX6 TNF AIP8L3, 5 Inh L1 SST NMBR(ADARB2+), 6 Inh L1-4 LAMP5 LCP2(Rosehip), 7 Inh L1-2 LAMP5 DBP, 8 Inh L2-6 LAMP5 CA1(lgtp), Inh L1 SST CHRNA4(ADARB2+), 14 Inh L1-2 GAD1 MC4R(ADARB2+), 15 Inh L1-2 SST BAGE2(ADARB2+), 17 Inh L1-3 PAX6 SYT6(Sncg), 19 Inh L1-2 VIP TSPAN12, 20 Inh L1-4 VIP CHRNA6, 21 Inh L1-3 VIP ADAMTSL1, 22 Inh L1-4 VIP PENK, 27 Inh L2-6 VIP QPCT, 28 Inh L3-6 VIP HS3ST3A1, 29 Inh L1-2 VIP PCDH20, 31 Inh L2-5 VIP SERPINF1, 32 Inh L2-5 VIP TYR, 37 Inh L1-3 VIP CHRM2, 38 Inh L2-4 VIP CBLN1, 39 Inh L1-3 VIP CCDC184, 40 Inh L1-3 VIP GGH, 42 Inh L1-2 VIP LBH, 43 Inh L2-3 VIP CASC6, 45 Inh L2-4 VIP<h2 style=";text-align:left;direction:ltr">SPAG17、46 Inh L1-4 VIP OPRM1、Inh L3-6 SST NPY(Chodl)、52 Inh L3-6 SST HPGD、55 Inh L4-6 SST B3GAT2、56 Inh L5-6 SST KLHDC8A、57 Inh L5-6 SST NPM1P10、58 Inh L4-6 SST GXYLT2、59 Inh L4-5 SST STK32A、62 Inh L1-3 SST CALB1、63 Inh L3-5 SST ADGRG6、64 Inh L2-4 SST FRZB、65 Inh L5-6 SST TH、66 Inh L5-6 GAD1 GLP1R(LHX6+)、68 Inh L5-6 PVALB LGR5, 71 Inh L4-5 PVALB MEPE, 73 Inh L2-4 PVALB WFDC2, 74 Inh L4-6 PVALB SULF1, 75 Inh L5-6 SST MIR548F2, 76 Inh L2-5 PVALB SCUBE3 (SCUBE3), 82 Exc L2-5 LAMP5 LTK, 83 Exc L2-4 LINC00507 GLP2R, 84 Exc L2-3 LINC00507 FREM3, 85 Exc L5-6 THEMIS C1QL3, 87 Exc L3-4 RORB CARM1P1, 89 Exc L3-5 RORB ESR1,90 Exc L3-5 RORB COL22A1,92 Exc L3-5 RORB FILIP1L,93 Exc L3-5 RORB TWIST2,96 Exc L4-5 RORB FOLH1B,98 Exc L4-6 RORB SEMA3E,99 Exc L4-5 RORB DAPK2、100 Exc L5-6 RORB TTC12、101 Exc L4-6 RORB C1R、104 Exc L5-6 THEMIS FGF10、105 Exc L4-6 FEZF2 IL26(NP)、106 Exc L5-6 FEZF2 ABO、107 ​​Exc L6 FEZF2 SCUBE1、108 Exc L5-6SLC17A7 IL15, 109 Exc L6 FEZF2 OR2T8, 110 Exc L5-6 FEZF2 EFTUD1P1, glial; OPC L1-6 PDGFRA, Astro L1-6 FGFR3 SLC14A1, Astro L1-2 FGFR3 GFAP, Oligo L1-6 OPALIN, Endo L2-6 NOSTRIN and Micro L1-3 TYROBP. [Figure 7] Rapid expression from CN1390 allows assessment of connectivity in human circuits. Human neocortical organotypic slices were transduced with CN1390 and AAV-hSyn1-dTomato for 2.5 days. After only 2.5 days in culture, GABAergic cells and all neurons can be labeled in culture using CN1390 and AAV-hSyn1-dTomato, respectively. Human synapsin 1 (hSyn1) is a well-known pan-neuronal promoter. This allows assessment of connectivity between prospectively virus-marked patched cells (labeled by Cascade Blue). Fluorescent dyes listed in the bottom left corner of the fluorescent images are (from top to bottom): pan-GABA-SYFP, hSyn1-tdTomato, and Fill-Blue. [Figure 8]Figures 8A, 8B. All major classes of human neocortical GABAergic neurons are marked by CN1390. (Figure 8A) Multiplexed FISH using HCR v3.0 reveals major classes of GABAergic neurons labeled by somatostatin (SST), parvalbumin (PVALB) or vasoactive intestinal peptide (VIP) genes. CN1390 labeling in 350 μm thick neocortical brain slice cultures is shown. Text on the left image of Figure 8A is as follows: (top left) pial surface; (top right) lipofuscin, PVALB, SST, VIP and SYFP; and (bottom left) Hu, 350 μm section, viral CN1390eB, 7 DIV / DPI. (Figure 8B) Marking of expected cell classes of physiology, connectivity and morphology. Multiplexed FISH reveals the molecular identity of cell classes labeled with CN1390 and a portion of patched cells backfilled with neurobiotin and visualized by streptavidin-BV421. The image on the left in Figure 8B is labeled with SYFP, SST, VIP, PVALB, and lipofuscin. The image on the right in Figure 8B is labeled with biocytin-BV421. All of these cells displayed GABAergic cell morphology and were mostly characterized by expression of SYFP2 from CN1390. [Figure 9]Figures 9A-9F. AAV vector reagents for reversing Dravet syndrome (DS) symptoms in Scn1a+ / - mice. (9A) Vectors for delivering epitope-tagged Nav genes (NavBacs) of bacterial origin. The Nav genes shown here are NavMs (from Magnetococcus marinus), NavBp (from Bacillus pseudofirmus) and NavSheP-D60N (from Shewanella putrifaciens with an engineered D60N mutation). All of these examples have an N-terminal epitope tag (hexahistidine in the case of CN1367, or 3xHA in the cases of CN1498, CN1499 and CN1500). hI56i refers to the full-length I56i enhancer of SEQ ID NO: 1; 3xhI56iCore refers to the concatemerized core of the I56i enhancer (SEQ ID NO: 3); (9B) Graded expression levels from NavBac vectors. (9C) Weak but detectable expression from vector CN1367 in Pvalb interneurons. (9D) Trend towards seizure protection with vector CN1367. (9E) Vector 1500 drives high level expression in Pvalb+ and Pvalb- interneurons throughout the cortex. (9F) Abundant production of HA-tagged NavBac in the cell body and proximal processes by vectors 1498 and 1500, but not 1499. [Figure 10] CN1500 rAAV vector substantially reverses febrile seizures in Scn1a+ / - mice. Febrile seizure assay shown as the internal body temperature at which seizures are first detected. (Top) Circles indicate Scn1a+ / - mice not transduced with AAV, diamonds represent animals transduced with CN1500. Large dots and error bars represent the mean + / - SEM for each group of animals. (Bottom) Using Kaplan-Meier curves, the trend of the same data is shown as the percentage of mice in each group that remain seizure-free at different body temperatures. [Figure 11]11A, 11B. Conservation of I56i enhancer sequences. (FIG. 11A) Alignment of human (SEQ ID NO: 1) I56i, mouse (SEQ ID NO: 4) I56i and zebrafish (SEQ ID NO: 5) I46i enhancer sequences. Residues shared by all three sequences are highlighted in light grey; residues shared by mouse and human sequences are highlighted in dark grey. The core sequence (SEQ ID NO: 2) corresponds to positions 268-398 of the human sequence shown. As this is an ultraconserved enhancer sequence, the mouse and human I56i enhancer core sequences are completely identical (100% sequence identity). It is also very similar to the zebrafish genomic sequence, and the orthologous zebrafish enhancer (called I46i) has been used in many contexts over the years to drive transgene expression in neocortical interneurons, including mouse neocortical interneurons. (FIG. 11B) Graph showing similarity between human, mouse and zebrafish enhancer sequences. The graphs show (labeled from right to left) similarity, absolute complexity and absolute complexity (human I56i). [Figure 12] Sequence and indicated features of construct CN1389 pAAV-hI56i(core)-minBG-SYFP2-WPRE3-BGHpA (SEQ ID NO:41). Selected restriction endonuclease sites are indicated, as are regions corresponding to different parts of the construct. [Figure 13] Sequence and indicated features of construct CN1390 pAAV-3xhI56i(core)-minBG-SYFP2-WPRE3-BGHpA (SEQ ID NO:42). Selected restriction endonuclease sites are indicated, as are regions corresponding to different parts of the construct. [Figure 14] Sequence and indicated features of construct CN1203 scAAV-hI56i-minbGlobin-SYFP2-WPRE3-BGHpA (SEQ ID NO: 43). Selected restriction endonuclease sites are indicated, as are regions corresponding to different parts of the construct. [Figure 15] Features of exemplary vectors disclosed herein. [Figure 16]Artificial expression constructs within the teachings of the present disclosure. Each construct begins with a concatemerized core of hI56i core (e.g., SEQ ID NO: 3 or 7), indicated as *. The following abbreviations are also used: β-globin minimal promoter (minB, referred to elsewhere herein as minBglobin), minimal cytomegalovirus promoter (minC, referred to elsewhere herein as minCMV), mutated minimal cytomegalovirus promoter (mut), minimal rhodopsin promoter (minR, referred to elsewhere herein as minRho), cytomegalovirus promoter (CMV), simian vacuolating virus 40 promoter (SV40), Hsp68 minimal promoter (H68, referred to elsewhere herein as proHSP68), Rous sarcoma virus long terminal repeat promoter (RSV), fluorescent protein (FP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), orange fluorescent protein (OFP), red fluorescent protein (RFP), ), far-red fluorescent protein (fRFP), yellow fluorescent protein (YFP), luciferase (Luc), enzyme (enz), transcription factor (TF), receptor (rec), cellular transport protein (CTP), signal transduction molecule (SM), neurotransmitter (NT), calcium reporter (CR), channelrhodopsin (ChR), guide RNA (gRNA), nuclease (Nuc), woodchuck hepatitis virus post-transcriptional response element (W, referred to elsewhere herein as WPRE3), bovine growth hormone polyadenylation signal (bG, referred to elsewhere herein as bGHpA), simian vacuolating virus 40 polyadenylation signal (S, referred to elsewhere herein as SV40pA), internal ribosome entry site 2 (I2, referred to elsewhere herein as IRES2) and 2A skipping elements (T2A, P2A, E2A and F2A). [Figure 17]Additional sequences supporting this disclosure: hI56i enhancer: (SEQ ID NO:1); hI56i enhancer core: (SEQ ID NO:2); 3xhI56iCore, triple concatemerized core of hI56i enhancer: (SEQ ID NO:3); Mouse I56i enhancer (core is the same as human): (SEQ ID NO:4); Zebrafish I46i enhancer: (SEQ ID NO:5); Zebrafish I46i enhancer core: (SEQ ID NO:6); Zebrafish I46i enhancer 3x concatemerized core: (SEQ ID NO:7); β globin minimal promoter (pBGmin / minBGlobin / minBGprom): (SEQ ID NO:8); minCMV promoter: (SEQ ID NO:9); Mutated minCMV promoter (SacI RE site removed): (SEQ ID NO:10); minRho promoter: (SEQ ID NO:11); Hsp68 minimal promoter (proHsp68): (SEQ ID NO:12); SYFP2: (SEQ ID NO:13); EGFP: (SEQ ID NO:14); Optimized Flp recombinase (FlpO): (SEQ ID NO:15); Improved Cre recombinase (iCre): (SEQ ID NO:16); NavMs, endogenous sequence: (SEQ ID NO:17); NavMs, codon optimized, with N-terminal 3xHA tag and linker: (SEQ ID NO:18); NavMs, codon optimized, with N-terminal His tag and linker: (SEQ ID NO:19); NavBp, endogenous sequence String: (SEQ ID NO:20); NavBp, codon optimized, with N-terminal 3xHA tag: (SEQ ID NO:21); NavSheP-D60N, codon optimized, with N-terminal 3xHA tag: (SEQ ID NO:22); NavSheP endogenous sequence: (SEQ ID NO:23); WPRE3: (SEQ ID NO:24); BGHpA: (SEQ ID NO:25); P2A coding sequence: (SEQ ID NO:26); P2A: (SEQ ID NO:27); T2A: (SEQ ID NO:28); E2A: (SEQ ID NO:29); F2A: (SEQ ID NO:30); N-terminal 3XHA tag: (SEQ ID NO:31); N-terminal 3XHA tag: (SEQ ID NO:32); PHP.eB capsid: (SEQ ID NO:90); AAV9 VP1 capsid protein: (SEQ ID NO:34); tet-transactivator version 2 (tTA2): (SEQ ID NO:35); CN1367-portion between L-ITR and R-ITR: positions 142 to 2984: (SEQ ID NO:36);The portion between CN1500-L-ITR and R-ITR: positions 142 to 2976: (SEQ ID NO: 37); the portion between CN1498-L-ITR and R-ITR: positions 142 to 2943: (SEQ ID NO: 38); the portion between CN1499-L-ITR and R-ITR: positions 142 to 2946: (SEQ ID NO: 39); the portion between CN1244-L-ITR and R-ITR: positions 142 to 2042: (SEQ ID NO: 40); the portion between CN1389-L-ITR and R-ITR corresponds to positions 142 to 1660: (SEQ ID NO: 41); The portion between N1390-L-ITR and R-ITR corresponds to positions 142 to 1897: (SEQ ID NO: 42); the portion between CN1203-L-ITR and R-ITR corresponds to positions 183 to 2052: (SEQ ID NO: 43); lactase (SEQ ID NO: 44); lipase (SEQ ID NO: 45); helicase (SEQ ID NO: 46); amylase (SEQ ID NO: 47); α-glucosidase (SEQ ID NO: 48); transcription factor SP1 (SEQ ID NO: 49); transcription factor AP-1 (SEQ ID NO: 50); heat shock factor protein 1 (SEQ ID NO: 51) ;CCAAT / enhancer binding protein (C / EBP) beta isoform a (SEQ ID NO:52);Octamer binding protein 1 (SEQ ID NO:53);Transforming growth factor receptor beta 1 (SEQ ID NO:54);Platelet-derived growth factor receptor (SEQ ID NO:55);Epidermal growth factor receptor (SEQ ID NO:56);Vascular endothelial growth factor receptor (SEQ ID NO:57);Interleukin 8 receptor alpha (SEQ ID NO:58);Caveolin (SEQ ID NO:59);Dynamin (SEQ ID NO:60);Classrin heavy chain 1 isoform 1 (SEQ ID NO:61);Classrin heavy chain 2 isoform 1 (SEQ ID NO:62);Classrin light chain A isoform a (SEQ ID NO:63);Classrin light chain B isoform a (SEQ ID NO:64);Ras-related protein Rab-4A isoform 1 (SEQ ID NO:65);Ras-related protein Rab-11A (SEQ ID NO:66);Platelet-derived growth factor (SEQ ID NO:67);Transforming growth factor-beta 3 (SEQ ID NO:68);Nerve growth factor (SEQ ID NO:69);Epidermal growth factor (SEQ ID NO:70);GTPase HRas (SEQ ID NO:71); cocaine- and amphetamine-regulated transcript (A chain) (SEQ ID NO:72); protachykinin-1 (SEQ ID NO:73); substance P (SEQ ID NO:74); oxytocin-neurophysin 1 (SEQ ID NO:75);Oxytocin (SEQ ID NO: 76); somatostatin (SEQ ID NO: 77); myosin light chain kinase, green fluorescent protein, calmodulin chimera (A chain) (SEQ ID NO: 78); genetically encoded green calcium indicator NTnC (A chain) (SEQ ID NO: 79); calcium indicator TN-XXL (SEQ ID NO: 80); BRET-based autoluminescent calcium indicator (SEQ ID NO: 81); calcium indicator protein OeNL(Ca2+)-18u (SEQ ID NO: 82); GCaMP6m (SEQ ID NO: 99); GCaMP6s (SEQ ID NO: 100); GCaMP6f (SEQ ID NO: 101); Channelopsin (Channelo psin)1 (SEQ ID NO: 83 and 102); channelrhodopsin-2 (SEQ ID NO: 84 and 103); CRISPR-associated protein (Cas) (SEQ ID NO: 85); Cas9 (SEQ ID NO: 86); CRISPR-associated endonuclease Cpf1 (SEQ ID NO: 87); ribonuclease 4 or ribonuclease L (SEQ ID NO: 88); deoxyribonuclease II beta (SEQ ID NO: 89); sodium channel protein type 1 subunit alpha (SEQ ID NO: 104); potassium voltage-gated channel subfamily KQT member 2 (SEQ ID NO: 105); and voltage-gated L-type calcium channel subunit alpha-1C (SEQ ID NO: 106). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] To fully understand the biology of the brain, different cell types need to be distinguished and defined. To identify and / or study these different cell types, vectors need to be identified that can selectively label and perturb different cell types. In mice, there is great effectiveness in using recombinase driver lines to label cell populations that share marker gene expression. However, the generation, maintenance, and use of such lines that label cell types with high specificity can be costly and often require triple transgenic crosses, resulting in low frequency of experimental animals. Moreover, these tools require germline transgenic animals and are therefore not applicable to humans, and recent advances in single-cell profiling such as single-cell RNA-seq (Tasic et al., Nature 563, 72-78 (2018); Tasic 2016, Nat Neurosci 19, 335-346) as well as investigations of neuronal electrophysiology and morphology (Gouwens 2019, Nat Neurosci 22, 1182-1195) have revealed that many recombinant driver lines label a heterogeneous mixture of cell types, often including cells of multiple subclasses. For example, the Rbp4-Cre mouse driver line, commonly used to label layer 5 (L5) neurons, also labels cells with dramatically different connectivity patterns: L5 intratelencephalic (IT, also called intercortical) and pyramidal tract (PT, also called cortico-subcortical) neurons.

[0020] Dimidschstein and colleagues (Nat Neurosci 19(12):1743-1749, 2016) developed a rAAV that allows highly selective gene expression in GABAergic interneurons within the telencephalon. This rAAV contains a 527 bp enhancer sequence (termed mI56i or mDlx) from the intergenic interval between the distal-less homeobox 5 and 6 genes (Dlx5 / 6), which is naturally expressed by forebrain GABAergic interneurons during embryonic development. The Dimidschstein et al. construct is available on Addgene under ID number 83900 (the enhancer drives eGFP expression). Additional constructs using mouse or human I56i enhancers to drive various transgenes are available through Addgene, for example, Plasmid ID Nos. 83899 (driving GCaMP6f expression), 83898 (driving ChR2 expression), 83895 (driving synthetic eGFP expression), 89897 (driving hM3DREADD expression), 83896 (driving hM4Di expression), and 83894 (driving synthetic tdTomato expression). See also U.S. Patent Application Publication No. 2018 / 0078658.

[0021] Furthermore, the mI56i enhancer has previously been used to reliably target reporter genes in a pattern highly similar to the normal pattern of Dlx5 / 6 expression during embryonic development (Zerucha et al., J Neuroscience 20:709-721, 2000; Stuhmer et al., Cerebral Cortex 12:75-85, 2002; Stenman et al., J Neuroscience 23:167-174, 2003; Monory et al., Neuron. 51:455-455, 2006; Miyoshi et al., J Neuroscience 30:1532-1594, 2010).

[0022] One major drawback of using rAAV as a gene delivery system is the limited packaging limit of AAV, which is particularly restrictive for the inclusion of long gene control and expression elements. Furthermore, many existing interneuron-specific rAAV expression constructs can result in weak gene expression, which reduces their usefulness in research and therapeutic applications.

[0023] The present disclosure overcomes the shortcomings of the prior art by providing artificial enhancer elements that contain a concatemerized core of the I56i enhancer. These artificial enhancer elements lead to unexpectedly strong peak transgene expression in forebrain GABAergic interneurons after viral transduction of mouse, monkey and human brain tissue (see Figures 2A, 2B, 3, 4, 5A, 5E, 7, 8A and 8B). Onset is also surprisingly rapid (see Figures 5A-5E), leading to fast, high expression, for example, in direct comparison with virus packaged in Addgene plasmid no. 83900. The increase in expression appears to be synergistically supra-linear and not simply 3-fold the levels driven by the original enhancer (Figure 2B).

[0024] In certain embodiments, the I56i enhancer core may be derived from, for example, human and mouse I56i enhancer, or zebrafish I46i enhancer (SEQ ID NO: 1, 4, and 5, respectively). The selected core of the I56i enhancer may include SEQ ID NO: 2 (core shared by human and mouse) or SEQ ID NO: 6 (zebrafish I46i core). In certain embodiments, the core is concatemerized. For example, SEQ ID NO: 3 provides a 3-copy concatemer of the selected human / mouse I56i core, and SEQ ID NO: 7 provides a 3-copy concatemer of the selected zebrafish I46i core.

[0025] Of particular interest, the synthetic 3x human / mouse core (herein referred to as 3xhl56iCore; SEQ ID NO: 3), despite being a 3x concatemer, is shorter than the original full-length enhancer sequence reported in Dimidschstein et al. (Nat Neurosci 19(12):1743-1749, 2016). When used to construct heterologous expression cassettes such as recombinant adeno-associated viruses (rAAV), this artificial enhancer element provides more locations for enhancer-linked cargo genes (heterologous coding sequences). This is highly desirable in many gene expression vectors. For example, many functional protein cargo genes (more commonly effector elements) are too long to fit into AAV vector designs, making space (length of sequence) critical throughout the vector.

[0026] The engineered concatemerized I56i core disclosed herein enables new and improved gene delivery vectors that are particularly useful for achieving selective transgene expression in neocortical GABAergic interneurons of various animal species, including humans and non-human primates. Importantly, GABAergic interneurons are deeply involved in central processing and development, and their dysfunction has been implicated in a variety of brain disorders. Thus, the enhancers and expression constructs described herein have many immediate applications in research and in the development of clinical treatments. The artificial enhancer can be used in experimental situations where the original enhancer hl56i proves insufficient (e.g., retro-orbital delivery of viruses encoding transgenes for functional perturbation experiments).

[0027] Aspects of the disclosure will now be described with additional options and details, including: (i) artificial expression constructs & vectors for selective expression of genes in selected cell types; (ii) compositions for administration; (iii) cell lines comprising the artificial expression constructs; (iv) transgenic animals; (v) methods of use; (vi) kits and commercial packages; (vii) exemplary embodiments; (viii) experimental examples; and (ix) concluding paragraphs.

[0028] (i) Artificial expression constructs & vectors for selective expression of genes in selected cell types. The artificial expression constructs disclosed herein contain (i) an enhancer sequence that provides selective expression of a coding sequence in a targeted central nervous system cell type, (ii) a coding sequence to be expressed, and (iii) a promoter. The expression construct may also contain other regulatory elements if necessary or beneficial.

[0029] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the level of transcription associated with a promoter and can function in either orientation with respect to the promoter and the coding sequence to be transcribed, and can be located upstream or downstream from the promoter or the coding sequence to be transcribed. There are art-recognized methods and techniques for measuring the function of enhancer element sequences. Particular examples of enhancer sequences utilized within the artificial expression constructs disclosed herein include the concatemerized cores of the I56i enhancer, such as the concatemerization of SEQ ID NO:2 and / or SEQ ID NO:6, including, by way of example, SEQ ID NO:3 and SEQ ID NO:7. Additional particular examples of the concatemerized cores of the I56i enhancer can include SEQ ID NO:2 and SEQ ID NO:6 within one sequence, such as SEQ ID NO:2-SEQ ID NO:2-SEQ ID NO:6; SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:2; and SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:6.

[0030] In certain embodiments, a targeted central nervous system cell type enhancer is an enhancer that is uniquely or preferentially used in the targeted central nervous system cell type. A targeted central nervous system cell type enhancer has neuron-specific transcriptional activity because it increases expression of a gene in the targeted central nervous system cell type but does not substantially direct expression of the gene in other non-targeted cell types.

[0031] If the coding sequence is selectively expressed in the selected neuronal cell and not substantially expressed in other neuronal cell types, the product of the coding sequence is preferentially expressed in the selected neuronal cell type. In certain embodiments, preferential expression is greater than 50% expression compared to the reference cell type; greater than 60% expression compared to the reference cell type; greater than 70% expression compared to the reference cell type; greater than 80% expression compared to the reference cell type; or greater than 90% expression compared to the reference cell type. In certain embodiments, the reference cell type refers to a non-targeted neuronal cell. The non-targeted neuronal cell can be in the same anatomical structure as the targeted cell and / or can project to a common anatomical region. In certain embodiments, the reference cell type is in an anatomical structure adjacent to the anatomical structure that contains the targeted cell type. In certain embodiments, the reference cell type is a non-targeted neuronal cell that has a different gene expression profile than the targeted cell.

[0032] In certain embodiments, the product of the coding sequence may be expressed at low levels in unselected cell types, for example, less than 1%, or 1%, 2%, 3%, 5%, 10%, 15%, or 20% of the level at which the product is expressed in selected neural cells. In certain embodiments, the targeted central nervous system cell type is the only cell type that expresses the correct 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.

[0033] In certain embodiments, targeted cell types (e.g., neural, neuronal and / or non-neuronal) can be identified based on transcriptional profiles, such as those described in Tasic et al., 2018 Nature. For reference, the following description of neural cell types and distinguishing characteristics is also provided: GABAergic interneurons: express the GABA synthesis genes Gad1 / GAD1 and / or Gad2 / GAD2.

[0034] GABAergic subclass: Lamp5: Found in many cortical layers, especially the upper ones (L1-L2 / 3), mainly with neurogliaform and single bouquet morphology.

[0035] Sncg: Found in many cortical layers, they have molecular overlap with Lamp5 and Vip cells, but expression of Lamp5 or Vip is inconsistent and expression of Sncg is more consistent. These neurons express the neurotransmitter Cck and have a predominantly multipolar or basket cell morphology.

[0036] Serpinf1: Found in many cortical layers and has molecular overlap with Sncg and Vip cells, although expression of Sncg or Vip is inconsistent and expression of Serpinf1 is more consistent.

[0037] Vip: Found in many cortical layers, but especially frequent in the upper layers (L1-L4), these cells highly express the neurotransmitter vasoactive intestinal peptide (Vip).

[0038] Sst: 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 horizontally projecting Sst Chodl (or Sst Nos1) neurons, which are quite distinct from other Sst neurons but express shared marker genes with Sst.

[0039] Pvalb: Found in many cortical layers, but especially frequent in the lower layers (L5-L6). They highly express the neurotransmitter parvalbumin (Pvalb), express Tac1, and frequently attenuate the output of postsynaptic neurons. 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.

[0040] Meis2: A distinct subclass defined by a single type found in L6b and subcortical white matter.

[0041] Lamp5, Sncg, Serpinf1 and VIP: developmentally derived from precursor neurons in the caudal ganglia eminence (CGE).

[0042] Sst and Pvalb: developmentally derived from precursor neurons of the medial ganglia eminence (MGE).

[0043] Glutamatergic subclasses: All: express the glutamatergic transmitters Slc17a6 and / or Slc17a7.

[0044] L2 / 3 IT: Predominantly present in layers 2 / 3, with primarily intratelencephalic (intercortical) projections.

[0045] L4 IT: Predominantly present in layer 4, with primarily intratelencephalic (intercortical) projections.

[0046] L5 IT: Predominantly present in layer 5, with primarily intratelencephalic (intercortical) projections. Also called L5a.

[0047] L5 PT: Predominantly present in layer 5, with predominantly cortico-subcortical (pyramidal or subcortical) projections. Also called L5b or L5 CF. These cells are located in the primary motor cortex and adjacent areas and are corticospinal projection neurons. They are associated with motor neuron / movement disorders such as ALS.

[0048] Neocortical L5 extratelencephalic (ET) projection pyramidal neurons (L5 ET): Thick tufted pyramidal neurons that contain distinctive subtypes found only in specialized regions, such as Betz cells, Meynert cells, and von Economo cells.

[0049] L5 NP: Present mainly in layer 5, with projections mainly nearby.

[0050] L6 CT: Predominantly present in layer 6, with mainly corticothalamic projections.

[0051] L6 IT: Predominantly present in layer 6, with mainly intratelencephalic (intercortical) projections. This subclass includes L6 IT Car3 cells, which are highly similar to the intracortical projection cells of the claustrum.

[0052] L6b: Present mainly in the cortical subplate (L6b), projections to local areas (close to the cell body), intracortical projections from VISp to the anterior cingulate cortex, and cortico-subcortical projections to the thalamus are observed.

[0053] A distinct subclass defined by the monotype of CR:L1, Cajal-Retzius cells, express distinct molecular markers Lhx5 and Trp73.

[0054] Non-neuronal subclasses: Astrocytes: Neuroectoderm-derived glial cells that express the marker Aqp4. They have a distinct stellate morphology and are involved in the metabolic support of other cells in the brain.

[0055] Oligodendrocyte: A neuroectoderm-derived glial cell that expresses the marker Sox10. This category includes oligodendrocyte precursor cells (OPCs). Oligodendrocytes are the subclass primarily responsible for myelination of neurons.

[0056] VLMCs: Vascular leptomeningeal cells (VLMCs) are part of the meninges that surround the outer layer of the cortex and express the marker genes Lum and Col1a1.

[0057] Pericytes: Blood vessel-associated cells, also called mural cells, that express the marker genes Kcnj8 and Abcc9. Pericytes surround endothelial cells and are important in regulating capillary blood flow and are involved in the permeability of the blood-brain barrier.

[0058] SMC: Blood vessel-associated cells, also called mural cells, that express the marker gene Acta2. SMCs line the arterioles in the brain and are involved in the permeability of the blood-brain barrier.

[0059] endothelium: cells that line the blood vessels of the brain. Endothelial cells express the markers Tek and PDGF-β.

[0060] Macrophages: Immune cells including macrophages that reside in the brain and perivascular macrophages (PVM) that may be transiently associated with brain tissue or included as a by-product of the brain dissection procedure.

[0061] 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 an intended effect and that actually achieves this. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0062] 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, 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(TM) (Thermo Fisher Scientific); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (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 those encoding tandem conjugates.

[0063] GFP consists of 238 amino acids (26.9 kDa) and was originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresces green when exposed to blue light. Aequorea victoria GFP has a major excitation peak at 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, the lower green part of the visible spectrum. Renilla reniformis GFP has a single major excitation peak at 498 nm. Many different variants of GFP have been engineered because of its potential for widespread use and the evolving needs of researchers. 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, increasing its fluorescence, photostability, and shifting the major excitation peak to 488 nm while maintaining peak emission at 509 nm. Addition of the 37°C folding efficiency (F64L) point mutant to this scaffold resulted in enhanced fluorescence GFP (EGFP). EGFP has a fluorescence intensity of 9.13X10-21 m, also referred to 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, superfolder GFP was reported, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folded peptides.

[0064] "Yellow fluorescent protein" (YFP) is a genetic variant of the green fluorescent protein from the jellyfish Aequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm.

[0065] Exemplary functional molecules include functional ion transporters, cellular transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, or designer receptors activated only by designer drugs (DREADDs).

[0066] Ion transporters are transmembrane proteins that mediate the transport of ions across cell membranes. These transporters are prevalent in most cell types and are important for regulating cellular excitability and homeostasis. Ion transporters are involved in numerous cellular processes, such as action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many important biological processes in living cells involve the translocation of cations, such as calcium (Ca2+), potassium (K+), and sodium (Na+) ions, through such ion channels. In certain embodiments, ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0067] Exemplary enzymes, transcription factors, receptors, membrane proteins, cellular transport proteins, signaling molecules and neurotransmitters include enzymes such as lactase, lipase, helicase, α-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 β1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor and interleukin 8 receptor α; membrane proteins such as clathrin, dynamin, caveolin, Rab-4A and Rab-11A, cellular transport proteins; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPase and HRas; and neurotransmitters such as cocaine- and amphetamine-regulated transcripts, substance P, oxytocin and somatostatin.

[0068] In certain embodiments, functional molecules include receptors of neuronal function and status, such as calcium reporters. Intracellular calcium concentration is an important predictor of numerous cellular activities, including neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels is through 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 fusion of the N- and C-termini of circularly permuted GFP with M13 and calmodulin proteins. Some GCaMPs produce distinct fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs with green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Additionally, GECIs with red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV8-CAG-GCaMP3 (catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog number: BS12-NXSAAV9), 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).

[0069] In certain embodiments, the calcium reporter comprises a 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).

[0070] In certain embodiments, the functional molecule comprises a modulator of neural activity, such as channelrhodopsin (e.g., channelrhodopsin-1, channelrhodopsin-2 and variants thereof). Channelrhodopsin is a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels. In addition to channelrhodopsin 1 (ChR1) and channelrhodopsin 2 (ChR2), several variants of channelrhodopsin have been developed. For example, Lin et al. (Biophys J, 2009, 96(5):1803-14) describe the creation of a chimera 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 variants include the ChR2 variant described in Nagel, et al., Proc Natl Acad Sci USA, 2003, 100(24):13940-5), which is activated by blue light (470 nm) but insensitive to orange / red light, 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). Additional variants are described in Lin, Experimental Physiology, 2010, 96.1:19-25, and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.

[0071] In certain embodiments, the functional molecule includes DNA and RNA editing tools such as CRISPR / CAS (e.g., guide RNA and nucleases such as Cas, Cas9 or cpf1). The functional molecule may also include engineered Cpf1s, such as those described in US2018 / 0030425, US2016 / 0208243, WO / 2017 / 184768 and Zetsche et al. (2015) Cell 163:759-771; single gRNAs (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 or homologous recombination donor cassettes.

[0072] For additional information regarding CRISPR-Cas systems and their components, see US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and and related applications thereto; as well as WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO201 4 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, W O2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 0893 54, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO2017 / 106657, WO2017 / 127807 and related applications.

[0073] In certain embodiments, the functional molecules include designer receptors that are only activated by designer drugs (DREADDs). Designer receptors that are only activated by designer drugs (DREADDs) can be used to regulate cell function (Rogan and Roth, Pharmacol. Rev. 2011, 63(2):291-315). This family of evolved muscarinic receptors has been shown to increase (Gs-DREADDs; Gq-DREADDs) or decrease (Gi / o-DREADDs) cell activity after administration of the inactive synthetic ligand clozapine-n-oxide (Armbruster et al., PNAS, 2007, 104(12):5163-5168). When packaged in viral vectors or expressed in transgenic mouse models, these tools allow cell activity to be controlled in a defined spatial and temporal manner. For example, activation of hippocampal neurons by Gq-DREADD receptors amplifies gamma rhythms and increases locomotor activity and stereotypic behavior in mice (Alexander et al., Neuron, 2009, 63(1):27-39). DREADDs are formed by point mutations in the third and fifth transmembrane domains of muscarinic receptors (Y149C and A239G in hM3). In addition, Gs-binding DREADDs contain the second and third intracellular loops of β1-AR instead of the loops of M3 muscarinic receptors. Some exemplary DREADDs include hM3DREADD (hM3D) and hM4DREADD (hM4D). Various plasmids containing DREADDs are commercially available. For example, at addgene, AAV plasmids containing DREADDs include: pAAV-hSyn-DIO-hM3D(Gq)-mCherry (plasmid no. 44361), pAAV-hSyn-DIO-hM4d(Gi)-mCherry) (plasmid no. 44362), pAAV-EF1a-DIO-hM4d(Gi)-mCherry) (plasmid no. 50461), pAAV-GFAP-HA-hM3D(Gq)-IRES)-mCitrine (plasmid no. 50470), and pAAV-CaMKIIa-hM4D(Gi)-mCherry (plasmid no. 50477).

[0074] Additional effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to codon-improved Cre. dgCre refers to an enhanced GFP / Cre recombinase fusion gene with an N-terminal fusion of the first 159 amino acids of the chromosomal dihydrofolate reductase gene (DHFR or folA) of Escherichia coli K-12 strain, which has a G67S mutation and has been modified to also contain an R12Y / Y100I destabilization domain mutation. FlpO refers to a codon-optimized form of FLPe that greatly increases protein expression and FRT recombination efficiency in mouse cells. Similar to the Cre / LoxP system, the FLP / FRT system is widely used for gene expression (and for the generation of conditional knockout mice mediated by the FLP / FRT system). tTA2 refers to the tetracycline transactivator.

[0075] Exemplary expressible elements are expression products that do not include effector elements, e.g., non-functional or defective proteins. In certain embodiments, expressible elements can provide a way to test the effect 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.

[0076] Exemplary self-cleaving peptides include 2A peptides, which result in the production of two proteins from one mRNA. 2A sequences are 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 expression construct includes an internal ribosome entry site (IRES) sequence. The IRES allows the ribosome to begin translation at a second internal site in the mRNA molecule, resulting in the production of two proteins from one mRNA.

[0077] Coding sequences encoding the molecules (e.g., RNA, proteins) described herein can be readily obtained from publicly available databases and publications. The coding sequences can further include various sequence polymorphisms, mutations, and / or sequence variants where such changes do not affect the function of the encoded molecule. The term "encode" or "encoding" refers 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.

[0078] The term "gene" may include not only coding sequences, but also regulatory regions such as promoters, enhancers, and termination regions. The term may further include all introns and other DNA sequences spliced ​​from the mRNA transcript, along with variants resulting from alternative splice sites. The sequence may also include degenerate codons of the reference sequence, which may be introduced to provide codon preference in a particular organism or cell type.

[0079] The promoter may include a general promoter, a tissue-specific promoter, a cell-specific promoter, and / or a cytoplasm-specific promoter. The promoter may include a strong promoter, a weak promoter, a constitutive expression promoter, and / or an inducible promoter. An inducible promoter directs 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 affect transcription from the promoter. Particular examples of promoters include minBglobin, CMV, minCMV, mutant minCMV, SV40 immediate early promoter, Hsp68 minimal promoter (proHSP68), and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. A minimal promoter does not have the activity to drive gene expression by itself, but can be activated to drive gene expression when linked to a proximal enhancer element.

[0080] 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 to a vector nucleic acid molecule, e.g., integrated into the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in the cell, or may contain a sequence that allows integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0081] Viral vector is used broadly to refer to a nucleic acid molecule that contains virus-derived nucleic acid elements that facilitate the transfer and expression of non-natural nucleic acid molecules in cells. The term adeno-associated viral vector refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from AAV. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from lentiviruses, etc. The term "hybrid vector" refers to a vector that contains structural and / or functional genetic elements from more than one type of virus.

[0082] Adenovirus. "Adenovirus vector" refers to a construct that contains sufficient adenovirus sequences to (a) support packaging of an expression construct and (b) express coding sequences that have been cloned into it in either the sense or antisense orientation. Recombinant adenovirus vectors include genetically engineered forms of adenovirus. The genetic organization of adenovirus is known to be a 36 kb, linear, double-stranded DNA virus, allowing replacement of large segments 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.

[0083] 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 inverted repeats (ITRs) of 100-200 base pairs, 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 separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and some cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shutoff. The products of the late genes, including most of the viral capsid proteins, are expressed only after significant processing of one primary transcript driven by the major late promoter (MLP). The MLP is particularly efficient during the late stages of infection, and all mRNAs transcribed from this promoter contain a 5'-triple leader (TPL) sequence, making them preferred mRNAs for translation.

[0084] Other than the requirement that the adenoviral vector be replication-deficient, 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 may 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-deficient adenoviral vector for use in certain embodiments, since it is the human adenovirus for which considerable biochemical and genetic information is known and which has been used historically in most constructs using adenovirus as a vector.

[0085] As shown, typical vectors are replication-defective and do not have the adenovirus E1 region. Therefore, it is most convenient to introduce the polynucleotide encoding the gene of interest from the position where the E1-coding sequence has been removed. However, the position of insertion of the construct into the adenovirus sequence is not critical. The polynucleotide encoding the gene of interest may also be inserted in place of the deleted E3 region in an E3 replacement vector or the E4 region when a helper cell line or helper virus complements the E4 deficiency.

[0086] Adeno-associated virus (AAV) is a parvovirus that was discovered as a contaminant of adenovirus strains. It is a ubiquitous virus (antibodies are present in 85% of the US human population) and has not been associated with any disease. It is also classified as a dependovirus because its replication is dependent 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 encapsidated within the capsid proteins VP1, VP2, and VP3 to form icosahedral virions of 20-24 nm in diameter.

[0087] 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 responsible for viral replication, and cap codes for the capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential AAV cis components for chromosomal integration. Thus, AAV can be used as a vector in which all viral coding sequences have been removed and replaced by a cassette of genes for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their map location. Transcription from p5 and p19 leads to the production of rep proteins, and transcription from p40 produces capsid proteins.

[0088] AAV stands out for use within the present disclosure because of its excellent safety profile and because its capsid and genome can be tailored to allow expression in selected cell populations. scAAV refers to self-complementary AAV. pAAV refers to plasmid adeno-associated virus. rAAV refers to recombinant adeno-associated virus.

[0089] Other viral vectors can also be used, for example vectors derived from viruses such as vaccinia virus, poliovirus and herpes virus, which offer several attractive features for a variety of mammalian cells.

[0090] Retroviruses. 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 its 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 direct the expression of RNA molecules that code for the structural proteins and enzymes required to produce new viral particles.

[0091] Examples of 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.

[0092] "Lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary viruses include HIV (human immunodeficiency virus; including HIV types 1 and 2); Visna-Maedi virus (VMV); Caprine 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) may be used.

[0093] Enhanced safety for the use of some vectors may be provided 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 viral Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV) and Herpes Simplex Virus (HSV) (thymidine kinase) promoters. Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. This replacement reduces the likelihood that recombination will result in a replicative virus due to the absence of a complete U3 sequence in the viral production system. In certain embodiments, heterologous promoters have the added advantage of controlling how the viral genome is transcribed. For example, heterologous promoters may be inducible such that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include one or more compounds or physiological conditions such as temperature or pH under which the host cells are cultured.

[0094] 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 increase viral replication. However, this element is not necessary in embodiments in which the U3 region of 5'LTR is replaced by a heterologous promoter.

[0095] "R region" refers to the region within the retroviral LTR that begins at the beginning of the capping group (i.e., the beginning of transcription) and ends just before the beginning of the poly(A) tail. The R region is also defined by being flanked by the U3 and U5 regions. The R region is responsible for moving the nascent DNA from one end of the genome to the other during reverse transcription.

[0096] In certain embodiments, expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally transcription termination signals into the vector. A variety of post-transcriptional regulatory elements can increase 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 others (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.

[0097] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts can increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors contain polyadenylation sequences 3' of the polynucleotide encoding the molecule (e.g., 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 the initial RNA transcript by RNA polymerase II. Polyadenylation sequences promote mRNA stability by the addition of a poly(A) tail to the 3' end of the coding sequence and thus can contribute to increased transcription efficiency. Certain embodiments may use BGHpA or SV40pA. In certain embodiments, preferred embodiments of the expression construct include termination elements. These elements can contribute to increasing transcription levels and minimizing read-through from the construct to other plasmid sequences.

[0098] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may contribute to protecting the viral vector expression sequences, such as effector elements or expressible elements, from integration site effects (i.e., position effects; see, for example, Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001), which are mediated by cis-acting elements present in genomic DNA and may result in deregulated expression of the transferred sequences. In certain embodiments, the viral transport 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 (see 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).

[0099] Beyond the foregoing, a wide variety of suitable expression vector types will be known to those skilled in the art. These may include commercially available expression vectors (e.g., plasmids containing one or more reporter genes and the necessary regulatory elements for expression of the reporter genes in cells) designed for general recombinant procedures. Many vectors are commercially available, for example from Invitrogen, Stratagene, Clontech, etc., and are described in many of the accompanying manuals. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct capable of supporting expression of an encoded gene in mammalian cells (e.g., pUC or Bluescript plasmid series).

[0100] Particular embodiments of the vectors disclosed herein include:

[0101] [Table 1]

[0102] In certain embodiments, SYFP2 in CN1390, CN1244, CN1389, CN1203, CN1367, CN1498, CN1499, CN1500 and CN1838 can be replaced with a channelrhodopsin or calcium reporter, such as ChR2 or GCaMP. In certain embodiments, SYFP2 in CN1390 is replaced with ChR2 or GCaMP. 3XzI46i in CN1838 refers to a 3x concatemer of zebrafish I46iCore. See also FIG. 16, which provides additional exemplary vector components and combinations of the present disclosure.

[0103] In certain embodiments, a viral vector (e.g., AAV) having a capsid that crosses the blood-brain barrier (BBB) ​​is selected. In certain embodiments, the vector is modified to include a capsid that crosses the BBB. Examples of AAVs with viral capsids that 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) and AAV-PPS (Chen et al., Nat Med. 2009;15:1215). The PHP.eB capsid differs from AAV9 such that, when AAV9 is used as a reference, the amino acid starting at residue 586: S-AQ-A (SEQ ID NO:98) is changed to S-DGTLAVPFK-A (SEQ ID NO:33).

[0104] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, is able to cross the BBB after intravenous injection. It transduces large nodes of the central nervous system (CNS), thereby allowing minimally invasive procedures (Naso et al., BioDrugs. 2017;31(4):317), and has been described in the context of clinical trials, for example, for the treatment of superior mesenteric artery (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and for the treatment of CLN3 gene-associated neuronal ceroid lipofuscinosis (NCT03770572).

[0105] AAVrh.10 was originally isolated from rhesus macaques, shows low seropositivity in humans when compared to other common serotypes used for gene delivery applications (Selot et al., Front Pharmacol. 2017;8:441), and is being evaluated in clinical trials LYS-SAF302, LYSOGENE, and NCT03612869.

[0106] Two variants isolated from a library of chimeric AAV vectors (replacing the AAV1 capsid domain within AAVrh.10), AAV1R6 and AAV1R7, retain the ability to cross the BBB and transduce the CNS, while showing significantly reduced liver and vascular endothelial transduction.

[0107] rAAVrh.8 has also been isolated from rhesus macaques and has demonstrated comprehensive transduction of glial and neuronal cell types in clinically important areas following peripheral administration, as well as reduced peripheral tissue tropism compared to other vectors.

[0108] AAV-BR1 is an AAV2 variant that displays the NRGTEWD ​​(SEQ ID NO: 91) epitope, isolated during in vivo screening of a random AAV-display peptide library. It exhibits high specificity with high transgene expression in the brain with minimal off-target affinity (including the liver) (Korbelin et al., EMBO Mol Med. 2016; 8(6): 609).

[0109] AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence QAVRTSL (sequence number 92) that potently transduces neurons in the enteric nervous system and peripheral sensory afferents to enter the spinal cord and brainstem.

[0110] AAV-PHP.B (Addgene, Watertown, Mass.) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO:93), which delivers genes throughout the CNS more efficiently than AAV9 and transduces the majority of astrocytes and neurons across multiple CNS regions.

[0111] AAV-PPS is an AAV2 variant created by insertion of the DSPAHPS (SEQ ID NO: 94) epitope into the capsid of AAV2, and displays dramatically improved brain tropism relative to AAV2.

[0112] For additional information regarding capsids crossing the blood-brain barrier, see Chan et al., Nat. Neurosci. 2017 Aug:20(8):1172-1179.

[0113] (ii) Compositions for Administration. The artificial expression constructs and vectors (herein referred to as physiologically active components) of the present disclosure may 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 may be prepared in neutral form, as free base, or as pharmacologically acceptable salts.

[0114] Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0115] The carrier of the physiologically active ingredient includes solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption retarding agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active ingredients is well known in the art. Except for conventional media or agents that are incompatible with the physiologically active ingredient, they can be used with the compositions described herein.

[0116] The phrase "pharmacologically acceptable carrier" refers to a carrier that does not produce an allergic or similar adverse reaction when administered to humans, and in certain embodiments, when administered intravenously (e.g., retro-orbital plexus).

[0117] In certain embodiments, the compositions may be formulated for intravenous, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, oral, intraperitoneal, oral or nasal inhalation, or for direct injection or application to one or more cells, tissues or organs.

[0118] The composition may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres and / or nanoparticles.

[0119] The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulation half-life have been developed (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).

[0120] The present disclosure also provides pharma- ceutically acceptable nanocapsule formulations of physiologically active components. Nanocapsules can generally encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al., Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119, 1998; Douglas et al., Crit Rev Ther Drug Carrier Syst 3(3):233-261, 1987). To avoid side effects due to intracellular polymer overload, ultrafine particles can be designed with in vivo degradable polymers. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be readily prepared as described in Couvreur et al., J Pharm Sci 69(2):199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlen et al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998; and U.S. Pat. No. 5,145,684.

[0121] 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 via 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 may optionally contain one or more preservative compounds against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, 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. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and / or by the use of surfactants. Prevention of the action of microorganisms may be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation includes an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be accomplished by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Injectable compositions should be suitably buffered if necessary, and the diluent should first be rendered isotonic with sufficient saline or glucose.

[0122] Dispersions may also be prepared in glycols, liquid polyethylene glycols and mixtures thereof, and in oils.As indicated, under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0123] Sterile compositions can be prepared by incorporating physiologically active components in an appropriate amount of solvent together with other optional components (e.g., as listed above), followed by filtration sterilization.Generally, dispersions are prepared by incorporating various sterilized physiologically active components into a sterile vehicle that contains a basic dispersion medium and other desired components (e.g., as listed above).In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation may be vacuum drying and freeze-drying techniques, which produce powders of physiologically active components and any additional desired components from previously sterilized filtered solutions.

[0124] Oral compositions can be in liquid form, for example, solution, syrup or suspension, or can be presented as a preparation that is reconstituted with water or other suitable vehicle before use.Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoate or sorbic acid). The compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients such as binding agents (e.g., pregelatinized maize 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.

[0125] Inhalable compositions may be delivered in the form of aerosol spray preparations from pressurized packs or nebulizers by using suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of pressurized aerosols, dosage units can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0126] Compositions may 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).

[0127] Supplementary active ingredients can also be incorporated into the compositions.

[0128] Typically, the composition will contain at least 0.1% or more of the physiologically active component, although the percentage of the physiologically active component 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. Typically, the amount of the physiologically active component in each physiologically useful composition may be prepared in any given unit dose of the compound in such a way that a suitable dosage is obtained. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and 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.

[0129] 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 appropriate regulatory authorities in other countries.

[0130] (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.

[0131] Although various host cell lines can be used, in certain embodiments, the cells are mammalian neuronal cells. In certain embodiments, the enhancer sequence of the artificial expression construct is SEQ ID NO: 3 and / or 7 and / or CN1390, CN1244, CN1389, CN1203, CN1367, CN1498, CN1499, CN1500, CN1838, or a combination of components shown in FIG. 16, and the cell line is a human, primate, or mouse neuronal cell. Cell lines that can be utilized for gene transfer 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 several neuronal marker proteins in response to nerve growth factor (NGF). The PC12 cell line is considered to be a neuronal cell line and is applicable for use 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 in the embodiments described herein.

[0132] WO91 / 13150 describes various cell lines, including neuronal cell lines, and methods for making them. Similarly, WO97 / 39117 describes neuronal cell lines and methods for making such cell lines. The neuronal cell lines disclosed in these patent applications are applicable for use in the present disclosure.

[0133] In certain embodiments, "neuronal cell" refers to one or more cells located within the central nervous system, including neurons and glia, and cells derived from neurons and glia, including neoplastic and tumor cells derived from neurons or glia. "Neuronal derived cells" refers to cells that are derived from, originate from, or differentiate from neurons.

[0134] In certain embodiments, "neuronal" describes something that is of, relating to, or including a neuronal cell. A neuronal cell is defined by the presence of an axon and a dendrite. The term "neuronal-specific" refers to something or activity that is found or occurs in a neuronal cell or a cell derived from a neuronal cell, but is not found or occurs or does not substantially occur in a non-neuronal cell or a cell that is not derived from a neuronal cell, for example, astrocytes or glial cells such as oligodendrocytes.

[0135] 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, allowing the function of enhancer sequences to be evaluated (see, for example, Fiskerstrand et al., FEBS Lett 458:171-174, 1999).

[0136] A method for differentiating stem cells into neural cells includes replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenite), laminin, and polyornithine. A method for generating myelinating oligodendrocytes from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. Bibel, et al., 2007, Nat. Protoc. 2:1034-43, describes a protocol for generating glutamatergic neurons from stem cells, and Chatzi, et al., 2009, Exp. Neurol. 217:407-16, describes a procedure for generating 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, 95% of GABA neurons develop.

[0137] US Patent Application Publication No. 2012 / 0329714 describes the use of prolactin to increase neural stem cell numbers, and US Patent Application 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 toxoid; and transforming growth factor-α and TGF-β (U.S. Pat. Nos. 5,851,832 and 5,753,506).

[0138] In certain embodiments, the yeast one-hybrid system may also be used to identify compounds that inhibit specific protein / DNA interactions, such as the I56i enhancer, its core and / or the transcription factors of SEQ ID NOs: 3 and / or 7.

[0139] Transgenic animals are described below. Cell lines can be derived from such transgenic animals. For example, primary tissue culture from transgenic mice (e.g., also described below) can provide cell lines with expression constructs already integrated into genome (for example, see MacKenzie&Quinn, Proc Natl Acad Sci USA 96:15251-15255,1999).

[0140] (iv) Transgenic Animals. Another aspect of the present disclosure includes transgenic animals whose genome comprises an artificial expression construct comprising a concatemerization of I56i enhancer cores, such as SEQ ID NO:2 and / or 6 (e.g., SEQ ID NO:3 and / or 7), operably linked to a heterologous coding sequence. In certain embodiments, the genome of the transgenic animal comprises CN1390, CN1244, CN1389, CN1203, CN1367, CN1498, CN1499, CN1500, CN1838, or a combination of components shown in FIG. 16. In certain embodiments, when a non-integrative vector is utilized, the transgenic animal comprises within one or more of its cells an artificial expression construct comprising a concatemerization of I56i enhancer cores such as SEQ ID NO:2 and / or 6 (e.g., SEQ ID NO:3 and / or 7) and / or CN1390, CN1244, CN1389, CN1203, CN1367, CN1498, CN1499, CN1500, CN1838, or a combination of components shown in FIG. 16.

[0141] Detailed methods for making 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.

[0142] In certain embodiments, the construction of transgenic animals results in organisms with engineered constructs present in all cells at the same genomic integration site. Thus, cell lines derived from such transgenic animals will be consistent as long as the engineered construct is at the same genomic integration site in all cells, and therefore will suffer from the same position effect variegation. In contrast, introducing genes into cell lines or primary cell cultures can lead to heterologous expression of the construct. The drawback of this approach is that the expression of introduced DNA can be affected by the specific genetic background of the host animal.

[0143] As indicated above with respect 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 blastocysts from a host mother, and the host embryo is reimplanted into the mother. This results in chimeric mice whose tissues are composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Typically, the mouse from which the cultured ES cells used for gene transfer are derived is selected to have a different coat color than the host mouse whose embryo is injected with the transformed cells. Thus, the chimeric mice have a variety of coat colors. As long as the germline tissue is at least partially derived from the genetically modified cells, the chimeric mice are bred with the appropriate line to generate offspring carrying the transgene.

[0144] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods of delivering artificial expression constructs to target cells or selected tissues and organs of animals, particularly cells, organs or tissues of vertebrate mammals: sonophoresis (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); and feedback controlled delivery (U.S. Pat. No. 5,697,899).

[0145] (v) Methods of Use. In certain embodiments, compositions comprising the physiologically active components described herein are administered to a subject to produce a physiological effect.

[0146] In certain embodiments, the disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a heterologous gene partially or completely encoded in an engineered sequence downstream of an enhancer. Accordingly, provided herein are methods of using the disclosed artificial expression constructs in the research, testing and potential development of pharmaceuticals to prevent, treat or ameliorate symptoms of a disease, dysfunction or disorder.

[0147] Certain embodiments include methods of administering to a subject an artificial expression construct comprising SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:3 and / or SEQ ID NO:7 described herein to drive selective expression of a gene in a selected neural cell type.

[0148] Certain embodiments include methods of administering to a subject an artificial expression construct comprising CN1390, CN1244, CN1389, CN1203, CN1367, CN1498, CN1499, CN1500, CN1838, or a combination of components shown in FIG. 16 described herein, to drive selective expression of a gene in a selected neural cell type, where the subject can be an isolated cell, a network of cells, a tissue section, an experimental animal, a veterinary animal, or a human.

[0149] As is well known in the medical field, the dosage for any one subject will depend on many factors, including the subject's size, surface area, age, the particular compound being administered, sex, time and route of administration, general condition, and other drugs being administered concomitantly. Dosages of the compounds of the present disclosure will vary, but in certain embodiments, the dose will be within the range of 10 to 20 mg of the artificial expression construct of the present disclosure. 5 ~10 100 In certain embodiments, patients receiving intravenous, intraspinal, retroorbital or intrathecal administration may receive 10 copies of the artificial expression construct. 6 ~10 22 A copy may be injected.

[0150] An "effective amount" is the amount of a composition necessary to produce a desired physiological change in a subject. Effective amounts are typically administered for research purposes. Effective amounts disclosed herein may produce statistically significant effects in animal models or in vitro assays.

[0151] In certain embodiments, the constructs disclosed herein can be utilized to treat Dravet syndrome. In certain embodiments, the methods reduce or prevent seizures or symptoms thereof in a patient in need thereof. In certain embodiments, the methods provided can reduce or prevent one or more different types of seizures. Ideally, the methods of the present disclosure result in complete prevention of seizures. However, the present disclosure also encompasses 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%.

[0152] In general, seizures may include convulsions, repetitive movements, paresthesia, and combinations thereof. Seizures may be classified as focal seizures (also called partial seizures) and generalized seizures. Focal seizures occur only on one side of the brain, while generalized seizures occur on both sides of the brain. Specific types of focal seizures include simple focal seizures, complex focal seizures, and secondary generalized seizures. Simple focal seizures may be limited or concentrated in a particular lobe (e.g., temporal, frontal, parietal, or occipital lobe). Complex focal seizures generally occur in a larger part of one hemisphere than simple focal seizures, but generally occur in the temporal or frontal lobe. If a focal seizure spreads from one side (hemisphere) of the brain to both sides, the seizure is called a secondary 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.

[0153] 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 an alternative conventional treatment.

[0154] The amount of expression construct and the time of administration of such compositions are within the purview of one of ordinary skill in the art having the benefit of the present teachings. However, it appears that administration of an effective amount of the disclosed compositions may be accomplished by a single administration, such as, for example, a single injection of a sufficient number of infectious particles to produce an effect in the subject. Instead, in some situations, it may be desirable to provide multiple or successive administrations of the artificial expression construct composition or other genetic construct over a relatively short or 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 as a single dose or divided into two or more doses, as may be required to achieve the intended effect. 7 , 10 8, 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or even more 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.

[0155] In certain circumstances, it may be desirable to deliver the artificial expression constructs of the appropriately formulated compositions disclosed herein by pipette, retro-orbital injection, subcutaneous, intraocular, intravitreal, parenteral, subcutaneous, intravenous, intracerebroventricular, intramuscular, intrathecal, intraspinal, oral, intraperitoneal, by oral or nasal inhalation, or by direct application or injection into one or more cells, tissues or organs. Methods of administration also include those described in U.S. Patent No. 5,543,158; U.S. Patent No. 5,641,515 and U.S. Patent No. 5,399,363.

[0156] (vi) Kits and commercial packages. Kits and commercial packages include the artificial expression constructs described herein. The expression constructs may be isolated. In certain embodiments, the components of the expression product may be isolated from one another. In certain embodiments, the expression product may be in a vector, a viral vector, a cell, a tissue section or sample, and / or a transgenic animal. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or a means for delivery of the composition, such as a syringe, injectable, etc.

[0157] Kit or commercial package embodiments also include instructions for use of the included components, e.g., in basic research, electrophysiological studies, neuroanatomical studies, and / or in the study and / or treatment of a disorder, disease or condition.

[0158] The following illustrative embodiments and experimental examples are included to illustrate specific embodiments of the present disclosure. Those of skill in the art should recognize in light of this 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.

[0159] (vii) Exemplary embodiments.

[0160] 1. I56i enhancer core, I56i enhancer concatemerized core, or concatemerized I56i enhancer.

[0161] 2. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of embodiment 1, wherein the I56i enhancer is human, mouse, or zebrafish (I46i).

[0162] 3. The I56i enhancer core, the concatemerized I56i enhancer core, or the concatemerized I56i enhancer of embodiment 1 or 2, wherein the concatemerized core comprises SEQ ID NO: 2 or 6.

[0163] 4. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of any of embodiments 1 to 3, wherein the concatemerized core comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the I56i core.

[0164] 5. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of embodiment 4, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of SEQ ID NO:2 and / or SEQ ID NO:6 (e.g., SEQ ID NO:2-SEQ ID NO:2-SEQ ID NO:6; SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:6; SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2; and SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:6).

[0165] 6. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of embodiment 4 or 5, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of SEQ ID NO:2.

[0166] 7. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of embodiment 4 or 5, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of SEQ ID NO:6.

[0167] 8. The I56i enhancer core, the concatemerized I56i enhancer core, or the concatemerized I56i enhancer of embodiment 4 or 5, comprising 3 copies of SEQ ID NO:2.

[0168] 9. The I56i enhancer core, the concatemerized I56i enhancer core, or the concatemerized I56i enhancer of embodiment 4 or 5, comprising 3 copies of SEQ ID NO:6.

[0169] 10. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of embodiment 8, wherein the concatemerized core comprises SEQ ID NO:3.

[0170] 11. The I56i enhancer core, concatemerized I56i enhancer core, or concatemerized I56i enhancer of embodiment 9, wherein the concatemerized core comprises SEQ ID NO:7.

[0171] 12. An artificial expression construct comprising: (i) an I56i enhancer core, a concatemerized I56i enhancer core, or a concatemerized I56i enhancer of any of embodiments 1 to 11; (ii) a promoter; and (iii) a heterologous coding sequence.

[0172] 13. The artificial expression construct of embodiment 12, wherein the heterologous coding sequence encodes an effector element or an expressible element.

[0173] 14. The artificial expression construct of embodiment 12 or 13, wherein the effector element comprises a reporter protein or functional molecule.

[0174] 15. The artificial expression construct of embodiment 14, wherein the reporter protein is a fluorescent protein.

[0175] 16. The artificial expression construct of embodiment 14 or 15, wherein the effector element is a functional molecule selected from Cre, iCre, dgCre, FlpE, FlpO or tTA2, or a functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signaling molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a designer receptor activated only by a homologous recombination donor cassette or a designer drug (DREADD).

[0176] 17. The artificial expression construct of any of embodiments 13, wherein the expressible element is a non-functional molecule.

[0177] 18. The artificial expression construct of embodiment 17, wherein the non-functional molecule is a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette or DREADD.

[0178] 19. The artificial expression construct of any of embodiments 12-18, wherein the expression construct is associated with a capsid that crosses the blood-brain barrier.

[0179] 20. The artificial expression construct of embodiment 19, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS.

[0180] 21. The artificial expression construct of any of embodiments 12 to 20, wherein the expression construct comprises or encodes a skipping element.

[0181] 22. The artificial expression construct of embodiment 21, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES).

[0182] 23. The artificial expression construct of embodiment 22, wherein the 2A peptide is selected from T2A, P2A, E2A or F2A.

[0183] 24. The artificial expression construct of any of embodiments 12-23, wherein the expression construct comprises a set of features selected from 3XhI56Core, minBglobin, minCMV, SYFP2, His, 3xHA, NavMs, NavBp, NavSheP-D60N, WPRE3, BGHpA, or a combination of features selected from the constructs shown in FIG. 16.

[0184] 25. A vector comprising the artificial expression construct of any of embodiments 12 to 24.

[0185] 26. A vector containing a combination of components shown in FIG.

[0186] 27. The vector of embodiment 26, wherein the vector is a viral vector.

[0187] 28. The vector of embodiment 26 or 27, wherein the viral vector is a recombinant adeno-associated viral (AAV) vector.

[0188] 29. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is under the control of a promoter and enhancer selected from SEQ ID NO: 3 and / or 7.

[0189] 30. The AAV vector of embodiment 29, which is replicable.

[0190] 31. A transgenic cell comprising the expression construct or vector of any of the preceding embodiments.

[0191] 32. The transgenic cell of embodiment 31, which is a GABAergic interneuron.

[0192] 33. A non-human transgenic animal comprising the expression construct, vector or transgenic cell of any of the preceding embodiments.

[0193] 34. The non-human transgenic animal of embodiment 33, which is a mouse or a non-human primate.

[0194] 35. An administrable composition comprising the expression construct, vector or transgenic cell of any of the preceding embodiments.

[0195] 36. A kit comprising the expression construct, vector, transgenic cell, transgenic animal, and / or administrable composition of any of the preceding embodiments.

[0196] 37. A method for selectively expressing a heterologous gene in a population of neuronal cells in vivo or in vitro, comprising providing to a sample or subject comprising the population of neuronal cells an administrable composition of embodiment 35 in a sufficient dosage and for a sufficient period of time, thereby selectively expressing the gene in the population of neuronal cells.

[0197] 38. The method of embodiment 37, wherein the heterologous gene encodes an effector element or an expressible element.

[0198] 39. The method of embodiment 38, wherein the effector element comprises a reporter protein or a functional molecule.

[0199] 40. The method of embodiment 39, wherein the reporter protein is a fluorescent protein.

[0200] 41. The method of embodiment 39 or 40, wherein the effector element is a functional molecule selected from Cre, iCre, dgCre, FlpE, FlpO or tTA2, or a functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signaling molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a homologous recombination donor cassette or a DREADD.

[0201] 42. The method of embodiment 38, wherein the expressible element is a non-functional molecule.

[0202] 43. The method of embodiment 42, wherein the non-functional molecule is a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette or DREADD.

[0203] 44. The method of any of embodiments 37-43, wherein providing comprises pipetting.

[0204] 45. The method of embodiment 44, wherein the pipetting is to a brain slice.

[0205] 46. ​​The method of embodiment 45, wherein the brain slice comprises GABAergic interneurons.

[0206] 47. The method of any of embodiments 45 or 46, wherein the brain slice is murine, human or non-human primate.

[0207] 48. The method of any of embodiments 37-43, wherein providing comprises administering to a living subject.

[0208] 49. The method of embodiment 48, wherein the living subject is a human, a non-human primate, or a mouse.

[0209] 50. The method of any of embodiments 48 or 49, wherein administering to a living subject is by injection.

[0210] 51. The method of embodiment 50, wherein the injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternomagnathus (ICM) injection or intrathecal injection.

[0211] 52. An artificial expression construct consisting of, or consisting essentially of, a combination of the features shown in FIG.

[0212] 53. An ion transporter selected from a voltage-gated sodium channel (e.g., SCN1A), a potassium channel (e.g., KCNQ2) or a calcium channel (e.g., CACNA1C); a cellular transport protein selected from clathrin, dynamin, caveolin, Rab-4A or Rab-11A; an enzyme selected from lactase, lipase, helicase, α-glucosidase and amylase; a transcription factor selected from SP1, AP-1, heat shock factor protein 1, C / EBP (CCAA-T / enhancer binding protein) and Oct-1; a receptor selected from transforming growth factor receptor β1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor and interleukin-8 receptor α; nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF) and a GTPase. signaling molecules selected from HRas; neurotransmitters selected from cocaine and amphetamine regulated transcripts, substance P, oxytocin and somatostatin; genetically encoded calcium indicators (GECI, NTnC, GCaMP6s, GCaMP6f, GCaMP6m, jGCaMP7s, jGCaMP7f, jGCaMP7b, jGCaMP7c, jRGECO1a, jRGECO1b), myosin light chain kinase, green fluorescent protein, calmodulin chimera, calcium indicator TN-XXL, BRET-based autoluminescent calcium indicator Any of the above embodiments, comprising a calcium reporter selected from a calcium indicator and a calcium indicator protein OeNL(Ca2+)-18u); a channelrhodopsin selected from channelrhodopsin-1 and channelrhodopsin-2 or a variant thereof; a guide RNA; a nuclease selected from Cas, Cas9, Cpf1, ribonuclease 4 and deoxyribonuclease IIβ; and / or an effector element or expressible element which is a DREADD (e.g., hM3DREADD, hM4DREADD).

[0213] In this disclosure, where the context describes reference or use of the zebrafish forms of the enhancers described herein, I56i should be interpreted as I46i.

[0214] (viii) Experimental Examples Dravet syndrome (DS) is a drug-resistant and life-threatening form of epilepsy. It typically begins in the first year of life with fever- or temperature-induced seizures that progress to generalized clonic, tonic-clonic and unilateral seizures. These seizures are usually resistant to current antiepileptic drugs that are the first-line treatment for this syndrome, and complete seizure control is typically not achieved. As the disease progresses, most affected children also suffer from comorbidities including developmental delay, intellectual disability, impaired motor control and coordination, autistic behavior, sleep disorders, and many die early.

[0215] Heterozygous loss-of-function mutations in SCN1A, the gene encoding the pore-forming subunit of the voltage-gated sodium channel Nav1.1, are the most common cause of DS, occurring in approximately 1 / 16,000 newborns.

[0216] Mouse models generated by knockout of Scn1a recapitulate several key phenotypic features of this epilepsy, including infantile (P21)-onset epilepsy, high susceptibility to fever seizures, ataxia, spontaneous seizures, sleep disorders, autistic behavior, and premature death. Seizures and several comorbidities result from impaired interneuron function in these mice.

[0217] This mouse model was used to investigate the efficacy of new viral vectors against DS. The virus was delivered by retro-orbital injection using an insulin syringe, and its ability to suppress seizures was evaluated using a thermal seizure test. In this test, a thermostat and heat lamp are used to slowly increase the core body temperature of the mouse until seizures occur or until a temperature of 42.5°C is reached. The body temperatures at seizure onset of treated and control mice are compared to determine the efficacy of the intervention. In additional studies, the efficacy of the treatment on spontaneous seizures and early mortality is evaluated using video and electroencephalogram monitoring.

[0218] The viral vector is a novel AAV viral vector named CN1500. This viral vector is a recombinant AAV expressing the transgene SYFP2-P2A-NavSheP-D60N to rescue the defect of the voltage-gated sodium channel Nav1.1. NavSheP-D60N is a bacterially derived modified voltage-gated sodium channel modified to improve kinetics and expression in mammalian cells. The expression level of the transgene is increased by the addition of a WPRE3 element and transcription is terminated with a bovine growth hormone polyadenylation sequence. Transgene expression is high and restricted to inhibitory cells of forebrain structures including the cortex and hippocampus via the 3xhI56iCore synthetic enhancer (SEQ ID NO: 3) immediately 5' of the CMV minimal promoter. Additionally, the therapeutic transgene NavSheP-D60N is tagged with an HA epitope tag to confirm correct protein localization.

[0219] To test the efficacy of therapeutic AAV viral vectors, the CN1500 package with the PHP.eB serotype was used. + / - Cohorts of mice were dosed with 2x10 per animal. 11vg or left uninjected. AAV was introduced intravenously using a retro-orbital delivery route. Two weeks after virus administration, treated and control animals were assessed for susceptibility to febrile seizures. As shown before, febrile seizures were measured by steadily raising the mouse's temperature under a heat lamp at 0.5 degrees Celsius every 2 minutes and measuring the mouse's internal body temperature with a rectal probe. The temperature at which the mouse had a seizure is recorded.

[0220] The novel therapeutic vector CN1500 was highly expressed in GABAergic cells in both the mouse cortex and hippocampus, but not in Scn1a cells that had undergone febrile seizures. + / - The mean body temperature of the mice also increased from 38.7° C. to 41° C. These data indicate that CN1500 can substantially rescue the deficiency of Scn1a.

[0221] References for Example 1 include: Catterall et al. (2010) The Journal of physiology 588:1849-1859; Cheah et al. (2012) Proceedings of the National Academy of Sciences of the United States of America 109:14646-14651; Kalume (2013) Respir Physiol Neurobiol. 189(2):324-8;Kalume et al.,(2007)J Neurosci 27:11065-11074;Kalume et al.,(2013)The Journal of clinical investigation 123:1798-1808;Oakley et al.,(2009)Proceedings of the National Academy of Sciences of the United States of America 106:3994-3999.

[0222] (ix) Concluding paragraph. The nucleic acid sequences described herein are shown using standard letter abbreviations for the nucleotide bases as defined in 37 C.F.R. 1.822. Only a single strand of each nucleic acid sequence is shown, although it is understood that the complementary strand is included in the embodiments, where appropriate.

[0223] Variants of the sequences disclosed and referred to herein are also included. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without impairing biological activity can be found using computer programs such as DNASTAR™ (Madison, Wisconsin) software, which are well known in the art. 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 include the substitution of a family of amino acids that are related in their side chains.

[0224] In a peptide or protein, suitable conservative amino acid substitutions 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 as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, non-polar residues): isoleucine ( Group 10 (small aliphatic, non-polar or slightly polar residues): Ala, Ser, Thr, Pro and Gly; and Group 12 (sulfur-containing): Met and Cys. Further information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0225] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are: 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).

[0226] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophobicity indexes and scores and still result in proteins with similar biological activity, i.e., still obtain biologically functional equivalent proteins. In making such changes, substitution of amino acids with hydrophobicity indexes of ±2 is 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 substitution of similar amino acids can be effectively made on the basis of hydrophilicity.

[0227] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Aspartate (+3.0±1); Glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular an immunologically equivalent, protein. In such changes, substitutions of amino acids with hydrophilicity values ​​of ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0228] As outlined 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.

[0229] As noted elsewhere, variants of a gene sequence may include codon-optimized variants, sequence polymorphisms, splicing variants, and / or mutations that do not affect the function of the encoded product to a statistically significant degree.

[0230] 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 and gene sequences disclosed herein.

[0231] "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" (also often referred to as "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and percent identity calculations can 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 alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)).Suitable programs 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). York, NY. Within the context of this disclosure, when sequence analysis software is used, it will be understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" refers to any set of values ​​or parameters that are initially loaded with the software when the software is first initialized.

[0232] Variants also include nucleic acids that hybridize to sequences disclosed herein under stringent hybridization conditions and provide the same function as the reference sequences. Exemplary stringent hybridization conditions include: 50% formamide, 5X SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured sheared salmon sperm DNA, incubated overnight at 42°C, followed by washing the filter with 0.1X SSC at 50°C. Alteration of the stringency of hybridization and signal detection is achieved initially through manipulation of formamide concentration (low percentage of formamide results in low stringency); salt conditions or temperature. For example, moderately high stringency conditions include overnight incubation at 37° C. in a solution containing 6XSSPE (20XSSPE=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 a wash with 1XSSPE, 0.1% SDS at 50° C. In addition, to achieve lower stringency, stringent hybridization may be followed by a wash with a higher salt concentration (e.g., 5X SSC). Variations of the above conditions may 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.

[0233] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein may comprise, consist essentially of, or consist of the specific referenced elements, steps, ingredients, or components. Thus, the term "include" or "including" should be interpreted to enumerate: "comprise, consist of, or consist essentially of." The transition term "comprise" or "comprises" means including, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in large amounts. The transitional phrase "consisting of" excludes all unspecified elements, steps, ingredients, or components. 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 materially affect the embodiment. A substantial effect would result in a statistically significant reduction in selective expression in the targeted cell population as determined by scRNA-Seq and the following enhancer / targeted cell population pairing: concatemerized core of I56i enhancer (e.g., SEQ ID NO:3) / GABAergic interneurons.

[0234] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, properties such as molecular weights, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without intending to limit the application of the doctrine of equivalents to the scope of the claims, all numerical parameters should at least 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", when used in conjunction with a stated numerical value or range, has the meaning reasonably ascribed to the term by a person of ordinary skill in the art, i.e., indicating something more or something less than the stated numerical value or range, and indicates within the range of ±20% of the stated numerical value; ±19% of the stated numerical value; ±18% of the stated numerical value; ±17% of the stated numerical value; ±16% of the stated numerical value; ±15% of the stated numerical value; ±14% of the stated numerical value; ±13% of the stated numerical value; ±12% of the stated numerical value; ±11% of the stated numerical value; ±10% of the stated numerical value; ±9% of the stated numerical value; ±8% of the stated numerical value; ±7% of the stated numerical value; ±6% of the stated numerical value; ±5% of the stated numerical value; ±4% of the stated numerical value; ±3% of the stated numerical value; ±2% of the stated numerical value; or ±1% of the stated numerical value.

[0235] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0236] Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," "the," and similar referents used in the context of describing the present invention (particularly in the context of the claims that follow) are intended to cover both the singular and the plural. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate numerical 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 may 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., "such as") presented herein is intended only to better illuminate the invention and is not intended to pose limitations on the scope of the invention as claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0237] The categorization of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be incorporated into or removed from a group for convenience and / or patentability. When any such inclusion or exclusion occurs, the specification is deemed to include the group as modified to fulfill all Markush group descriptions used in the appended claims.

[0238] Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Naturally, modifications of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such modifications will be employed by those skilled in the art as appropriate, and the inventors intend the invention to be practiced otherwise 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.

[0239] Additionally, numerous references are made throughout this specification to patents, publications, journal articles, and other writings (the "references" herein). Each of the references is individually incorporated herein by reference in its entirety for the teachings referenced thereto.

[0240] Finally, it is to be understood that the embodiments of the invention disclosed herein illustrate the principles of the invention. Other modifications that may be used 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.

[0241] The details shown in this specification are by way of example and are merely for the purpose of illustrative discussion of preferred embodiments of the present invention, and are presented to provide what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, there is no intention to show the structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention, and the present description, taken together with the figures and / or examples, will make clear to those skilled in the art how some forms of the present invention may be actually realized.

[0242] The definitions and explanations used in this disclosure are meant and intended to govern any future constructions unless explicitly and unambiguously modified in the following examples, or if the application of the meanings gives rise to any structural or substantive contradictions. If the construction of a term becomes meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition, or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. (i) A concatemer that is 2, 3, 4, 5, or 6 copies of SEQ ID NO: 2 or SEQ ID NO: 6, or A concatemer that is 2, 3, 4, 5, or 6 copies of a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2 or SEQ ID NO: 6 and having the same enhancer activity as a concatemer that is 2, 3, 4, 5, or 6 copies of SEQ ID NO: 2 or SEQ ID NO: 6, (ii) A promoter, and (iii) An artificial expression construct comprising a coding sequence. 】

2. The artificial expression construct according to claim 1, wherein the coding sequence encodes a fluorescent protein or an ion transporter selected from a potassium channel, a calcium channel, or a voltage-gated sodium channel. 】

3. The artificial expression construct according to claim 1, wherein the artificial expression construct is bound to a nucleic acid sequence encoding a capsid comprising PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 】

4. The artificial expression construct according to claim 1, wherein the artificial expression construct comprises or encodes a skipping element comprising T2A, P2A, E2A, F2A, or an internal ribosome entry site (IRES). 】

5. The artificial expression construct according to claim 1, which is within a vector for delivery to a cell. 】

6. The artificial expression construct according to claim 5, wherein the vector is a viral vector. 】

7. The artificial expression construct according to claim 6, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector. 】

8. A transgenic cell comprising the artificial expression construct according to claim 1. 】

9. The transgenic cell according to claim 8, which is a GABAergic interneuron. 】

10. The transgenic cell according to claim 8, which is a lysosomal membrane protein 5 (LAMP5) neuron, a vasoactive intestinal peptide (Vip) neuron, a somatostatin (Sst) neuron, or a parvalbumin (Pvalb) neuron. 】

11. The transgenic cell according to claim 8, which is derived from a mouse, a human, or a non-human primate. 】

12. A composition for use in a method for selectively expressing a coding sequence within a population of nerve cells in vivo or in vitro, The method comprises providing an administrable composition comprising an artificial expression construct to a sample or subject comprising the population of neurons, at a sufficient dosage and for a sufficient time, thereby selectively expressing the coding sequence within the population of neurons, wherein the artificial expression construct comprises (i) a concatamer that is 2, 3, 4, 5, or 6 copies of SEQ ID NO: 2 or SEQ ID NO: 6, or a concatamer that is 2, 3, 4, 5, or 6 copies of a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 6 and having the same enhancer activity as a concatamer that is 2, 3, 4, 5, or 6 copies of SEQ ID NO: 2 or SEQ ID NO: 6, (ii) a promoter, and (iii) a coding sequence and the composition. **Claim 13**: The composition according to claim 12, wherein the coding sequence encodes a fluorescent protein or an ion transporter selected from a potassium channel, a calcium channel, or a voltage-gated sodium channel. **Claim 14**: The composition according to claim 12, wherein the providing comprises pipetting to a brain slice comprising GABAergic interneurons. **Claim 15**: The composition according to claim 14, wherein the brain slice comprises LAMP5 neurons, VIP neurons, Sst neurons, or Pvalb neurons. **Claim 16**: The composition according to claim 12, wherein the providing comprises administering to a living subject. **Claim 17**: The composition according to claim 16, wherein the living subject is a human, a non-human primate, or a mouse. **Claim 18**: The composition according to claim 16, wherein administering to the living subject is by injection. **Claim 19**: The composition according to claim 18, wherein the injection comprises intravenous injection, intracerebral parenchymal injection, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection. **Claim 20**: An artificial enhancer comprising 1, 2, 3, 4, 5, or 6 copies of the sequence consisting of SEQ ID NO: 2 or SEQ ID NO: 6, or 1, 2, 3, 4, 5, or 6 copies of a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 2 or SEQ ID NO: 6 and having the same enhancer activity as 1, 2, 3, 4, 5, or 6 copies of SEQ ID NO: 2 or SEQ ID NO: 6 ​

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