Artificial expression constructs for modulating gene expression in astrocytes using autoregulators for the assessment and / or treatment of SLC6a1 disorders

Abstract

Artificial expression constructs for modulating gene expression in astrocytes and / or neurons are described. The artificial expression constructs can be used to express synthetic genes or modify gene expression in for the assessment and / or treatment of SLC6A1-related disorders, such as epilepsy, seizures, and autism spectrum disorder. In particular embodiments, the artificial expression constructs include an autoregulator, an enhancer, a promoter, and an SLC6A1 coding sequence.

Description

ARTIFICIAL EXPRESSION CONSTRUCTS FOR MODULATINGGENE EXPRESSION IN ASTROCYTES USING AUTOREGULATORS FOR THE ASSESSMENT AND / OR TREATMENT OF SLC6A1 DISORDERSCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 776,849 filed on March 24, 2025, which is incorporated herein by reference in its entirety as if fully set forth herein.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3LK8976.xml. The file is 159,234 bytes, was created March 24, 2026, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The current disclosure provides artificial expression constructs for modulating gene expression in astrocytes and / or neurons. The artificial expression constructs can be used to express synthetic genes or modify gene expression in astrocytes and / or neurons for the assessment and / or treatment of SLC6A1 -related disorders, such as epilepsy, seizures, and autism spectrum disorder. The artificial expression constructs generally include an autoregulator, an enhancer, a promoter, and an SLC6A1 coding sequence.BACKGROUND OF THE DISCLOSURE

[0004] Solute carrier family 6 member 1 (SLC6A1) encodes for the gamma-aminobutyric acid (GABA) transporter protein type 1, GAT-1, which is responsible for the removal of GABA from the synaptic cleft. GABA is the primary inhibitory neurotransmitter and GABA ion channel is primarily localized to axons and nerve terminals of GABAergic interneurons. As a member of the neurotransmitter sodium symporters family of proteins, GAT-1 couples the transport of GABA with ion exchange through the GAT-1 channel via the exchange of 1 GABA molecule for 2 sodium ions and 1 chloride ion. The GAT-1 transporter is specifically responsible for the reuptake of GABA into the presynapse following the firing of the interneuron. Without a functional GAT-1 channel, GABA builds up in the synaptic cleft, which can increase the inhibitory activity of the interneuron.

[0005] Although mutations in SLC6A1 are not considered diagnostic hallmarks of epilepsy or autism spectrum disorder, recent studies have found that the gene may play a significant role in these disorders. In patients with diagnosed mutations in the SLC6A1 gene, the variability in the mutational positions are striking including missense mutations, splice-site variants, frameshift mutations, nonsense mutations, and in-frame deletions.SUMMARY OF THE DISCLOSURE

[0006] The current disclosure provides artificial expression constructs for modulating gene expression in astrocytesand / or neurons. The artificial expression constructs can be used to express synthetic genes or modify gene expression in astrocytes and / or neurons for the assessment and / or treatment of SLC6A1 -related disorders, such as epilepsy, seizures, and autism spectrum disorder. In particular embodiments, the artificial expression constructs include an autoregulator, an enhancer, a promoter, and an SLC6A1 coding sequence.

[0007] In particular embodiments, the autoregulator includes a microRNA (miRNA) scaffold, a miRNA guide strand, and a miRNA binding site. In particular embodiments, the miRNA scaffold includes a miR9-1 scaffold, a mir30d scaffold, or a mir181 a2 scaffold. In particular embodiments, the miRNA guide strand includes an AI2 miRNA guide strand, an AI3 miRNA guide strand, or an AI4 miRNA guide strand. In particular embodiments, the miRNA binding site includes an AI2 miRNA binding site, an AI3 miRNA binding site, or an AI4 miRNA binding site.

[0008] In particular embodiments, the enhancer includes eHGT_380h. In particular embodiments, the promoter includes a minimal promoter (e.g., minBG promoter). In particular embodiments, the SLC6A1 coding sequence includes intronSLC6A1. In particular embodiments, intronSLC6A1 includes an SLC6A1 coding sequence including introns. In particular embodiments, the miRNA scaffold and miRNA guide strand are within an intron of the SLC6A1 coding sequence.

[0009] Particular embodiments provide artificial expression constructs including the features of vectors described herein including vectors: CN5517, CN5518, CN5519, CN5520, CN5521, CN5522, CN5523, CN5524, and CN5525.BRIEF DESCRIPTION OF THE FIGURES

[0010] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0011] FIGs. 1A-1D. Developing autoregulatory vectors to control expression and improve safety. (1A) Small RNA-seq study to identify candidate miRNA scaffolds for synthetic autoregulatory cassettes. Somatosensory cortex samples from twelve mice were analyzed for small RNA content using a direct adapter library preparation technique and sequencing via the Illumina platform. The twelve mice consisted of Slc6a1+ / +, Slc6a1+ / ~, and Slc6a1-'- mice, which were injected at postnatal day 21 (P21) with either phosphate buffered saline (PBS) or a human SLC6A1 expression adeno-associated virus (AAV) vector (1 e11 vector genomes, vg), and analyzed at P56 in technical duplicate (3 genotypes x 2 treatments x 2 replicates = 12 mice). Overall little variation in miRNA content was observed due to genotype or treatment. Candidate scaffold miRNA species for developing a synthetic miRNA autoregulatory cassette were: highly expressed regardless of experimental conditions, with strong preferences for guide strand over passenger strand processing (>1000x guide strand selectivity), and with well-defined guide-strand 22mer species (sharp cutoffs in sequencing coverage). Mir9-1, Mir30d, Mir181a2, Mir181a1, Mir30a, and Mir9-2 were top candidates, and the first three of these were cloned and tested (see FIGs. 3A-3C). (1B) Autoregulatory expression vector design. The autoregulatory cassette includes: an intron flanked by splice donor (SD) and splice acceptor (SA) sites, an engineeredMir30d scaffold (hairpin) containing an artificial AI2 guide and an engineered artificial opposite passenger strand that preserves the same base-pairing as the native Mir30d sequence, and finally an Al 2-binding site (1X AI2 BS) with perfect complementarity in the 3'UTR of the transgene. Other elements of this vector include: eHGT_380h (an astrocytespecific enhancer), a minimal promoter (minP), a codon-optimized artificial human SLC6A1 transgene (hSLC6A1) with the internally placed intron and artificial miRNA cassette, WPRE3 and BGHpA elements for transcript stability and maturation, and left and right inverted terminal repeats (ITRs) (L-ITR and R-ITR) for packaging into AAVs. This construct represents a functional SLC6A1 transgene containing a fully self-contained autoregulatory sequence (‘v2.0” in FIG. 1D). (1C) Testing regimen for determining functionality of autoregulators. Constructs were generated and packaged into AAV, here using the PHP.eB capsid for intravenous (IV) delivery in C57BI6 mice. Vectors were injected at P21 at 1e11 vg per animal dose, and at P42 brain tissue was analyzed for construct expression levels using flow cytometry / fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), western blot, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). (1D) Data summary for testing autoregulatory cassettes. The metrics used for validating functional autoregulation were: lowered expression at high dose of vector (measured by FACS, IHC, western, and RT-qPCR), a shallowed dose-expression curve (sub-proportionality of increased expression with increased dose, measured by FACS, IHC, western, and RT-qPCR), and a narrowed range of expression at high dose of vector (among cells within a sample, measured by FACS). The initial construct design (“v1.0”) utilized strong miRNA scaffold and guide RNA sequences that were published previously. The v1.0 was used first in experiments to test expression of an enhanced green fluorescent protein (EGFP) reporter in human embryonic kidney (HEK) cells, which showed functional autoregulation on all three metrics (see FIGs. 2A-2C). Similarly, the v1.0 design showed functional autoregulation of SLC6A1 in HEK cells, and of SLC6A1 in brain astrocytes. A weaker autoregulator was designed (“v2.0”) as shown in FIG. 1B. This resulted in less potent lowering of expression at high dose, while maintaining the shallowed dose-expression curve and narrowed range of expression with v2.0. Data on v1.0 are shown below in FIGs. 2A-2C. Data on v2.0 are shown in FIGs. 3- 7. Values are reported as the range of measured values across replicate experiments (n=2-3 experiments each).

[0012] FIGs. 2A-2C. A functional autoregulatory cassette that strengthens autoregulation through miRNA binding site multiplicity. (2A) Experiment to measure functional autoregulation in HEK cells. Cells were transfected with chicken actin promoter (CAG)-EGFP or modified CAG-EGFP vectors containing autoregulatory control elements, along with polyethyleneimine-MAX (PEI). After 48 hours the cells were collected and analyzed for EGFP reporter expression by flow cytometry. Empty vector shows little GFP fluorescence (left). Positive control CN3837 CAG-EGFP, as well as negative control autoregulatory elements containing miRNA alone (CN5103) or miRNA binding site (BS) alone (CN5105), all show high levels of EGFP fluorescence. However, combining the miRNA with the BS together in CN5111 results in a strong diminishment of expression levels, indicating functional autoregulation. The autoregulatory elements in CN5111 represent “v1.0” in Figure 1D. Data shown here at high vector dose (1000ng). Lower doses (1 OOng or 10ng) used balance empty carrier DNA for 1000ng constant total DNA delivered. (2B) Autoregulation demonstrated bylowered expression levels at high dose, and shallowed dose-response curve. EGFP signal levels were measured in cells transfected with varying doses of various vectors. Parent CAG-EGFP vector (CN3837) and all negative control autoregulators (CN5103-CN5110) all show high expression levels, as well as high slope and pearson correlation coefficient (PCC) of dose-expression curve relating levels to dose (ng DNA transfected). Functional autoregulators (CN5111-CN5116) show much lower slope of the dose-expression curve, as well as much less expression at high dose (1000ng). Furthermore, 2x and 4x BS constructs (CN5112 and CN5113) show lowered expression relative to 1XBS (CN5111), suggesting multiplicity of binding can strengthen autoregulation further. EGFP signal levels were transformed as logio(signal + 1), where the signal is the geometric mean of all the positively expressing cells in the transfected cell population. Each dot represents an independent experiment. From left to right (i.e., CN3837 to CN5116), the slope of dose expression curve is: 7.29, 6.56, 6.61, 4.32, 6.91, 6.13, 8.12, 6, 8.13, 0.35, 0.02, -0.03, 0.22, 0.21, and 0.27; and the PCC of dose-expression curve is: 0.9946, 0.9895, 0.9904, 0.9831, 0.9931, 0.9932, 0.9931, 0.9959, 0.9988, 0.9979, 0.2955, -0.2939, 0.9977, 0.999, and 0.9995. (2C) Autoregulation demonstrated by diminished dispersion of expression within populations of expressing cells. EGFP signal levels in populations of transfected cells were used to compute the robust coefficient of variation (rCV), here calculated as the robust standard deviation scaled to the median, which is an outlier-proof scalar metric of dispersion within a dataset. Autoregulated constructs (CN5111-CN5116) showed lowered robust coefficients of variation as compared to the negative control constructs (CN3837 and CN5103-CN5110), suggesting tighter distribution of expression values.

[0013] FIGs. 3A-3C. Autoregulation of SLC6A1 expression levels in mouse brain astrocytes. (3A) Testing regimen for determining functionality of autoregulators in astrocytes. SLC6 1-expressing constructs were generated and packaged into AAV using astrocyte-specific enhancer elements along with a FLAG epitope for detection, into PHP.eB capsid for intravenous delivery in C57BI6 mice. Vectors were injected at P21 at a dose of 1e11 vg per animal delivered into the bloodstream retro-orbitally (RO). At P42 brain tissue was analyzed for construct expression levels using fixed cell flow cytometry / FACS, IHC, western blot, and RT-qPCR. (3B) Flow cytometry to measure levels of transgenic SLC6A1 expression in astrocytes. Phycoerythrin-anti Neuronal antigen (PE-NeuN) was used to identify neurons, and Fluorescein isothiocarbonate anti-FLAG (FITC-FLAG) was used to measure transgene expression levels in nonneurons. Cells from motor cortex were dissociated, fixed, stained, and analyzed. Uninjected animals show background levels of FLAG expression, and positive control parental non-autoregulated CN4419-injected animals show high levels of FLAG expression in non-neurons. Nine different candidate v2.0 autoregulatory cassettes were tested (CN5517-CN5525) with three different miRNA scaffolds and three different synthetic guide sequences. Some vectors showed little autoregulatory activity, resulting in expression like the parental CN4419 (CN5517, CN5520, CN5523). Some vectors showed very high autoregulatory activity which precluded transgene detection (CN5522, CN5524, CN5525). Some vectors showed a moderate amount of autoregulatory activity which could constrain expression to a therapeutic level (CN5518, CN5519, and CN5521 which is starred as exemplary). (3C) Quantification of expression levels of autoregulated SLC6A1 vectors. These reflect the data shown in FIG. 3B, one mouse each condition, confining analysisonly to FLAG+ cells in the gate in FIG. 3B. Top', the expression levels of FLAG-tagged SLC6A1 protein in astrocytes of these animals. Each dot represents a single cell (single astrocyte), crossbars represent means and standard deviations of expression levels, y-axis is on logarithmic scale, and mean expression values are given. Autoregulated constructs show lowered mean expression values than the parental CN4419 vector, as well as more narrow ranges of expression values. Exemplary CN5519 shows 3-fold lower mean expression than CN4419. Bottom', the rCV of expression values across expressing astrocytes, here calculated as the robust standard deviation scaled to the median, and used as a scalar metric of expression spread / dispersion. Functional autoregulators show reduced rCV values compared to parental CN4419. Exemplary CN5519 shows 2-fold lower rCV as compared to CN4419.

[0014] FIGs. 4A, 4B. Autoregulators reduce intensity and variability of SLC6A f-myc-FLAG transgene expression in cortical astrocytes, as measured by IHC. (4A) Reduced expression and variability of myc expression as assessed by IHC. Mice were injected RO at P22 with astrocyte-specific vectors delivering myc- and FLAG-tagged SLC6A1 transgene at 1e11 or 3e10vg per animal, and expression levels were assessed with anti-myc IHC at P44. Shown is a cortical column of primary visual cortex (VI Sp) in one of two representative animals. Compared to the parental CN4419 vector, the autoregulated vectors (CN5518, CN5519, and CN5521) show less and more even expression levels among transduced cells, as well as more similar levels between 3e10 and 1e11vg doses. (4B) Reduced expression and shallowed dose-expression curve from autoregulated constructs, as assessed by quantitative IHC. Cortical IHC as shown in FIG. 4A was used for quantification by finding the mean pixel intensity over an RO 0.5 x 1 mm in size, covering hundreds of gray matter astrocytes. These mean pixel intensities were background subtracted using uninjected negative control tissue. Each dot represents one animal (n = 2 per condition), and the fold-change of expression between doses and between vectors at the high 1 e11 vg dose was calculated. Parental vector CN4419 shows a strong diminishment in expression strength when doses are decreased between the 1e11 and 3e10vg doses, but the doseexpression diminishment is much less in the autoregulated vectors (CN5518, CN5519, and CN5521). Furthermore, the maximal expression levels at the high dose is much less for the autoregulated vectors. Negative control non-injected animals show minimal levels of detection.

[0015] FIGs. 5A, 5B. Autoregulators reduce intensity and variability of SLC6A f-myc-FLAG transgene expression in cortical astrocytes, as measured at the bulk RNA and protein levels. (5A) Reduced expression and shallowed doseexpression curve from autoregulated constructs, measured at the transcript level by digital droplet reverse transcri ption-polymerase chain reaction (ddRT-PCR), a form of RT-qPCR. Visual cortex (VISp) tissue was collected from the contralateral hemisphere of the animals shown in FIG. 4B, and RNA was extracted and prepared for ddPCR analysis of transgenic transcript levels. Parental vector CN4419 shows a strong diminishment in expression strength when doses are decreased between the 1 e11 and 3e10vg doses, but the dose-expression diminishment is much less in the autoregulated vectors (CN5518, CN5519, and CN5521). Furthermore, the maximal expression levels at the high dose is much less for the autoregulated vectors than for CN4419. Negative control non-injected animals show minimal levels of detection. (5B) Reduced expression and shallowed dose-expression curve from autoregulated constructs, measuredat the bulk protein level using western blot (WB) of immunoprecipitated protein products. Somatosensory cortex was harvested from the contralateral hemisphere of the animals shown in FIG 4B, crude protein was prepared, and immunoprecipitated (IP) with anti-FLAG-conjugated paramagnetic microbeads. The IP protein was then analyzed by WB using anti-GAT-1 antibodies (the protein produced by the SLC6A1 gene) to measure abundance. Parental vector CN4419 shows a strong diminishment in expression strength when doses are decreased between the 1e11 and 3e10vg doses, but the dose-expression diminishment is less in the autoregulated vectors (CN5518 and CN5521). Furthermore, the maximal expression levels at the high dose is much less for the autoregulated vectors than for CN4419. Negative control non-injected animals show minimal levels of detection.

[0016] FIGs. 6A-6C. Autoregulators reduce intensity and variability of SLC6A f-myc-FLAG transgene expression in cortical astrocytes, as measured using flow cytometry. (6A) Reduced expression and shallowed dose-expression curve from autoregulated constructs, measured at the single cell level using anti-FLAG flow cytometry. Mice were injected RO at P22 with astrocyte-specific vectors delivering myc- and FLAG-tagged SLC6A1 transgene at 1 e11 or 3e10vg per animal (the same mice as shown in FIGs. 4A-5B). Motor cortex cells were harvested and prepared for fixed cell flow cytometry with PE-NeuN and FITC-FLAG as well as rabbit anti-GAT-1 detected with 647-goat anti rabbit secondary antibody. Uninjected negative control animals show minimal detection of FLAG+ astrocytes as expected, but mice injected with parental vector CN4419 show dose-dependent expression of FLAG, and CN5518-injected mice show relatively dose-independent levels of expression. (6B) Reduced FLAG expression, and less dispersion of FLAG expression, in transduced astrocytes using fixed cell flow cytometry. Each dot represents a single cell (single astrocyte), crossbars represent means and standard deviations of expression levels, y-axis is on logarithmic scale. Each column represents an individual animal. (6C) Reduced GAT-1 expression, and less dispersion of GAT-1 expression, in transduced astrocytes using fixed cell flow cytometry. Each dot represents a single cell (single astrocyte), crossbars represent means and standard deviations of expression levels. Each pair of columns represents an individual animal, divided into non-transduced (neg) and transduced (pos) fractions of astrocytes (assessed by FLAG positivity). The mean expression levels of GAT-1 in each fraction are given, and the rates of transduction within astrocytes are shown. Astrocytes are defined as GAT-1 +NeuN- cells for these analyses. Overall the autoregul atory vectors diminish maximal expression and mean expression at high dose, and shallow the dose-response curve for expression. The % Transduction for each vector (left to right) reads: 0.1%, 93.9%, 93.3%, 57.1%, 79.8%, 82.9%, 90.5%, 66.5%, 52.2%, 87.9%, 91.3%, 69.5%, 64%, 64.6%, 51.2%, 60.1%, and 41.8%. The mean expression for each vector (left to right) reads: 6419, 3000, 4475, 15339, 7737, 14374, 6567, 9283, 6238, 15628, 5063, 10560, 4660, 9212, 5805, 10216, 6470, 9590, 4685, 10978, 4882, 14415, 6502, 11118, 6177, 10598, 4858, 13627, 6095, 9441, 5590, 9179, 6643, and 10716.

[0017] FIGs. 7A, 7B. Autoregulators reduce intensity and variability of SLC6A 1-myc-FLAG transgene expression in cortical astrocytes, as measured using flow cytometry (7 A) Reduced expression and shallowed dose-expression curve from autoregulated constructs, measured at the single cell level using anti-FLAG flow cytometry. Expression levels from all transduced (FLAG+) motor cortex astrocytes from an animal were averaged to the median, and each dotrepresents one animal, and dot shapes represent independent experiments. Summarizing over multiple animals and experiments, autoregulatory cassettes decrease expression at high dose, and also lead to a shallowed dose-response curve. Uninjected animals have small numbers of cells falling into the FLAG+ gate at low levels by chance (see FIGs.6A and 6B). (7 B) Autoregulation demonstrated by diminished dispersion of expression within populations of expressing cells. FLAG signal levels in populations of transfected cells were used to compute the rCV, here used as a scalar metric of dataset dispersion. Overall the rCV values were much lower in autoregulated constructs than that seen in the parental vectors. Uninjected animals have small numbers of cells falling into the FLAG+gate at low levels by chance, making their rCV values unreliable and uninformative.

[0018] FIG. 8. Sequences supporting the disclosure. mir9-1-AI2 (SEQ ID NO: 1); mir30d-AI2 (SEQ ID NO: 2); mir181a2-AI2 (SEQ ID NO: 3); mir9-1-AI3 (SEQ ID NO: 4); mir30d-AI3 (SEQ ID NO: 5); mir181a2-AI3 (SEQ ID NO: 6); mir9-1-AI4 (SEQ ID NO: 7); mir30d-AI4 (SEQ ID NO: 8); mir131 a2-AI4 (SEQ ID NO: 9); AI2 guide strand (SEQ ID NO: 10); AI3 guide strand (SEQ ID NO: 11); AI4 guide strand (SEQ ID NO: 12); AI2 binding site (SEQ ID NO: 13); AI3 binding site (SEQ ID NO: 14); AI4 binding site (SEQ ID NO: 15); eHGT_380h (SEQ ID NO: 16); Beta-Globin Minimal Promoter (SEQ ID NO: 17); minCMV Promoter (SEQ ID NO: 18); Mutated minCMV Promoter (SEQ ID NO: 19); minRho Promoter (SEQ ID NO: 20); minRho* Promoter (SEQ ID NO: 21); Hsp68 minimal Promoter (SEQ ID NO: 22); SYFP2 (SEQ ID NO: 23); EGFP (SEQ ID NO: 24); Optimized Flp recombinase (SEQ ID NO: 25); Improved Ore recombinase (SEQ ID NO: 26); Spacer coding sequence (SEQ ID NO: 27); SP10 insulator (SEQ ID NO: 28); 3xSP10ins (SEQ ID NO: 29); c-Myc tag coding sequence (SEQ ID NO: 30); Flag tag coding sequence (SEQ ID NO: 31); Myc-Flag tag coding sequence (SEQ ID NO: 32); 3XFLAG coding sequence (SEQ ID NO: 33); 10aa coding sequence (SEQ ID NO: 34); H2B coding sequence (SEQ ID NO: 35); WPRE3 (SEQ ID NO: 36); WPRE (SEQ ID NO: 37); BGHpA (SEQ ID NO: 38); hGHpA (SEQ ID NO: 39); P2A (SEQ ID NO: 40); T2A (SEQ ID NO: 41); E2A (SEQ ID NO: 42); F2A (SEQ ID NO: 43); Exemplary Plasmid Backbone 1 - Left ITR (SEQ ID NO: 44); Exemplary Plasmid Backbone 1 - Right ITR (SEQ ID NO: 45); Exemplary Plasmid Backbone 2 - Right ITR (SEQ ID NO: 46); Exemplary Plasmid Backbone 3 -Left ITR (SEQ ID NO: 47); Exemplary Plasmid Backbone 3 - Right ITR (SEQ ID NO: 48); PHP.eB capsid (SEQ ID NO: 49); AAV9 VP1 capsid protein (SEQ ID NO: 50); tet-Transactivator version 2 (SEQ ID NO: 51); SLC6A1 part 1 (SEQ ID NO: 52); SLC6A1 part 2 (SEQ ID NO: 53); hHB-3 intron v1. part 1 (SEQ ID NO: 54); hHB-3 intron v1. Part 2 (SEQ ID NO: 55); hHB-3 intron v2. Part 1 (SEQ ID NO: 56); hHB-3 intron v2. Part 2 (SEQ ID NO: 57); intronSLC6A1 and autoregulator coding sequences (SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66); GTPase HRas (SEQ ID NO: 67); Substance P (SEQ ID NO: 68); Oxytocin (SEQ ID NO: 69); GCaMP6m (SEQ ID NO: 70); GCaMP6s (SEQ ID NO: 71); GCaMP6f (SEQ ID NO: 72); CN5517 (SEQ ID NO: 73); CN5518 (SEQ ID NO: 74); CN5519 (SEQ ID NO: 75); CN5520 (SEQ ID NO: 76); CN5521 (SEQ ID NO: 77); CN5522 (SEQ ID NO: 78); CN5523 (SEQ ID NO: 79); CN5524 (SEQ ID NO: 80); and CN5525 (SEQ ID NO: 81).DETAILED DESCRIPTION

[0019] Solute carrier family 6 member 1 (SLC6A1) encodes for the gamma-aminobutyric acid (GABA) transporter protein type 1, GAT-1, which is responsible for the removal of GABA from the synaptic cleft. GABA is the primary inhibitory neurotransmitter and the GABA ion channel is primarily localized to axons and nerve terminals of GABAergic interneurons. As a member of the neurotransmitter sodium symporters family of proteins, GAT-1 couples the transport of GABA with ion exchange through the GAT-1 channel via the exchange of 1 GABA molecule for 2 sodium ions and 1 chloride ion. The GAT-1 transporter is specifically responsible for the reuptake of GABA into the presynapse following the firing of the interneuron. Without a functional GAT-1 channel, GABA builds up in the synaptic cleft, which can increase the inhibitory activity of the interneuron.

[0020] Although mutations in SLC6A1 are not considered diagnostic hallmarks of epilepsy or autism spectrum disorder, recent studies have found that the gene may play a significant role in these disorders. In patients with diagnosed mutations in the SLC6A1 gene, the variability in the mutational positions are striking including missense mutations, splice-site variants, frameshift mutations, nonsense mutations, and in-frame deletions.

[0021] The current disclosure provides artificial expression constructs for modulating gene expression in astrocytes and / or neurons. The artificial expression constructs can be used to express synthetic genes or modify gene expression in astrocytes and / or neurons for the assessment and / or treatment of SLC6A1 -related disorders, such as epilepsy, seizures, and autism spectrum disorder. In particular embodiments, delivery of artificial expression constructs to astrocytes and / or neurons is sufficient to rescue SLC6A1 expression (e.g., in a subject with an SLC6A1 -related disorder). In particular embodiments, the artificial expression constructs include an autoregulator, an enhancer, a promoter, and an SLC6A1 coding sequence.

[0022] In particular embodiments, the autoregulator includes a microRNA (miRNA) scaffold, a miRNA guide strand, and a miRNA binding site. In particular embodiments, the miRNA scaffold includes a miR9-1 scaffold, a mir30d scaffold, or a mir181 a2 scaffold. In particular embodiments, the miRNA guide strand includes an AI2 miRNA guide strand, an AI3 miRNA guide strand, or an AI4 miRNA guide strand. In particular embodiments, the miRNA binding site includes an AI2 miRNA binding site, an AI3 miRNA binding site, or an AI4 miRNA binding site.

[0023] In particular embodiments, the enhancer includes eHGT_380h. In particular embodiments, the eHGT_380h enhancer provides level SLC6A1 expression in astrocytes across regions of the brain and spinal cord.

[0024] In particular embodiments, the promoter includes a minimal promoter (e.g., minBG promoter). In particular embodiments, the SLC6A1 coding sequence includes intronSLC6A1. In particular embodiments, intronSLC6A1 includes an SLC6A1 coding sequence including introns. In particular embodiments, the miRNA scaffold and miRNA guide strand are within an intron of the SLC6A1 coding sequence.

[0025] Particular embodiments provide artificial expression constructs including the features of vectors described herein including vectors: CN5517, CN5518, CN5519, CN5520, CN5521, CN5522, CN5523, CN5524, and CN5525.

[0026] Aspects of the disclosure are now described with the following additional options and detail: (i) ArtificialExpression Constructs & Vectors for Targeted Expression of Genes in Astrocytes and / or Neurons; (ii) Compositions for Administration; (iii) Cell Lines Including Artificial Expression Constructs; (iv) Transgenic Animals; (v) Methods of Use; (vi) Kits and Commercial Packages; (vii) Exemplary Embodiments; and (viii) Closing Paragraphs. These headings are provided for organization purposes only and do not limit the scope or interpretation of the disclosure.(I) Artificial Expression Constructs & Vectors for Targeted Expression of Genes in Astrocytes and / or Neurons. Artificial expression constructs disclosed herein include (I) an autoregulator; (II) an enhancer that leads to targeted expression of a coding sequence within astrocytes and / or neurons, (iii) an SLC6A1 coding sequence, and (iv) a promoter. The artificial expression construct can also include other regulatory elements if necessary or beneficial.

[0027] An autoregulator regulates the expression of a coding sequence (e.g., a therapeutic coding sequence) within a vector. Autoregulatory miRNAs, for example, regulate their own expression through a positive feedback loop. The autoregulators described herein include a miRNA scaffold, a miRNA guide strand, and a miRNA binding site. In particular embodiments, the components of the autoregulator are located at different locations within the artificial expression construct. In particular embodiments, the autoregulator is not contiguous.

[0028] miRNAs are non-coding microRNAs of 21 to 25 bases (nt) in length that are widely found in the genome of eukaryotes. miRNAs are generally transcribed from the miRNA gene located in the intergenic region and intron to form the original miRNA (pri-miRNA), which is processed into a 70 nt miRNA precursor (pre-miRNA) in the nucleus of the animal, and then transported to the cytoplasm where they are processed into mature miRNAs. The mature miRNA enters the miRNA-induced silencing complex (miRISC) and is paired with the target mRNA to negatively regulate gene expression by degrading the target mRNA or hindering protein translation.

[0029] The miRNA scaffold provides for the processing and maturation of synthetic miRNAs. In particular embodiments, the miRNA scaffold is embedded within the intron of the SLC6A1 coding sequence. In particular embodiments, the miRNA scaffold includes mir9-1 , mir30d, or mir181 a2.

[0030] The miRNA guide strand is the strand of the miRNA that gets loaded into the RNA-induced silencing complex (RISC). The miRNA guide strand targets specific sequences, such as the mRNA of the encoded gene (e.g., SLC6A1) or regulatory elements within the cell. In particular embodiments, the miRNA guide strand targets SLC6A1 mRNA, enabling autoregulation. In particular embodiments, autoregulation includes downregulation. In particular embodiments, the miRNA guide strand includes an AI2 guide strand, an AI3 guide strand, or an AI4 guide strand. In particular embodiments, the miRNA scaffold and miRNA guide strand are within the intron of the SLC6A1 coding sequence to allow for co-expression of the miRNA scaffold, miRNA guide strand, and SLC6A1.

[0031] The miRNA binding site is a complementary sequence within the encoded gene’s (e.g., SLC6A1) mRNA sequence. In particular embodiments, the miRNA binding site is the in the 3’UTR of the encoded gene’s mRNA sequence. In particular embodiments, the miRNA guide strand is complementary and binds to miRNA binding site. In particular embodiments, the binding of the miRNA guide strand to the miRNA binding site leads to mRNA degradation or inhibition of translation. In particular embodiments, the miRNA binding site includes an AI2 binding site, an AI3binding site, or an AI4 binding site.

[0032] The miRNA binding site multiplicity refers to the number of miRNA binding sites present in the target mRNA In particular embodiments, the level of expression downregulation can be modulated by changing the miRNA binding site multiplicity. In particular embodiments, the miRNA binding site multiplicity can be 1x, 2x, 3x, 4x, or 5x. In particular embodiments, the miRNA binding site multiplicity is 1x (i.e., one copy).

[0033] Herein, each autoregulator is described as the type of miRNA scaffold and the type of miRNA guide strand / binding site. For example, a miR9-1 scaffold, an AI2 miRNA guide strand, and an AI2 miRNA binding site form a miR9-1 -AI2 autoregulator. Likewise, the mir30d scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site is referred to as a mir30d-AI2 autoregulator; the mir181a2 scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site is referred to as a mir181 a2-AI2 autoregulator; the miR9-1 scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site is referred to as a mir9-1 -AI3 autoregulator; the mir30d scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site is referred to as a mir30d-AI3 autoregulator; the mir181a2 scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site is referred to as a mir181a2-AI3 autoregulator; the miR9-1 scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site is referred to as a mir9-1 -AI4 autoregulator; the mir30d scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site is referred to as a mir30d-AI4 autoregulator; and the mir181 a2 scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site is referred to as a mir181a2-AI4 autoregulator.

[0034] In particular embodiments, an “enhancer” or an “enhancer element” is a cis-acting sequence that increases the level of transcription associated with a promoter and can function in either orientation relative to the promoter and the coding sequence that is to be transcribed and can be located upstream or downstream relative to the promoter or the coding sequence to be transcribed. There are art-recognized methods and techniques for measuring function(s) of enhancer element sequences. Particular examples of enhancer sequences utilized within artificial expression constructs disclosed herein include an eHGT_380h enhancer.

[0035] In particular embodiments, a targeted central nervous system cell type enhancer comprises an astrocyte enhancer and is an enhancer that is uniquely or predominantly utilized by the astrocyte. An astrocyte enhancer enhances expression of a gene in the astrocyte. In certain embodiments, an astrocyte enhancer is also an astrocyte enhancer that enhances expression of a gene in the astrocyte and does not substantially direct expression of genes in other non-targeted cell types, thus having cell type specific transcriptional activity.

[0036] In particular embodiments, a targeted central nervous system cell type enhancer comprises a neuron enhancer and is an enhancer that is uniquely or predominantly utilized by a neuron. A neuron enhancer enhances expression of a gene in the neuron. In certain embodiments, a neuron enhancer is also a neuron enhancer that enhances expression of a gene in the neuron and does not substantially direct expression of genes in other non-targeted cell types, thus having cell type specific transcriptional activity.

[0037] When a coding sequence (or heterologous coding sequence) operatively linked to an enhancer disclosed hereinleads to expression in astrocytes and / or neurons, it leads to expression of the administered coding sequence in the astrocytes and / or neurons.

[0038] When a coding sequence is selectively expressed in astrocytes and / or neurons, it leads to expression of the administered coding sequence in astrocytes and / or neurons and is not substantially expressed in other cell types, as explained in additional detail below. In particular embodiments, not substantially expressed in other cell types is less than 50% expression in a reference cell type as compared to selected cell types (e.g. , astrocytes and / or neurons); less than 40% expression in a reference cell type as compared to selected cell types; less than 30% expression in a reference cell type as compared to selected cell types; less than 20% expression in a reference cell type as compared to selected cell types; or less than 10% expression in a reference cell type as compared to selected cell types. In particular embodiments, a reference cell type refers to non- selected cell types . The non-selected cell types can be within the same anatomical structure as the selected cell types. In particular embodiments, a reference cell type is within an anatomical structure that is adjacent to an anatomical structure that includes the selected cell types. In particular embodiments, a reference cell type has a different gene expression profile than selected cell types.

[0039] In particular embodiments, the product of the coding sequence may be expressed at low levels in non-selected cell types, for example at less than 1% or 1%, 2%, 3%, 5%, 10%, 15% or 20% of the levels at which the product is expressed in selected cell types. In particular embodiments, selected cell types are the only cell type that expresses the right combination of transcription factors that bind an enhancer disclosed herein to drive gene expression. Thus, in particular embodiments, expression occurs exclusively within selected cell types.

[0040] In particular embodiments, targeted cell types (e.g. neuronal, and / or non-neuronal) can be identified based on transcriptional profiles, such as those described in Tasic et al., Nature 563, 72-78 (2018) and Hodge et al., Nature 573, 61-68 (2019). For reference, the following description of cell types and distinguishing features is also provided:

[0041] Neocortical GABAergic neuron Subclasses:• All: Express GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2.• Lamp5, Sncg, Serpinfl, and Vip GABAergic neurons: Developmentally derived from neuronal progenitors from the caudal ganglionic eminence (CGE) or preoptic area (POA).• Ssf and Pvalb GABAergic neurons: Developmentally derived from neuronal progenitors in the medial ganglionic eminence (MGE).• Lamp5 GABAergic neurons: Found in many neocortical layers, especially upper (L1-L2 / 3), and have mainly neurogliaform and single bouquet morphology.• Lamp5_Lhx6 GABAergic neurons: A subset of Lamp5 GABAergic neurons that co-express Lamp5 and Lhx6.• Sncg GABAergic neurons: Found in many neocortical layers, and have molecular overlaps with Lamp5 and Vip cells, but inconsistent expression of Lamp5 or vasoactive intestinal peptide (Vip), with more consistent expression of Sncg.• Serpinfl GABAergic neurons: Found in many neocortical layers, and have molecular overlaps with Sncg andVip cells, but inconsistent expression of Sncg or Vip, with more consistent expression of Serpinfl.• Vip GABAergic neurons: Found in many neocortical layers, but especially frequent in upper layers (L1-L4), and highly express the neurotransmitter Vip.• Sst GABAergic neurons: Found in many neocortical layers, but especially frequent in lower layers (L5-L6).They highly express the neurotransmitter somatostatin (Sst), and frequently block dendritic inputs to postsynaptic neurons. Included in this subclass are sleep-active Sst Chodl neurons (which also express Nos1 and Tacrl) that are highly distinct from other Sst neurons but express some shared marker genes including Sst. In human, SST gene expression is often detected in layer 1 LAMP5+ GABAergic neuron subtypes. • Pvalb GABAergic neurons: Found in many neocortical layers, but especially frequent in lower layers (L5-L6).They highly express the calcium-binding protein parvalbumin (Pvalb), express neuropeptide Tad, and frequently dampen the output of postsynaptic neurons. Most fast-spiking GABAergic neurons express Pvalb strongly. Included in this subclass are chandelier cells, which have distinct, chandelier-like morphology and express the markers Cpne5 and Vipr2 in mouse, and NOG and UNC5B in human.• Meis2. A distinct subclass defined by a single type, only neocortical GABAergic neuron type that expresses Meis2 gene, and does not express some other genes that are expressed by all other neocortical GABAergic neuron types (for example, Thy1 and Scn2b). This type is found in L6b and subcortical white matter.

[0042] Neocortical glutamatergic neuron subclasses:• All: Express glutamate transmitters Slc17a6 and / or Sid 7a7. They all express Snap25 and lack expression of Gad1 / Gad2.• L2 / 3 IT glutamatergic neurons: Primarily reside in Layer 2 / 3 and have mainly intratelencephalic (cortico- cortical) projections.• L4 IT glutamatergic neurons: Primarily reside in Layer 4 and mainly have either local or intratelencephalic (cortico-cortical) projections.• L5 IT glutamatergic neurons: Primarily reside in Layer 5 and have mainly intratelencephalic (cortico-cortical) projections. Also called L5a.• L5 PT glutamatergic neurons: Primarily reside in Layer 5 and have mainly cortico-subcortical (pyramidal tract or corticofugal) projections. Also called L5b or L5 CF (corticofugal) or L5 ET (extratelencephalic). This subclass includes cells that are located in the primary motor cortex and neighboring areas and are corticospinal projection neurons, which are associated with motor neuron / movement disorders, such as ALS. This subclass includes thick-tufted pyramidal neurons, including distinctive subtypes found only in specialized regions, e.g., Betz cells, Meynert cells, and von Economo cells.• L5 NP glutamatergic neurons: Primarily reside in Layer 5 and have mainly nearby projections.• L6 CT glutamatergic neurons: Primarily reside in Layer 6 and have mainly cortico-thalamic projections. • L6 IT glutamatergic neurons: Primarily reside in Layer 6 and have mainly intratelencephalic (cortico-cortical)projections.• L6 IT Car3 glutamatergic neurons: Most densely present in claustrum and endopyriform nucleus, and sparsely throughout L6 in many cortical areas including the primary visual cortex. These cells have mainly intratelencephalic (cortico-cortical) projections. Additional marker genes for claustrum enriched neurons include Gnb4 and Ntng2.• L6b glutamatergic neurons: Primarily reside in the neocortical subplate (L6b), with local (near the cell body) projections and some cortico-cortical projections from VISp to anterior cingulate, and cortico-subcortical projections to the thalamus.• CR neurons: A distinct subclass defined by a single type in L1 , Cajal-Retzius cells express distinct molecular markers Lhx5 and Trp73.

[0043] Non-Neuronal Astrocytes:• Distinguishing features of astrocytes are that they are neuroectoderm-derived glial cells which express the marker Aqp4 and often GFAP, but do not express the neuronal marker SNAP25. They can have a distinct star-shaped morphology and are involved in metabolic support of other cells in the brain. Multiple astrocyte morphologies are observed in mouse and human.

[0044] In particular embodiments, the artificial expression construct includes a coding sequence. In particular embodiments, the coding sequence is a coding sequence that encodes an effector element. An effector element is a sequence that is expressed to achieve, and that in fact achieves, an intended effect. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0045] In particular embodiments, afunctional molecule includes an SLC6A1 gene product. In particular embodiments, a nucleotide sequence encoding SLC6A1 includes Accession No. NM_003042.4, NM_001348250.2, NM_001348251.2, NM_001348252.2, NM_001348253.2, or the sequence as set forth in SEQ ID NO: 31. In particular embodiments, the amino acid sequence of SLC6A1 includes the sequence (GenBank: AAH33904.1):MATNGSKVADGQISTEVSEAPVANDKPKTLWKVQKKAADLPDRDTWKGRFDFLMSCVGYAIGLGNVWRFPYLCGK NGGGAFLIPYFLTLIFAGVPLFLLECSLGQYTSIGGLGVWKLAPMFKGVGLAAAVLSFWLNIYYIVIISWAIYYLYNSFTT TLPWKQCDNPWNTDRCFSNYSMVNTTNMTSAWEFWERNMHQMTDGLDKPGQIRWPLAITLAIAWILVYFCIWKGV GWTGKWYFSATYPYIMLIILFFRGVTLPGAKEGILFYITPNFRKLSDSEVWLDAATQIFFSYGLGLGSLIALGSYNSFHN NVYRDSIIVCCINSCTSMFAGFVIFSIVGFMAHVTKRSIADVAASGPGLAFLAYPEAVTQLPISPLWAILFFSMLLMLGID SQFCTVEGFITALVDEYPRLLRNRRELFIAAVCIISYLIGLSNITQGGIYVFKLFDYYSASGMSLLFLVFFECVSISWFYGV NRFYDNIQEMVGSRPCIWWKLCWSFFTPIIVAGVFIFSAVQMTQLTMGNYVFPKWGQGVGWLMALSSMVLIPGYMA YMFLTLKGSLKQRIQVMVQPSEDIVRPENGPEQPQAGSSTSKEAYI (SEQ ID NO: 82).

[0046] In particular embodiments, SLC6A1 is encoded by the combination of SEQ ID NO: 52 and SEQ ID NO: 53. In particular embodiments, the SLC6A1 coding sequence further includes an intron including the combination of SEQ ID NO: 54 and SEQ ID NO: 55, or SEQ ID NO: 56 and SEQ ID NO: 57. In particular embodiments, components of theautoregulator are within the intron. In particular embodiments, the miRNA scaffold and miRNA guide strand are within the intron. In particular embodiments, a sequence including an SLC6A1 coding sequence, intron, miRNA scaffold, and miRNA guide strand includes a sequences selected from SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66.

[0047] Exemplary reporter genes / proteins include those expressed by Addgene ID#s 83894 (pAAV-hDIx-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-mDIx-GFP-Fishell-1), and 89897 (pcDNA3-FLAG-mTET2 (N500)). Exemplary reporter genes particularly can include those which encode an expressible fluorescent protein, or expressible biotin; blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™ (Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0048] GFP is composed of 238 amino acids (26.9 kDa), originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea that fluoresces green when exposed to blue light. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered. The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability and a shift of the major excitation peak to 488 nm with the peak emission kept at 509 nm. The addition of the 37°C folding efficiency (F64L) point mutant to this scaffold yielded enhanced GFP (EGFP). EGFP has an extinction coefficient (denoted E), also known as its optical cross section of 9.13X10-21 mVmolecule, also quoted as 55,000 L / (mol*cm). Superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.

[0049] The "yellow fluorescent protein" (YFP) is a genetic mutant of green fluorescent protein, derived from Aequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm.

[0050] In particular embodiments, functional molecules include DNA and RNA editing tools such CRISPR / Cas (e.g., guide RNA and a nuclease, such as Cas, Cas9 orcpf1). Functional moleculescan also include engineered Cpflssuchas those described in US 2018 / 0030425, US 2016 / 0208243, WO / 2017 / 184768 and Zetsche et al. (2015) Cell 163: 759-771; single gRNA (see e.g., Jinek et al (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563) or editase, guide RNA molecules, microRNA, or homologous recombination donor cassettes.

[0051] In particular embodiments, functional molecules include a localizing cassette. In particular embodiments, localizing cassettes are used to localize a molecule (e.g., a vector, a protein, a sensor) to a specific subcellular compartment such as the soma, axon, or dendrite(s) of a neuron. In particular embodiments, localizing cassettes include a soma tag (e.g., soma (EE-RR)) to localize at the soma; an axon tag (e.g. derived from GAP43) or synaptophysin (sy) to localize at the axon; hydrophobic tails to localize at the plasma membrane; and hydrophobicity or alkyl chain to localize at the endoplasmic reticulum. In particular embodiments, localizing cassettes are fused to a sensor molecule such as a GECI. In particular embodiments, fusion proteins of a GECI and a localizing cassette includes soma-jGCaMP8s, axon-jRGECO1a, syGCaMP5G, and soma-jGCaMP7s.

[0052] In particular embodiments, functional molecules include tag cassettes. A tag cassette includes His tag (HHHHHH; SEQ ID NO: 83), Flag tag (DYKDDDDK; SEQ ID NO: 84), Xpress tag (DLYDDDDK; SEQ ID NO: 85), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 86), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 87), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 88), Myctag (EQKLISEEDL; SEQ ID NO: 89), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 90), STREP® tag II (WSHPQFEK SEQ ID NO: 91 (I BA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 92), Softag 3 (TQDPSRVG; SEQ ID NO: 93), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 94). In particular embodiments, a tag cassette includes a fusion of tag cassettes such as 3XFLAG. In particular embodiments, 3XFLAG includes the sequence set forth in SEQ ID NO: 33. In particular embodiments, a tag cassette includes a Myc-Flag tag. In particular embodiments, the Myc-Flag tag is encoded by the sequence set forth in SEQ ID NO: 32.

[0053] Additional effector elements include Ore, ICre, dgCre, FlpO, and tTA2. ICre refers to a codon-improved Ore. dgCre refers to an enhanced GFP / Cre recombinase fusion gene with an N terminal fusion of the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring a G67S mutation and modified to also include the R12Y / Y100I destabilizing domain mutation. FlpO refers to a codon-optimized form of FLPe that greatly increases protein expression and FRT recombination efficiency in mouse cells. Like the Cre / LoxP system, the FLP / FRT system has been widely used for gene expression (and generating conditional knockout mice, mediated by the FLP / FRT system). tTA2 refers to tetracycline transactivator.

[0054] Exemplary expressible elements are expression products that do not include effector elements, for example, a non-functioning or defective protein. In particular embodiments, expressible elements can provide methods to study the effects of their functioning counterparts. In particular embodiments, expressible elements are non-functioning or defective based on an engineered mutation that renders them non-functioning. In these aspects, non-expressible elements are as similar in structure as possible to their functioning counterparts.

[0055] Exemplary self-cleaving peptides include the 2A peptides which lead to the production of two proteins from onemRNA. The 2A sequences are short (e.g., 20 amino acids), allowing more use in size-limited constructs. Particular examples include P2A, T2A, E2A, and F2A. In particular embodiments, the artificial expression constructs include an internal ribosome entry site (IRES) sequence. IRES allow ribosomes to initiate translation at a second internal site on a mRNA molecule, leading to production of two proteins from one mRNA.

[0056] Artificial expression constructs can encode nuclear localization proteins, such as Histone H1, Histone H2A, Histone H2B, Histone H3, Histone H4, histone-like protein HPhA, or H2B*. Artificial expression construct can include sequences encoding 10 amino acid (10aa) linkers. In particular embodiments, a 10 linker is encoded by the sequence as set forth in SEQ ID NO: 34.

[0057] Coding sequences encoding molecules (e.g., RNA, proteins) described herein can be obtained from publicly available databases and publications. Coding sequences can further include various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not affect the function of the encoded molecule. The term “encode" or “encoding” refers to a property of sequences of nucleic acids, such as a vector, a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of other molecules such as proteins.

[0058] The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or post-regulatory elements, such as termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. The sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type.

[0059] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or promoters specific for the cytoplasm. Promoters may include strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters. Inducible promoters direct expression in response to certain conditions, signals or cellular events. For example, the promoter may be an inducible promoter that requires a particular ligand, small molecule, transcription factor or hormone protein in order to effect transcription from the promoter. Particular examples of promoters include minBglobin (also referred to as minBGprom), CMV, minCMV, minCMV* (minCMV* is minCMV with a Sacl restriction site removed), minRho, minRho* (minRho* is minRho with a Sacl restriction site removed), SV40 immediately early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous Sarcoma Virus (RSV) long-terminal repeat (LTR) promoter. Minimal promoters have no activity to drive gene expression on their own but can be activated to drive gene expression when linked to a proximal enhancer element.

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

[0061] Viral vector is widely used to refer to a nucleic acid molecule that includes virus-derived components that facilitate transfer and expression of non-native nucleic acid molecules within a cell. The term adeno-associated viral vector refers to a viral vector or plasmid containing 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 containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term "lentivi ral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a lentivirus, and so on. The term "hybrid vector" refers to a vector including structural and / or functional genetic elements from more than one virus type In particular embodiments, the AAV includes a recombinant adeno-associated viral vector (rAAV). In particular embodiments, the rAAV includes a plasmid AAV (pAAV) or a self-complementary AAV (scAAV).

[0062] Adenovirus vectors refer to those constructs containing adenovirus sequences sufficient to (a) support packaging of an artificial expression construct and (b) to express a coding sequence that has been cloned therein in a sense or antisense orientation. A recombinant Adenovirus vector includes a genetically engineered form of an adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequencesup to 7 kb. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

[0063] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.

[0064] Other than the requirement that an adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of particular embodiments disclosed herein. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. In particular embodiments, adenovirus type 5 of subgroup C is the preferred starting material in order to obtain a conditional replication-defective adenovirus vector for use in particular embodiments, since Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

[0065] As indicated, the typical vector is replication defective and will not have an adenovirus E1 region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical. The polynucleotide encoding the gene of interest may also be inserted in lieu of a deleted E3 region in E3 replacement vectors or in the E4 region where a helper cell line or helper virus complements the E4 defect.

[0066] Adeno-Associated Virus (AAV) is a parvovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to 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 singlestranded linear DNA that is encapsidated into capsid proteins VP1 , VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter.

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

[0068] AAVs stand out for use within the current disclosure because of their superb safety profile and because their capsids and genomes can be tailored to allow expression in targeted cell populations, such as astrocytes. scAAV refers to a self-complementary AAV. pAAV refers to a plasmid adeno-associated virus. rAAV refers to a recombinant adeno-associated virus.

[0069] Other viral vectors may also be employed. For example, vectors derived from viruses such as vaccinia virus, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells.

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

[0071] Illustrative retroviruses suitable for use in particular embodiments, include: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemiavirus, Murine Stem Cell Virus (MSCV), Rous Sarcoma Virus (RSV), and lentivirus.

[0072] "Lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV); the caprine arthritisencephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In particular embodiments, HIV based vector backbones (i.e., HIV cis-acting sequence elements) can be used.

[0073] A safety enhancement for the use of some vectors can be provided by replacing the U3 region of the 5' LTR with a promoter to drive transcription of the viral genome during production of viral particles Examples of promoters which can be used for this purpose include, for example, 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 able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system. In particular embodiments, the promoter has additional advantages in controlling the manner in which the viral genome is transcribed. For example, the promoter can be inducible, such that transcription of all or part of the viral genome will occur only when the induction factors are present. Induction factors include one or more chemical compounds or the physiological conditions such as temperature or pH, in which the host cells are cultured.

[0074] In particular embodiments, viral vectors include a TAR element. The term "TAR" refers to the "trans-activation response" genetic element located in the R region of lentiviral LTRs. This element interacts with the lentiviral transactivator (tat) genetic element to enhance viral replication However, this element is not required in embodiments wherein the U3 region of the 5' LTR is replaced by a promoter.

[0075] The "R region" refers to the region within retroviral LTRs beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly(A) tract. The R region is also defined as being flanked by the U3 and U5 regions. The R region plays a role during reverse transcription in permitting the transfer of nascent DNA from one end of the genome to the other.

[0076] In particular embodiments, expression of sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can increase expression of a nucleic acid. Examples include the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Smith etal., Nucleic Acids Res. 26(21):4818-4827, 1998); and the like (Liu etal., 1995, Genes Dev., 9:1766). In particular embodiments, vectors include a posttranscriptional regulatory element such as a WPRE or HPRE. In particular embodiments, vectors lack or do not include a posttranscriptional regulatory element such as a WPRE or HPRE.

[0077] Elements directing the efficient termination and polyadenylation of a nucleic acid transcript can increase geneexpression. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors include a polyadenylation signal 3' of a polynucleotide encoding a molecule (e.g., protein) to be expressed. The term "poly(A) site" or "poly (A) sequence" denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly (A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Particular embodiments may utilize BGHpA, hGHpA, or SV40pA. In particular embodiments, a preferred embodiment of an expression construct includes a terminator element. These elements can serve to enhance transcript levels and to minimize read through from the construct into other plasmid sequences.

[0078] In particular embodiments, a viral vector further includes one or more insulator elements. Insulators elements may contribute to protecting viral vector-expressed sequences, e.g., effector elements or expressible elements, from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences ( / .e., position effect; see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001). In particular embodiments, viral transfer vectors include one or more insulator elements at the 3' LTR and upon integration of the provirus into the host genome, the provirus includes the one or more insulators at both the 5' LTR and 3' LTR, by virtue of duplicating the 3' LTR. Suitable insulators for use in particular embodiments include the chicken p-globin insulator (see Chung et al., Cell 7A’.5O5, 1993; Chung et al., PNAS SA 94:575, 1997; and Bell etal., Ce / / 98:387, 1999), SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer blocking insulators (Liu et al., Nature Biotechnology, 33:198, 2015).

[0079] Beyond the foregoing description, a wide range of suitable expression vector types will be known to a person of ordinary skill in the art. These can include commercially available expression vectors designed for general recombinant procedures, for example plasmids that contain one or more reporter genes and regulatory elements required for expression of the reporter gene in cells. Numerous vectors are commercially available, e.g., from Invitrogen, Stratagene, Clontech, etc., and are described in numerous associated guides. In particular embodiments, suitable expression vectors include any plasmid, cosmid or phage construct that is capable of supporting expression of encoded genes in mammalian cell, such as pUC or Bluescript plasmid series

[0080] Particular embodiments of vectors disclosed herein include:Expression FeaturesConstruct NameCN5517(AiP15517) pAAV:eHGT_380h-minBG-mir9-1-AI2-intronSLC6A1-Myc-Flag-1xAI2BS-WPRE3-BGHpA CN5518(ASP15518) pAAV:eHGT_380h-minBG-mir30d-A!2-intronSLC6A1-Myc-Flag-1xA!2BS-WPRE3-BGHpA CN5519(AiP15519) pAAV:eHGT_380h-minBG-mir181a2-AI2-infronSLC6A1-IVIyc-Flag-1xAI2BS-WPRE3-BGHpA CN5520(Af PI 5520) pAAV:eHGT_380h-minBG-mif9-1-AI3-intronSLC6A1-Myc-Flag-1xAI3BS-WPRE3-BGHpA CN5521(AiP15521) pAAV:eHGT_380h-minBG-mir30d-A!3-intronSLC6A1-Myc-Flag-1xA!3BS-WPRE3-BGHpA CN5522(AiP15522) pAAV:eHGT_380h-minBG-mir181a2-AI3-infronSLC6A1-Myc-Fiag-1xAI3BS-WPRE3-BGHpA CN5523(AIP15523) pAAV:eHGT_380h-minBG-mir9-1-AI4-intronSLC6A1-Myc-Flag-1xAI4BS-WPRE3-BGHpA CN5524(AiP15524) pAAV:eHGT_380h-minBG-mir30d-AI4-intronSLC6A1-Myc-Flag-1xAI4BS-WPRE3-BGHpA

[0081] Subcomponent sequences within the larger vector sequences can be readily identified by one of ordinary skill in the art and based on the contents of the current disclosure (see FIG. 8). Nucleotides between identifiable and enumerated subcomponents reflect restriction enzyme recognition sites used in assembly (cloning) of the constructs, and in some cases, additional nucleotides do not convey any identifiable function. These segments of complete vector sequences can be adjusted based on use of different cloning strategies and / or vectors. In general, short 6-nucleotide palindromic sequences reflect vector construction artifacts that are not important to vector function.

[0082] In particular embodiments vectors (e.g, AAV) with capsids that cross the blood-brain barrier (BBB) are selected. In particular embodiments, vectors are modified to include capsids that cross the BBB. Examples of AAV 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), AAV-PHP.B (Deverman et al, Nat Biotechnol. 2016; 34(2): 204), AAV-PPS (Chen et al, Nat Med. 2009; 15: 1215), and PHP.eB. In particular embodiments, the PHP.eB capsid differs from AAV9 such that, using AAV9 as a reference, amino acids starting at residue 586: S-AQ-A (SEQ ID NO: 95) are changed to S-DGTLAVPFK-A (SEQ ID NO: 96). In particular embodiments, PHP.eb refers to SEQ ID NO: 49.

[0083] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the BBB following intravenous injection. It transduces large sections of the central nervous system (CNS), thus permitting minimally invasive treatments (Naso et al, BioDrugs. 2017; 31(4): 317), for example, as described in relation to clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome by AveXis (AVXS-101, NCT03505099) and the treatment of CLN3 gene-Related Neuronal Ceroid-Lipofuscinosis (NCT03770572).

[0084] AAVrh.10, was originally isolated from rhesus macaques and shows low seropositivity in humans whencompared with other common serotypes used for gene delivery applications (Selot et al., Front Pharmacol. 2017; 8: 441) and has been evaluated in clinical trials LYS-SAF302, LYSOGENE, and NCT03612869

[0085] AAV1R6 and AAV1R7, two variants isolated from a library of chimeric AAV vectors (AAV1 capsid domains swapped into AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing significantly reduced hepatic and vascular endothelial transduction.

[0086] rAAVrh.8, also isolated from rhesus macaques, shows a global transduction of glial and neuronal cell types in regions of clinical importance following peripheral administration and also displays reduced peripheral tissue tropism compared to other vectors.

[0087] AAV-BR1 is an AAV2 variant displaying the NRGTEWD (SEQ ID NO: 97) epitope that was isolated during in vivo screening of a random AAV display peptide library. It shows high specificity accompanied by high transgene expression in the brain with minimal off-target affinity (including for the liver) (Kbrbelin et al., EMBO Mol Med. 2016; 8(6): 609).

[0088] AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated with the CREATE method that encodes the 7-mer sequence QAVRTSL (SEQ ID NO: 98), transduces neurons in the enteric nervous system, and strongly transduces peripheral sensory afferents entering the spinal cord and brain stem.

[0089] AAV-PHP. B (Addgene, Watertown, MA) is a variant of AAV9 generated with the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 99). It transfers genes throughout the CNS with higher efficiency than AAV9 and transduces the majority of astrocytes and neurons across multiple CNS regions.

[0090] AAV-PPS, an AAV2 variant crated by insertion of the DSPAHPS (SEQ ID NO: 100) epitope into the capsid of AAV2, shows a dramatically improved brain tropism relative to AAV2.

[0091] For additional information regarding capsids that cross the blood brain barrier, see Chan et al., Nat. Neurosci.2017 Aug: 20(8): 1172-1179.(II) Compositions for Administration. Artificial expression constructs and vectors of the present disclosure (referred to herein as physiologically active components) can be formulated with a carrier or more than one carrier that is suitable for administration to a cell, tissue slice, animal (e.g., mouse, non-human primate), or human. Physiologically active components within compositions described herein can be prepared in neutral forms, as freebases, or as pharmacologically acceptable salts.

[0092] Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. 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.

[0093] Carriers of physiologically active components can include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, and the like. The use ofsuch carriers for physiologically active components is well known in the art. Except insofar as any conventional media or agent is incompatible with the physiologically active components, it can be used with compositions as described herein.

[0094] The phrase "pharmaceutically-acceptable carriers" refer to carriers that do not produce an allergic or similar untoward reaction when administered to a human, and in particular embodiments, when administered intravenously (e.g. at the retro-orbital plexus).

[0095] In particular embodiments, compositions can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular (ICV), intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or by direct injection in or application to one or more cells, tissues, or organs.

[0096] Compositions may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles.

[0097] The formation and use of liposomes is generally known to those of skill in the art. Liposomes have been developed with improved serum stability and circulation half-times (see, for instance, U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (see, for instance U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

[0098] The disclosure also provides for pharmaceutically acceptable nanocapsule formulations of the physiologically active components. Nanocapsules can generally entrap compounds in a stable and reproducible way (Quintanar-Guerrero eta / ., Drug Dev Ind P / ia / m 24(12):1113-1128, 1998; Quintanar-Guerrero eta / ., Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et a / ., J. Microencapsul. 15(1):107-119, 1998; Douglas et a / ., Crit Rev Ther Drug Carrier Syst 3(3):233-261 , 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles can be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be easily made, as described in Couvreur eta / ., J Pharm Sc / 69(2): 199-202, 1980; Couvreur etal., 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.

[0099] Injectable compositions can 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 particular embodiments, it is stable under the conditions of manufacture and storage, and optionally contains one or more preservative compounds against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), 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 dispersion, and / or by the use of surfactants. The prevention of the action of microorganisms can be brought about byvarious antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation will include an isotonic agent(s), for example, sugar(s) or sodium chloride. Prolonged absorption of the injectable compositions can be accomplished by including in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin. Injectable compositions can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.

[0100] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. As indicated, under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0101] Sterile compositions can be prepared by incorporating the physiologically active component in an appropriate amount of a solvent with other optional ingredients (e.g., as enumerated above), followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized physiologically active components into a sterile vehicle that contains the basic dispersion medium and the required other ingredients (e.g., from those enumerated above). In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the physiologically active components plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0102] Oral compositions may be in liquid form, for example, as solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Tablets may be coated by methods well-known in the art.

[0103] Inhalable compositions can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0104] Compositions can 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. No. 5,770,219 and U.S. Pat. No.5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).

[0105] Supplementary active ingredients can also be incorporated into the compositions

[0106] Typically, compositions can include at least 0.1% of the physiologically active components or more, although the percentage of the physiologically active components may, of course, be varied and may conveniently be between 1 or 2% and 70% or 80% or more or 0.5-99% of the weight or volume of the total composition. Naturally, the amount of physiologically active components in each physiologically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of compositions and dosages may be desirable.

[0107] In particular embodiments, for administration to humans, compositions should meet sterility, pyrogenicity, and the general safety and purity standards as required by United States Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.

[0108] (iii) Cell Lines Including Artificial Expression Constructs. The present disclosure includes cells including an artificial expression construct described herein. A cell that has been transformed with an artificial expression construct can be used for many purposes, including in neuroanatomical studies, assessments of functioning and / or nonfunctioning proteins, and drug screens that assess the regulatory properties of autoregulators, enhancers, and / or coding sequences (e.g., therapeutic coding sequences).

[0109] Avariety of host cell lines can be used, but in particular embodiments, the cell is a mammalian cell. In particular embodiments, the artificial express construct includes an autoregulator and / or a vector sequence of mir9-1-AI2, mir30d-AI2, mir181 a2-AI2, mir9-1-AI3, mir30d-AI3, mir181a2-AI3, mir9-1-AI4, mir30d-AI4, or mir181 a2-AI4 and / or CN5517, CN5518, CN5519, CN5520, CN5521, CN5522, CN5523, CN5524, or CN5525, and the cell line is a human, primate, or murine cell. Cell lines which can be utilized for transgenesis in the present disclosure also include primary cell lines derived from living tissue such as rat or mouse brains and organotypic cell cultures, including brain slices from animals such as rats, mice, non-human primates, or human neurosurgical tissue. In particular embodiments, non-neuronal cell lines may be used, including mouse embryonic stem cells. Cultured mouse embryonic stem cells can be used to analyze expression of genetic 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 the embryonic stem cells. In culture, the stem cells form a variety of differentiated cell types. Differentiation is caused by the expression of tissue specific transcription factors, allowing the function of an enhancer sequence to be evaluated. (See for example Fiskerstrand et al., FEBS Left 458: 171-174, 1999).

[0110] In particular embodiments, yeast one-hybrid systems may also be used to identify compounds that inhibit specific protein / DNA interactions, such as transcription factors for enhancers or autoregulators described herein.

[0111] Transgenic animals are described below. Cell lines may also be derived from such transgenic animals. For example, primary tissue culture from transgenic mice (e.g., also as described below) can provide astrocyte cell lines with the artificial expression construct already integrated into the genome, (for an example see Mackenzie & Quinn, Proc Natl Acad Sci USA 96: 15251-15255, 1999).

[0112] (iv) Transgenic Animals. Another aspect of the disclosure includes transgenic animals, the genome of which contains an artificial expression construct including mir9-1-AI2, mir30d-AI2, mir181a2-AI2, mir9-1-AI3, mir30d-AI3, mir181a2-AI3, mir9-1-AI4, mir30d-AI4, and / or mir181a2-AI4 operatively linked to a coding sequence. In particular embodiments, the genome of a transgenic animal includes CN5517, CN5518, CN5519, CN5520, CN5521, CN5522, CN5523, CN5524, and / or CN5525. In particular embodiments, when a non-integrating vector is utilized, a transgenic animal includes an artificial expression construct including mir9-1-AI2, mir30d-AI2, mir181 a2-AI2, mir9-1-AI3, mir30d-AI3, mir181 a2-AI3, mir9-1-AI4, mir30d-AI4, and / or mir181 a2-AI4 and / or CN5517, CN5518, CN5519, CN5520, CN5521, CN5522, CN5523, CN5524, and / or CN5525 within one or more of its cells.

[0113] Detailed methods for producing transgenic animals are described in U.S. Pat. No. 4,736,866. Transgenic animals may be of any nonhuman species, but preferably include nonhuman primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens, and rodents such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.

[0114] In particular embodiments, construction of a transgenic animal results in an organism that has an engineered construct present in all cells in the same genomic integration site. Thus, cell lines derived from such transgenic animals will be consistent in as much as the engineered construct will be in the same genomic integration site in all cells and hence will suffer the same position effect variegation. In contrast, introducing genes into cell lines or primary cell cultures can give rise to expression of the construct. A disadvantage of this approach is that the expression of the introduced DNA may be affected by the specific genetic background of the host animal.

[0115] As indicated above in relation to cell lines, the artificial expression constructs of this disclosure can be used to genetically modify mouse embryonic stem (ES) cells using techniques known in the art. Typically, the artificial expression construct is introduced into cultured murine embryonic stem cells. Transformed ES cells are then injected into a blastocyst from a host mother and the host embryo re-implanted into the mother. This results in a chimeric mouse whose tissues are composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Usually the mice from which the cultured ES cells used for transgenesis are derived are chosen to have a different coat color from the host mouse into whose embryos the transformed cells are to be injected. Chimeric mice will then have a variegated coat color. As long as the germ-line tissue is derived, at least in part, from the genetically modified cells, then the chimeric mice crossed with an appropriate strain can produce offspring that will carry the transgene.

[0116] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering artificial expression constructs to selected cell types: 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. No. 5,770,219 and U.S. Pat. No. 5,783,208); feedback-controlled delivery (U.S. Pat. No. 5,697,899), and any other delivery method available and / or described elsewhere in the disclosure.

[0117] (v) Methods of Use. In particular embodiments, a composition including a physiologically active component described herein is administered to a subject to result in a physiological effect.

[0118] In particular embodiments, the disclosure includes the use of the artificial expression constructs described herein to modulate expression of a gene which is either partially or wholly encoded in a location downstream to that enhancer in an engineered sequence. Thus, there are provided herein methods of use of the disclosed artificial expression constructs in the research, study, and potential development of medicaments for preventing, treating or ameliorating the symptoms of a disease, dysfunction, or disorder.

[0119] Particular embodiments include methods of administering to a subject an artificial expression construct that includes mir9-1-AI2, mir30d-AI2, mir181a2-AI2, mir9-1-AI3, mir30d-AI3, mir181 a2-AI3, mir9-1-AI4, mir30d-AI4, and / or mir181 a2-AI4 and / or CN5517, CN5518, CN5519, CN5520, CN5521, CN5522, CN5523, CN5524, and / or CN5525 as described herein to drive expression of a gene in a targeted cell type. The subject can be an isolated cell, a network of cells, a tissue slice, an experimental animal, a veterinary animal, or a human.

[0120] As is well known in the medical arts, dosages for any one subject depends upon many factors, including the subject's size, surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages for the compounds of the disclosure will vary, but, in particular embodiments, a dose could be from 105to 10100copies of an artificial expression construct of the disclosure. In particular embodiments, a patient receiving intravenous, intraparenchymal, intraspinal, retro-orbital, or intrathecal administration can be infused with from 106to 1022copies of the artificial expression construct.

[0121] Certain exemplary uses include methods of treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with artificial expression constructs disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0122] An "effective amount" is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts disclosed herein can cause a statistically-significant effect in an animal model, human study, in vivo, or in vitro assay. For example, an effective amount can reduce a symptom of an SLC6A1 -related disorder in an animal model. Effective amounts are often administered for research purposes.

[0123] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of an SLC6A1 -related disorder or displays only early signs or symptoms of an SLC6A1 -related disorder such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the SLC6A1-related disorder further. Thus, a prophylactic treatment functions as a preventative treatment against an SLC6A1-related disorder.

[0124] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of an SLC6A1 -related disorder and is administered to the subject for the purpose of diminishing or eliminating the signs or symptoms of the SLC6A1 -related disorder. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the SLC6A1 -related disorder and / or reduce control or eliminate side effects of the SLC6A1 -related disorder.

[0125] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0126] In certain examples, an effective amount of the artificial expression construct can reduce the incidence and severity of an SLC6A1 -related disorder or disease.

[0127] Exemplary SLC6A1 -related disorders that can be treated include epilepsy (e.g., myoclonic-atonic epilepsy (MAE; also referred to as Doose syndrome), genetic generalized epilepsy, non-acquired focal epilepsy), epileptic encephalopathy, seizures (e.g., atypical absence seizures, atonic seizures, or myoclonic seizures), autism spectrum disorder (ASD), intellectual disability, schizophrenia, movement disorders, ataxia, tremors, behavior disorders, aggression, hyperactivity, and / or other SLC6A1 -related disorders described herein.

[0128] Many subjects with an SLC6A1 -related disorder are diagnosed with specific epilepsy syndromes including Doose syndrome, childhood absence epilepsy, epilepsy with eyelid myoclonias, or Lennox-Gastaut syndrome. In these cases, the epilepsy syndrome diagnosis is a description of the types of seizures the subject is experiencing, but the genetic diagnosis of SLC6A1 -related disorder is the primary diagnosis that explains why the subject has developed epilepsy.

[0129] In particular embodiments, methods to determine the efficacy of the treatments using constructs disclosed herein will be measured before treatment, during the first year after treatment, and at other times. In particular embodiments, efficacy of the treatments using constructs disclosed herein will be determined to be effective if the evaluated measurements can be maintained at a normal, non-SLC6A1 -related disorder level, reduced to a non-SLC6A1 -related disorder level, or reduced such that it is still elevated compared to a non-SLC6A1 -related disorder individual, but is still less than the level which would be expected in an individual without treatment.

[0130] Therapeutically effective amounts disclosed herein can improve motor control, muscle tone, behavior (e.g., aggression or hyperactivity), cognitive function, and / or occurrence of seizures.

[0131] Therapeutically effective amounts can be assessed using developmental tests for cognitive and motor function, neuropsychological tests, electroencephalogram (EEG), magnetic resonance imaging (MRI), computerized tomography (CT) scan, positron emission tomography (PET), single-photon emission computerized tomography (SPECT), statistical parametric mapping (SPM), electrical source imaging (ESI), and / or magnetoencephalography (MEG).

[0132] In a neuropsychological test, thinking, memory and speech skills are assessed.

[0133] An EEG is one of the most common tests used for diagnosis of epilepsy. For an EEG, electrodes are attached to a subject's scalp to record the electrical activity of the subject's brain. In some subjects, the EEG of a subject with epilepsy may look different than a subject without epilepsy. In particular embodiments, the EEG is used while the subject is having a seizure in order to diagnose the class of seizure or epilepsy. In particular embodiments, the EEG is a high-density EEG.

[0134] An MRI uses magnets and radio waves to create a detailed viewof a subject’s brain. In particular embodiments, lesions or abnormalities in the brain can be detected and can be useful in diagnosing seizures or epilepsy. In particular embodiments, an MRI includes a functional MRI (fMRI). A functional MRI measures the changes in blood flow that occur when specific parts of the brain are working. An fMRI is useful in determining the exact locations of critical functions of the brain, such as speech and movement.

[0135] A CT scan uses X-rays to obtain cross-sectional images of the brain. CT scans can reveal abnormalities in the structure of the brain that might be causing seizures, such as tumors, bleeding and cysts.

[0136] PET scans use a small amount of low-dose radioactive material that is injected into a vein to help visualize metabolic activity of the brain and detect abnormalities. Areas of the brain with low metabolism may indicate where seizures occur.

[0137] A SPECT test uses a small amount of low-dose radioactive material that's injected into a vein to create a detailed, 3D map of the blood flow activity in the brain during seizures. Areas of higher than normal blood flow during a seizure may indicate where seizures occur. In particular embodiments, a subtraction ictal SPECT coregistered to MRI (SISCOM) test can be performed. A SISCOM test may provide even more-detailed result by overlapping the SPECT results with a brain MRI.

[0138] SPM is a method of comparing areas of the brain that have increased blood flow during seizures to normal brains, which can give doctors an idea of where seizures begin.

[0139] ESI is a technique that takes EEG data and projects it onto an MRI of the brain to illustrate where seizures are occurring.

[0140] MEG measures the magnetic fields produced by brain activity to identify potential areas of seizure onset.

[0141] For autism spectrum disorder, therapeutically effective amounts can additionally be assessed using standard evaluations for this disease including the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). This guide was created by the American Psychiatric Association and is used by health care providers to diagnose mental disorders. Autism is a “spectrum" disorder because there is a wide variation in the type and severity of symptoms. Some symptoms can include difficulty with communication and interaction with people and / or restricted interests and repetitive behaviors.

[0142] Motor and developmental tests can be utilized to assess therapeutically effective amounts within this context. Examples include the Peabody Developmental Motor Scale (PDMS-II), Alberta Infant Motor Scale (AIMS), Bayley Scales of Infant and Toddler Development®-Third Edition (Bayley-Ill), or the Comprehensive Developmental Inventoryfor Infants and Toddlers (CDIIT).

[0143] The amount of expression constructs and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of effective amounts of the disclosed compositions may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide an effect in the subject. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the artificial expression construct compositions or other genetic constructs, either over a relatively short, or a relatively prolonged 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 107, 108, 109, 1010, 1011, 1012, 1013, or even higher, infectious particles / ml given either as a single dose or divided into two or more administrations as may be required to achieve an intended effect. In fact, in certain embodiments, it may be desirable to administer two or more different expression constructs in combination to achieve a desired effect.

[0144] In certain circumstances it will be desirable to deliver the artificial expression construct in suitably formulated compositions disclosed herein either by pipette, retro-orbital injection, subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intraparenchymally, intracerebro-ventricularly, intramuscularly, intrathecally, intraspinally, intraperitoneally, by oral or nasal inhalation, or by direct application or injection to one or more cells, tissues, or organs. The methods of administration may also include those modalities as described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363.

[0145] (vi) Kits and Commercial Packages. Kits and commercial packages contain an artificial expression construct described herein. The artificial expression construct can be isolated. In particular embodiments, the components of an expression product can be isolated from each other. In particular embodiments, the expression product can be within a vector, within a viral vector, within a cell, within a tissue slice or sample, and / or within a transgenic animal. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutics, pharmaceutical compounds, or means for delivery of the compositions such as syringes, injectables, and the like.

[0146] Embodiments of a kit or commercial package will also contain instructions regarding use of the included components, for example, in basic research, electrophysiological research, neuroanatomical research, and / or the research and / or treatment of a disorder, disease or condition.

[0147] The Exemplary Embodiments below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0148] (vii) Exemplary Embodiments.1. An artificial expression construct including:(i) an autoregulator including:(a) a miRNA scaffold selected from a miR9-1 scaffold, a mir30d scaffold, and a mir181a2 scaffold;(b) a miRNA guide strand selected from an AI2 miRNA guide strand, an AI3 miRNA guide strand, and an AI4 miRNA guide strand; and(c) a miRNA binding site selected from an AI2 miRNA binding site, an AI3 miRNA binding site, and an AI4 miRNA binding site;(ii) an eHGT_380h enhancer;(iii) a promoter; and(iv) an SLC6A1 coding sequence including intronSLC6A1.The artificial expression construct of embodiment 1, wherein the miR9-1 scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site form a miR9-1 -AI2 autoregulator.The artificial expression construct of embodiments 1 or 2, wherein the mir30d scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site form a mir30d-AI2 autoregulator.The artificial expression construct of any of embodiments 1 -3, wherein the mi r181 a2 scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site form a mir181a2-AI2 autoregulator.The artificial expression construct of any of embodiments 1-4, wherein the miR9-1 scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site forms a mir9-1 -AI3 autoregulator.The artificial expression construct of any of embodiments 1-5, wherein the mir30d scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site forms a mir30d-AI3 autoregulator.The artificial expression construct of any of embodiments 1 -6, wherein the mi r181 a2 scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site forms a mir181 a2-AI3 autoregulator.The artificial expression construct of any of embodiments 1-7, wherein the miR9-1 scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site forms a mir9-1 -AI4 autoregulator.The artificial expression construct of any of embodiments 1-8, wherein the mir30d scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site forms a mir30d-AI4 autoregulator.The artificial expression constructof any of embodiments 1-9, wherein the mir181a2 scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site forms a mir181 a2-AI4 autoregulator.The artificial expression construct of any of embodiments 1-10, wherein the AI2 miRNA guide strand includes the sequence of SEQ ID NO: 10.The artificial expression construct of any of embodiments 1-11, wherein the AI3 miRNA guide strand includes the sequence of SEQ ID NO: 11.The artificial expression construct of any of embodiments 1-12, wherein the AI4 miRNA guide strand includes the sequence of SEQ ID NO: 12.The artificial expression construct of any of embodiments 1-13, wherein the AI2 miRNA binding site includes the sequence of SEQ ID NO: 13.The artificial expression construct of any of embodiments 1-14, wherein the AI3 miRNA binding site includes the sequence of SEQ ID NO: 14.The artificial expression construct of any of embodiments 1-15, wherein the AI4 miRNA binding site includes the sequence of SEQ ID NO: 15.The artificial expression construct of any of embodiments 1-16, wherein the mir9-1 scaffold and AI2 miRNA guide strand includes the sequence of SEQ ID NO: 1.The artificial expression construct of any of embodiments 1-17, wherein the mir30d scaffold and AI2 miRNA guide strand includes the sequence of SEQ ID NO: 2.The artificial expression construct of any of embodiments 1-18, wherein the mir181 a2 scaffold and AI2 miRNA guide strand includes the sequence of SEQ ID NO: 3.The artificial expression construct of any of embodiments 1-19, wherein the mir9-1 scaffold and AI3 miRNA guide strand includes the sequence of SEQ ID NO: 4.The artificial expression construct of any of embodiments 1-20, wherein the mir30d scaffold and AI3 miRNA guide strand includes the sequence of SEQ ID NO: 5.The artificial expression construct of any of embodiments 1-21, wherein the mir181 a2 scaffold and AI3 miRNA guide strand includes the sequence of SEQ ID NO: 6.The artificial expression construct of any of embodiments 1-22, wherein the mir9-1 scaffold and AI4 miRNA guide strand includes the sequence of SEQ ID NO: 7.The artificial expression construct of any of embodiments 1-23, wherein the mir30d scaffold and AI4 miRNA guide strand includes the sequence of SEQ ID NO: 8.The artificial expression construct of any of embodiments 1-24, wherein the mir181 a2 scaffold and AI4 miRNA guide strand includes the sequence of SEQ ID NO: 9.The artificial expression construct of any of embodiments 1-25, wherein the eHGT_380h enhancer includes the sequence of SEQ ID NO: 16.The artificial expression construct of any of embodiments 1-26, wherein the promoter includes a minimal promoter. The artificial expression construct of embodiment 27, wherein the minimal promoter includes minBG, minCMV, minCMV*, minRho, orminRho*.The artificial expression construct of any of embodiments 1-28, wherein the SLC6A1 coding sequence includes SEQ ID NO: 52 and SEQ ID NO: 53.The artificial expression construct of any of embodiments 1-29, wherein the artificial expression construct is associated with a capsid that crosses a blood brain barrier.The artificial expression construct of any of embodiments 30, wherein the capsid includes PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS.The artificial expression construct of any of embodiments 1-31, further including a tag cassette.The artificial expression construct of embodiment 32, wherein the tag cassette encodes the sequence of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 33, or SEQ ID NO: 32. The artificial expression construct of embodiments 32 or 33, wherein the tag cassette includes the sequence of SEQ ID NO: 32.The artificial expression construct of any of embodiments 1-34, wherein the artificial expression construct includes or encodes a skipping element.The artificial expression construct of embodiment 35, wherein the skipping element includes a 2A peptide and / or an internal ribosome entry site (IRES).The artificial expression construct of embodiment 36, wherein the 2A peptide includes T2A, P2A, E2A, or F2A. The artificial expression construct of any of embodiments 1-37, wherein the artificial expression construct includes or encodes a set of features selected from: mir9-1 scaffold, mir30d scaffold, mir181 a2 scaffold, AI2 miRNA guide strand, AI3 miRNA guide strand, AI4 miRNA guide strand, AI2 miRNA binding site, AI3 miRNA binding site, AI4 miRNA binding site, hsA2, AAV, scAAV, rAAV, pAAV, minBG, hSynl, mGad2prom, CMV, minCMV, minRho, minRho*, intronSLC6A1, fluorescent protein (e.g., EGFP, SYFP2, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), tag cassette, 10 aa, nuclear localization proteins, cleavage peptide, WPRE, WPRE3, hGHpA and / or BGHpA.The artificial expression construct of any of embodiments 1-38, wherein the artificial expression construct includes or encodes a set of features selected from:eHGT_380h-minBG-mir9-1-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-WPRE3-BGHpA;eHGT_380h-minBG-mir30d-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-WPRE3-BGHpA;eHGT_380h-minBG-mir181a2-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-WPRE3-BGHpA;eHGT_380h-minBG-mir9-1-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-WPRE3-BGHpA;eHGT_380h-minBG-mir30d-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-WPRE3-BGHpA;eHGT_380h-minBG-mir181 a2-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-WPRE3-BGHpA;eHGT_380h-minBG-mir9-1-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-WPRE3-BGHpA;eHGT_380h-minBG-mir30d-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-WPRE3-BGHpA;eHGT_380h-minBG-mir9-1-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-[post-regulatory elements]; eHGT_380h-minBG-mir30d-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-[post-regulatory elements]; eHGT_380h-minBG-mir181a2-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-[post-regulatory elements]; eHGT_380h-minBG-mir9-1-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-[post-regulatory elements]; eHGT_380h-minBG-mir30d-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-[post-regulatory elements]; eHGT_380h-minBG-mir181a2-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-[post-regulatory elements]; eHGT_380h-minBG-mir9-1-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-[post-regulatory elements]; andeHGT_380h-minBG-mir30d-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-[post-regulatory elements],A vector including the artificial expression construct of any of embodiments 1-39.The vector of embodiment 40, wherein the vector includes a viral vector.The vector of embodiment 41 , wherein the viral vector includes a recombinant adeno-associated viral (AAV) vector. An adeno-associated viral (AAV) vector including an SLC6A1 coding sequence, wherein the SLC6A1 coding sequence is under transcriptional control of a promoter, an eHGT_380h enhancer, and an autoregulator selected from mir9-1-AI2, mir30d-AI2, mir181a2-AI2, mir9-1-AI3, mir30d-AI3, mir181a2-AI3, mir9-1-AI4, mir30d-AI4, and mir181 a2-AI4.A transgenic cell including the artificial expression construct of any of embodiments 1-39.The transgenic cell of embodiment 44, wherein the transgenic cell is murine, human, or non-human primate. A non-human transgenic animal including the artificial expression construct of any of embodiments 1-39.The non-human transgenic animal of embodiment 46, wherein the non-human transgenic animal is a mouse or a non-human primate.An administrable composition of the artificial expression construct of any of embodiments 1-39.A kit including the artificial expression construct of any of embodiments 1-39.A method for expressing SLC6A1 within astrocytes and / or neurons in vivo or in vitro, the method including providing the administrable composition of embodiment 48 in a sufficient dosage and for a sufficient time to a sample or subject including astrocytes and / or neurons thereby expressing SLC6A1 within astrocytes and / or neurons.The method of embodiment 50, wherein the SLC6A1 is encoded by intronSLC6A1.The method of embodiments 50 or 51, wherein the SLC6A1 is encoded by the sequence of SEQ ID NOs: 52 and 53.The method of any of embodiments 50-52, wherein the providing includes pipetting.The method of embodiment 53, wherein the pipetting is to a brain slice.The method of embodiment 54, wherein the brain slice includes an astrocyte and / or neuron.The method of embodiments 54 or 55, wherein the brain slice is murine, human, or non-human primate.The method of any of embodiments 50-52, wherein the providing includes administering to a living subject. The method of embodiment 57, wherein the living subject is a human, a non-human primate, or a mouse.The method of embodiments 57 or 58, wherein the administering provides a therapeutically effective amount. The method of embodiment 59, wherein the therapeutically effective amount treats an SLC6A1 -related disorder. The method of embodiment 60, wherein the SLC6A1 -related disorder includes epilepsy, epileptic encephalopathy, seizures, autism spectrum disorder (ASD), intellectual disability, schizophrenia, movement disorders, ataxia, tremors, behavior disorders, aggression, or hyperactivity.The method of embodiment 61, wherein epilepsy includes myoclonic-atonic epilepsy, genetic generalized epilepsy,or non-acquired focal epilepsy.63 The method of embodiments 61 or 62, wherein seizures include atypical absence seizures, atonic seizures, or myoclonic seizures.64. The method of any of embodiments 58-63, wherein the administering to a living subject is through injection. 65. The method of embodiment 64, wherein the injection includes intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, retro-orbital injection, intra-cisterna magna (ICM) injection, or intrathecal injection.66 A method of treating a subject with an SLC6A1 -related disorder including administering a therapeutically effective amount of the artificial expression construct of any of embodiments 1-39, thereby treating the subject.67. The method of embodiment 66, wherein the SLC6A1 -related disorder includes epilepsy, epileptic encephalopathy, seizures, autism spectrum disorder (ASD), intellectual disability, schizophrenia, movement disorders, ataxia, tremors, behavior disorders, aggression, or hyperactivity.68. The method of embodiment 67, wherein the subject is a mammal.69 The method of embodiments 67 or 68, wherein the subject is a human.70. An artificial expression construct having the sequence of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81, or a sequence having at least 90% sequence identity to the full length of the sequence of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81.71. A method of autoregulating SLC6A1 expression, the method including administering a therapeutically effective amount of the artificial expression construct of any of embodiments 1-39, thereby autoregulating SLC6A1 expression.72. The method of embodiment 71, wherein the administering is to a subject.73. The method of embodiment 72, wherein the subject is a mammal.74. The method of embodiments 72 or 73, wherein the subject is a murine, human, or non-human primate.75. The method of embodiment 71, wherein the administering is to a cell.76 The method of embodiment 75, wherein the cell includes an astrocyte.77. The method of embodiments 75 or 76, wherein the cell includes a neuron.

[0149] (viii) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0150] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programswell known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acidchanges in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0151] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, 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 divided 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 (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non-polar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0152] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic 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 on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+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); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0153] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±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 the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0154] As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+O.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicityvalue and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0155] 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.

[0156] As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistical ly-significant degree.

[0157] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0158] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part i (Griffin, A. M., and Griffin, H. G., 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 to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0159] Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to asequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pig / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0160] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms "include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant deregulation in expression of the artificial expression constructs disclosed herein.

[0161] In particular embodiments, artificial means not naturally occurring.

[0162] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth 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 attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, towithin a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0163] 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. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0164] The terms “a,” “an," “the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0165] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. 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 included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0166] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than 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 otherwise clearly contradicted by context.

[0167] Furthermore, numerous references have been made to patents, printed publications, journal articles and otherwritten text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching

[0168] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0169] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0170] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

CLAIMSWhat is claimed is:

1. An artificial expression construct comprising:(i) an autoregulator comprising:(a) a mir30d scaffold;(b) an AI2 miRNA guide strand; and(c) an AI2 miRNA binding site;(ii) an eHGT_380h enhancer;(iii) a promoter; and(iv) an SLC6A1 coding sequence comprising intronSLC6A1.

2. An artificial expression construct comprising:(i) an autoregulator comprising:(a) a miRNA scaffold selected from a miR9-1 scaffold, a mir30d scaffold, and a mir181a2 scaffold;(b) a miRNA guide strand selected from an AI2 miRNA guide strand, an AI3 miRNA guide strand, and an AI4 miRNA guide strand; and(c) a miRNA binding site selected from an AI2 miRNA binding site, an AI3 miRNA binding site, and an AI4 miRNA binding site;(ii) an eHGT_380h enhancer;(iii) a promoter; and(iv) an SLC6A1 coding sequence comprising intronSLC6A13. The artificial expression construct of claim 2, wherein the miR9-1 scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site form a miR9-1 -AI2 autoregulator.

4. The artificial expression construct of claim 2, wherein the mir30d scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site form a mir30d-AI2 autoregulator.

5. The artificial expression construct of claim 2, wherein the mir181 a2 scaffold, the AI2 miRNA guide strand, and the AI2 miRNA binding site form a mir181 a2-AI2 autoregulator.

6. The artificial expression construct of claim 2, wherein the miR9-1 scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site forms a mir9-1 -AI3 autoregulator.

7. The artificial expression construct of claim 2, wherein the mir30d scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site forms a mir30d-AI3 autoregulator.

8. The artificial expression construct of claim 2, wherein the mir181 a2 scaffold, the AI3 miRNA guide strand, and the AI3 miRNA binding site forms a mir181 a2-AI3 autoregulator.

9. The artificial expression construct of claim 2, wherein the miR9-1 scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site forms a mir9-1 -AI4 autoregulator.

10. The artificial expression construct of claim 2, wherein the mir30d scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site forms a mir30d-AI4 autoregulator.

11. The artificial expression construct of claim 2, wherein the mir181 a2 scaffold, the AI4 miRNA guide strand, and the AI4 miRNA binding site forms a mir181 a2-AI4 autoregulator.

12. The artificial expression construct of claim 2, wherein the AI2 miRNA guide strand comprises the sequence of SEQ ID NO: 10.

13. The artificial expression construct of claim 2, wherein the AI3 miRNA guide strand comprises the sequence of SEQ ID NO: 1114. The artificial expression construct of claim 2, wherein the AI4 miRNA guide strand comprises the sequence of SEQ ID NO: 12.

15. The artificial expression construct of claim 2, wherein the AI2 miRNA binding site comprises the sequence of SEQ ID NO: 13.

16. The artificial expression construct of claim 2, wherein the AI3 miRNA binding site comprises the sequence of SEQ ID NO: 14.

17. The artificial expression construct of claim 2, wherein the AI4 miRNA binding site comprises the sequence of SEQ ID NO: 15.

18. The artificial expression construct of claim 2, wherein the mir9-1 scaffold and AI2 miRNA guide strand comprises the sequence of SEQ ID NO: 1.

19. The artificial expression construct of claim 2, wherein the mir30d scaffold and AI2 miRNA guide strand comprises the sequence of SEQ ID NO:

220. The artificial expression construct of claim 2, wherein the mir181a2 scaffold and AI2 miRNA guide strand comprises the sequence of SEQ ID NO: 3.

21. The artificial expression construct of claim 2, wherein the mir9-1 scaffold and AI3 miRNA guide strand comprises the sequence of SEQ ID NO: 4.

22. The artificial expression construct of claim 2, wherein the mir30d scaffold and AI3 miRNA guide strand comprises the sequence of SEQ ID NO:

523. The artificial expression construct of claim 2, wherein the mir181a2 scaffold and AI3 miRNA guide strand comprises the sequence of SEQ ID NO: 6.

24. The artificial expression construct of claim 2, wherein the mir9-1 scaffold and AI4 miRNA guide strand comprises the sequence of SEQ ID NO: 7.

25. The artificial expression construct of claim 2, wherein the mir30d scaffold and AI4 miRNA guide strand comprises the sequence of SEQ ID NO:

826. The artificial expression construct of claim 2, wherein the mir181a2 scaffold and AI4 miRNA guide strand comprises the sequence of SEQ ID NO: 9.

27. The artificial expression construct of claim 2, wherein the eHGT_380h enhancer comprises the sequence of SEQ ID NO: 16.

28. The artificial expression construct of claim 2, wherein the promoter comprises a minimal promoter.

29. The artificial expression construct of claim 28, wherein the minimal promoter comprises minBG, minCMV, minCMV*, minRho, or minRho*30. The artificial expression construct of claim 28, wherein the minimal promoter comprises minBG.

31. The artificial expression construct of claim 2, wherein the SLC6A1 coding sequence comprises SEQ ID NO: 52 and SEQ ID NO: 5332. The artificial expression construct of claim 2, wherein the artificial expression construct is associated with a capsid that crosses a blood brain barrier.

33. The artificial expression construct of claim 32, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, orAAV-PPS.

34. The artificial expression construct of claim 2, further comprising a tag cassette.35 The artificial expression construct of claim 34, wherein the tag cassette encodes the sequence of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 33, or SEQ ID NO:

32.

36. The artificial expression construct of claim 34, wherein the tag cassette comprises the sequence of SEQ ID NO:32.

37. The artificial expression construct of claim 3, wherein the artificial expression construct comprises or encodes a skipping element.

38. The artificial expression construct of claim 37, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES).

39. The artificial expression construct of claim 38, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.

40. The artificial expression construct of claim 2, wherein the artificial expression construct comprises or encodes a set of features selected from: mir9-1 scaffold, mir30d scaffold, mir181 a2 scaffold, AI2 miRNA guide strand, AI3 miRNA guide strand, AI4 miRNA guide strand, AI2 miRNA binding site, AI3 miRNA binding site, AI4 miRNA binding site, hsA2, AAV, scAAV, rAAV, pAAV, minBG, hSynl, mGad2prom, CMV, minCMV, minRho, minRho*, intronSLC6A1 , fluorescent protein (e.g., EGFP, SYFP2, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), tag cassette, 10 aa, nuclear localization proteins, cleavage peptide, WPRE, WPRE3, hGHpA and / or BGHpA.

41. The artificial expression construct of claim 2, wherein the artificial expression construct comprises or encodes a set of features selected from:eHGT_380h-minBG-mir9-1-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-WPRE3-BGHpA;eHGT_380h-minBG-mir30d-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-WPRE3-BGHpA;eHGT_380h-minBG-mir181a2-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-WPRE3-BGHpA;eHGT_380h-minBG-mir9-1-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-WPRE3-BGHpA;eHGT_380h-minBG-mir30d-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-WPRE3-BGHpA;eHGT_380h-minBG-mir181a2-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-WPRE3-BGHpA;eHGT_380h-minBG-mir9-1-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-WPRE3-BGHpA;eHGT_380h-minBG-mir30d-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-WPRE3-BGHpA;eHGT_380h-minBG-mir9-1-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-[post-regulatory elements]; eHGT_380h-minBG-mir30d-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-[post-regulatory elements]; eHGT_380h-minBG-mir181a2-AI2-intronSLC6A1-[tag cassette]-1xAI2BS-[post-regulatory elements]; eHGT_380h-minBG-mir9-1-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-[post-regulatory elements]; eHGT_380h-minBG-mir30d-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-[post-regulatory elements]; eHGT_380h-minBG-mir181a2-AI3-intronSLC6A1-[tag cassette]-1xAI3BS-[post-regulatory elements]; eHGT_380h-minBG-mir9-1-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-[post-regulatory elements]; and eHGT_380h-minBG-mir30d-AI4-intronSLC6A1-[tag cassette]-1xAI4BS-[post-regulatory elements],42 A vector comprising the artificial expression construct of claim 2.

43. The vector of claim 42, wherein the vector comprises a viral vector.

44. The vector of claim 43, wherein the viral vector comprises a recombinant adeno-associated viral (AAV) vector.

45. An adeno-associated viral (AAV) vector comprising an SLC6A1 coding sequence, wherein the SLC6A1 coding sequence is under transcriptional control of a promoter, an eHGT_380h enhancer, and an autoregulator selected from mir9-1-AI2, mir30d-AI2, mir181 a2-AI2, mir9-1-AI3, mir30d-AI3, mir181 a2-AI3, mir9-1-AI4, mir30d-AI4, and mir181 a2-AI4.

46. A transgenic cell comprising the artificial expression construct of claim 2.

47. The transgenic cell of claim 46, wherein the transgenic cell is murine, human, or non-human primate.

48. A non-human transgenic animal comprising the artificial expression construct of claim 2.

49. The non-human transgenic animal of claim 48, wherein the non-human transgenic animal is a mouse or a non- human primate.50 An administrable composition of the artificial expression construct of claim 2.

51. A kit comprising the artificial expression construct of claim 2.

52. A method for expressing SLC6A1 within astrocytes and / or neurons in vivo or in vitro, the method comprising providing the administrable composition of claim 50 in a sufficient dosage and for a sufficient time to a sample or subject comprising astrocytes and / or neurons thereby expressing SLC6A1 within astrocytes and / or neurons.

53. The method of claim 52, wherein the SLC6A1 is encoded by intronSLC6A1.54 The method of claim 52, wherein the SLC6A1 is encoded by the sequence of SEQ ID NOs: 52 and 53.

55. The method of claim 52, wherein the providing comprises pipetting.

56. The method of claim 55, wherein the pipetting is to a brain slice.

57. The method of claim 56, wherein the brain slice comprises an astrocyte and / or neuron.58 The method of claim 56, wherein the brain slice is murine, human, or non-human primate.

59. The method of claim 52, wherein the providing comprises administering to a living subject.

60. The method of claim 59, wherein the living subject is a human, a non-human primate, or a mouse.

61. The method of claim 59, wherein the administering provides a therapeutically effective amount.

62. The method of claim 61, wherein the therapeutically effective amount treats an SLC6A1 -related disorder.

63. The method of claim 62, wherein the SLC6A1 -related disorder comprises epilepsy, epileptic encephalopathy, seizures, autism spectrum disorder (ASD), intellectual disability, schizophrenia, movement disorders, ataxia, tremors, behavior disorders, aggression, or hyperactivity.

64. The method of claim 63, wherein epilepsy comprises myoclonic-atonic epilepsy, genetic generalized epilepsy, or non-acquired focal epilepsy.

65. The method of claim 63, wherein seizures comprise atypical absence seizures, atonic seizures, or myoclonic seizures.66 The method of claim 60, wherein the administering to a living subject is through injection.

67. The method of claim 66, wherein the injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, retro-orbital injection, intra-cisterna magna (ICM) injection, or intrathecal injection.

68. A method of treating a subject with an SLC6A1 -related disorder comprising administering a therapeutically effective amount of the artificial expression construct of claim 2, thereby treating the subject.69 The method of claim 68, wherein the SLC6A1 -related disorder comprises epilepsy, epileptic encephalopathy, seizures, autism spectrum disorder (ASD), intellectual disability, schizophrenia, movement disorders, ataxia, tremors, behavior disorders, aggression, or hyperactivity.

70. The method of claim 69, wherein the subject is a mammal.

71. The method of claim 69, wherein the subject is a human.

72. An artificial expression construct having the sequence of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81, or a sequence having at least 90% sequence identity to the full length of the sequence of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81.

73. A method of autoregulating SLC6A1 expression, the method comprising administering a therapeutically effective amount of the artificial expression construct of claim 2, thereby autoregulating SLC6A1 expression.

74. The method of claim 73, wherein the administering is to a subject.75 The method of claim 74, wherein the subject is a mammal.

76. The method of claim 74, wherein the subject is a murine, human, or non-human primate.

77. The method of claim 73, wherein the administering is to a cell.

78. The method of claim 77, wherein the cell comprises an astrocyte.79 The method of claim 77, wherein the cell comprises a neuron.

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