Artificial expression constructs for selectively regulating gene expression in interneurons

Abstract

Artificial expression constructs are described for selectively regulating gene expression in selected central nervous system cell types. The artificial expression constructs can be used to selectively express synthetic genes or modify gene expression in GABAergic interneurons.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 742,835, filed Oct. 8, 2018; U.S. Provisional Patent Application No. 62 / 749,012, filed Oct. 22, 2018; and U.S. Provisional Patent Application No. 62 / 810,281, filed Feb. 25, 2019, each of which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0002] Statement Regarding Federally Sponsored Research or Development This invention was made with government support under grant number RF1MH114126 from the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to a Sequence Listing The Sequence Listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is A166 - 0006PCT_ST25.txt. The text file is 379 KB, was created on Oct. 3, 2019, and is electronically transmitted via EFS - Web.

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

Background Art

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

[0006] Gene delivery specific to a cell type or cell class using non-pathogenic recombinant adeno-associated virus (rAAV) has shown increasing support for the treatment of a wide range of diseases. Incorporating one or more cis-acting DNA control elements, such as a specific promoter or enhancer, into rAAV has been beneficial to provide specificity of expression within specific target cells, including specific cell types or cell classes in the brain.

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

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

[0009] One major drawback of using rAAV as a gene delivery system is the limited packaging capacity of AAV. This is particularly restrictive for inserting long gene control elements and expression elements. Furthermore, many existing intervening neuron-specific rAAV expression constructs can result in weak gene expression that reduces their usefulness in research and therapeutic applications.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0012] The present disclosure overcomes the disadvantages of the prior art by providing engineered enhancer elements that result in rapid and robust cell-specific expression of heterologous coding sequences in forebrain GABAergic interneurons.

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

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

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

[0016] The engineered concatenated I56i core disclosed herein enables a new and improved gene delivery vector that is particularly useful for achieving selective transgene expression in forebrain GABAergic interneurons of diverse animal species, including humans.

[0017] Many of the drawings presented herein are better understood as color drawings. The applicant considers the color versions of the drawings to be part of the original application and reserves the right to present color images of the drawings in later proceedings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] To fully understand brain biology, it is necessary to distinguish and define different cell types. To identify and / or study these different cell types, it is necessary to identify vectors that can selectively label and perturb different cell types. In mice, the use of recombinase driver lines has a great effect on labeling cell populations that share marker gene expression. However, the generation, maintenance, and use of such lines to label cell types with high specificity can be costly, often require triple transgenic crosses, and result in low-frequency experimental animals. Furthermore, since these tools require germline transgenic animals, they are not applicable to humans, and recent advances in single-cell profiling such as single-cell RNA-seq (Tasic et al., Nature 563, 72-78 (2018); Tasic 2016, Nat Neurosci 19, 335-346) as well as investigations of neuroelectrophysiology and morphology (Gouwens 2019, Nat Neurosci 22, 1182-1195) have revealed that many recombinant driver lines label heterogeneous mixtures of cell types and often contain multiple subclasses of cells. For example, the Rbp4-Cre mouse driver line commonly used to label layer 5 (L5) neurons also labels cells with dramatically different connectivity patterns, namely intratelencephalic (IT, also called corticocortical) and pyramidal tract (PT, also called corticocortical) neurons within L5 telencephalon.

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

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

[0022] One significant drawback of using rAAV as a gene delivery system is the limited packaging capacity of AAV. This is particularly restrictive for incorporating long gene control and expression elements. Additionally, many existing intervening neuron-specific rAAV expression constructs can result in weak gene expression that reduces their usefulness in research and therapeutic applications.

[0023] The present disclosure overcomes the drawbacks of the prior art by providing artificial enhancer elements that include concatemerized cores of the I56i enhancer. These artificial enhancer elements result in unexpectedly strong peak transgene expression in forebrain GABAergic interneurons after viral transduction of mouse, monkey, and human brain tissues (see FIGS. 2A, 2B, 3, 4, 5A, 5E, 7, 8A, and 8B). The onset is also surprisingly rapid (see FIGS. 5A - 5E), resulting in fast and high expression when directly compared to, for example, the virus packaged in Addgene plasmid number 83900. The increase in expression is synergistically supra-linear and does not appear to be a simple three-fold of the level driven by the original enhancer (FIG. 2B).

[0024] In certain embodiments, the I56i enhancer core can be derived from, for example, the human and mouse I56i enhancers, or the zebrafish I46i enhancer (SEQ ID NOs: 1, 4, and 5, respectively). The selected core of the I56i enhancer can include SEQ ID NO: 2 (the core shared by humans and mice) or SEQ ID NO: 6 (zebrafish I46icore). In certain embodiments, the core is concatemerized. For example, SEQ ID NO: 3 provides a 3-copy concatemer of the selected human / mouse I56i core, and SEQ ID NO: 7 provides a 3-copy concatemer of the selected zebrafish I46i core.

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

[0026] The engineered concatemerized I56i core disclosed herein enables a new and improved gene delivery vector that is particularly useful for achieving selective transgene expression in neocortical GABAergic interneurons of diverse animal species, including humans and non-human primates. Importantly, GABAergic interneurons are deeply involved in central processing and development, and their dysfunction is associated with various brain disorders. Thus, the enhancers and expression constructs described herein have many immediate uses in research and the development of clinical treatments. The artificial enhancer can be used in experimental situations where the original enhancer hl56i has been found to be insufficient (e.g., retro-orbital delivery of a virus encoding a transgene for a loss-of-function experiment).

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

[0028] (i) Artificial expression constructs & vectors for the selective expression of genes in selected cell types. The artificial expression constructs disclosed herein comprise (i) enhancer sequences that effect selective expression of coding sequences within targeted central nervous system cell types, (ii) coding sequences to be expressed, and (iii) promoters. The expression constructs may also include other regulatory elements if necessary or beneficial.

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

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

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

[0032] In certain embodiments, the product of the coding sequence is expressed at low levels in non-selected cell types, e.g., less than 1% of the level at which the product is expressed in the selected neurons, or 1%, 2%, 3%, 5%, 10%, 15% or 20%. In certain embodiments, the targeted central nervous system cell type is the only cell type that expresses the correct combination of transcription factors that bind to the enhancers disclosed herein and drive gene expression. Thus, in certain embodiments, expression occurs only within the targeted cell type.

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

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

[0035] Sncg: Found in many cortical layers, having molecular overlap with Lamp5 and Vip cells, but the expression of Lamp5 or Vip is not consistent, while the expression of Sncg is more consistent. These neurons express the neurotransmitter Cck and mainly have a multipolar or cage cell morphology.

[0036] Serpinf1: Found in many cortical layers, having molecular overlap with Sncg and Vip cells, but the expression of Sncg or Vip is not consistent, while the expression of Serpinf1 is more consistent.

[0037] Vip: Found in many cortical layers, especially frequently in the upper layers (L1 - L4), highly expressing the neurotransmitter vasoactive intestinal peptide (Vip).

[0038] Sst: Found in many cortical layers, especially frequently in the lower layers (L5 - L6). These highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic inputs to postsynaptic neurons. This subclass includes sleep - active horizontal projection Sst Chodl (or Sst Nos1) neurons that express a shared marker gene containing Sst, which are very different from other Sst neurons.

[0039] Pvalb: Found in many cortical layers, especially frequently in the lower layers (L5 - L6). These highly express the neurotransmitter parvalbumin (Pvalb), express Tac1, and frequently attenuate the output of postsynaptic neurons. This subclass includes chandelier cells that have a distinct chandelier - like morphology and express the markers Cpne5 and Vipr2 in mice and NOG and UNC5B in humans.

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

[0041] Lamp5, Sncg, Serpinf1, and VIP: Developmentally derived from progenitor neurons of the caudal ganglionic eminence (CGE).

[0042] Sst and Pvalb: Developmentally derived from progenitor neurons of the medial ganglionic eminence (MGE).

[0043] Glutamatergic subclass: All: Express the glutamate neurotransmitters Slc17a6 and / or Slc17a7.

[0044] L2 / 3 IT: Mainly present in layer 2 / 3, mainly having intracerebral (intercortical) projections.

[0045] L4 IT: Mainly present in layer 4, mainly having intracerebral (intercortical) projections.

[0046] L5 IT: Mainly present in layer 5, mainly having intracerebral (intercortical) projections. Also called L5a.

[0047] L5 PT: Mainly present in layer 5, mainly having corticocortical (pyramidal tract or corticofugal) projections. Also called L5b or L5 CF. These cells are located in the primary motor cortex and adjacent regions and are corticospinal projection neurons. These are associated with motor neuron / movement disorders such as ALS.

[0048] Neocortical layer 5 extracerebral (ET) projection pyramidal neurons (L5 ET): Thick fusiform pyramidal neurons including characteristic subtypes found only in special regions such as Betz cells, Meynert cells, and von Economo cells.

[0049] L5 NP: Mainly present in layer 5, mainly having projections nearby.

[0050] L6 CT: Mainly present in layer 6, mainly having corticothalamic projections.

[0051] L6 IT: Mainly present in layer 6, it mainly has intracortical (intercortical) projections within the telencephalon. This subclass includes L6 IT Car3 cells that are very similar to intracortical projection cells in the prefrontal cortex.

[0052] L6b: Mainly present in the cortical subplate (L6b), local area (near the cell body) projections, intercortical projections from VISp to the anterior cingulate gyrus, and corticocortical projections to the thalamus are observed.

[0053] CR: A distinct subclass defined by a single type in L1, Cajal-Retzius cells express distinct molecular markers Lhx5 and Trp73.

[0054] Non-neuronal subclasses: Astrocytes: Glial cells of neuroectodermal origin that express the marker Aqp4. They have a distinct star-shaped morphology and are involved in the metabolic support of other cells in the brain.

[0055] Oligodendrocytes: Glial cells of neuroectodermal origin that express the marker Sox10. This category includes oligodendrocyte precursor cells (OPCs). Oligodendrocytes are the main subclass responsible for myelinating neurons.

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

[0057] Pericytes: Vascular-related cells also called mural cells that express the marker genes Kcnj8 and Abcc9. Pericytes wrap around endothelial cells, are important for regulating blood flow in capillaries, and are involved in the permeability of the blood-brain barrier.

[0058] SMC: Vascular-related cells also called mural cells that express the marker gene Acta2. SMC covers the arterioles in the brain and is involved in the permeability of the blood-brain barrier.

[0059] Endothelium: Cells lining the blood vessels of the brain. Endothelial cells express the markers Tek and PDGF-β.

[0060] Macrophages: Immune cells including macrophages that are present in the brain and perivascular macrophages (PVMs) that may be temporarily associated with brain tissue or included as a byproduct of a brain dissection method.

[0061] In certain embodiments, the coding sequence is a heterologous coding sequence encoding an effector element. An effector element is a sequence that is expressed to achieve an intended effect and that achieves this in practice. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0062] Exemplary reporter genes / proteins include those expressed by Addgene ID numbers 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), 83900 (pAAV-mDlx-GFP-Fishell-1), and 89897 (pcDNA3-FLAG-mTET2(N500)).Exemplary reporter genes can include, inter alia, an expressible fluorescent protein, or an expressible biotin; blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green(TM) (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomer 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(TM) (Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and those encoding tandem conjugates can be included.

[0063] GFP is composed of 238 amino acids (26.9 kDa) and was originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea and emits green fluorescence when exposed to blue light. The GFP of A. victoria has a major excitation peak at a wavelength of 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, which is in the lower green part of the visible spectrum. The GFP of Renilla reniformis has a single major excitation peak at 498 nm. Due to its wide range of potential uses and the evolving needs of researchers, many different variants of GFP have been designed. The first major improvement was a single-point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the spectral properties of GFP, enhancing fluorescence, photostability, shifting the major excitation peak to 488 nm, and maintaining peak emission at 509 nm. Adding the 37 °C folding efficiency (F64L) point mutant to this scaffold resulted in enhanced green fluorescent protein (EGFP). EGFP has an extinction coefficient (denoted by ε), also cited as 55,000 L / (mol●cm), of 9.13X10-21 m 2 / molecule, also known as the optical cross-section area. Superfolder GFP, a series of mutations that allow GFP to fold and mature rapidly even when fused to poorly folding peptides, was reported in 2006.

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

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

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

[0067] Exemplary enzymes, transcription factors, receptors, membrane proteins, cell transport proteins, signaling molecules, and neurotransmitters include enzymes such as lactase, lipase, helicase, α-glucosidase, amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAAT / enhancer-binding protein), and Oct-1; receptors such as transforming growth factor receptor β1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, and interleukin 8 receptor α; membrane proteins, cell transport proteins such as clathrin, dynamin, caveolin, Rab-4A, and Rab-11A; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPase, and HRas; and neurotransmitters such as cocaine- and amphetamine-regulated transcript, substance P, oxytocin, and somatostatin.

[0068] In certain embodiments, the functional molecule includes receptors for nerve function and status, such as calcium reporters. Intracellular calcium concentration is an important predictor of a number of cellular activities, including neuron activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels is by means of a genetically encoded calcium indicator (GECI). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor called GCaMP is an efficient and widely used tool. GCaMP is formed by the fusion of M13 and calmodulin proteins to the N-terminus and C-terminus of circularly permuted GFP. Some GCaMPs produce different fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs with green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Furthermore, GECIs with red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV products including AAV8-CAG-GCaMP3 (Catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (Catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (Catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (Catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (Catalog number: BS12-NXSAAV9), AAV9-CAG-FLEX-jGCaMP7f-WPRE (Catalog number: BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (Catalog number: BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (Catalog number: BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (Catalog number: BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (Catalog number: BS7-NXBAAV8).

[0069] In certain embodiments, the calcium reporter includes a genetically encoded calcium indicator GECI, NTnC; myosin light chain kinase, GFP, calmodulin chimera; calcium indicator TN-XXL; a BRET-based bioluminescent calcium indicator; and / or calcium indicator protein OeNL(Ca2+)-18u).

[0070] In certain embodiments, the functional molecule includes a modulator of neural activity, such as channelrhodopsin (e.g., channelopsin-1, channelrhodopsin-2, and variants thereof). Channelrhodopsin is a subfamily of rhodopsin proteins that function as light-gated ion channels. In addition to channelrhodopsin 1 (ChR1) and channelrhodopsin 2 (ChR2), several variants of channelrhodopsin have been developed. For example, Lin et al. (Biophys J, 2009, 96(5):1803-14) describe creating chimeras of the transmembrane domains of ChR1 and ChR2 in combination with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6):631-3) describe VChR1, a red-shifted channelrhodopsin variant. VChR1 has low light sensitivity and insufficient membrane trafficking and expression. Other known channelrhodopsin variants include the ChR2 variant described by Nagel, et al., Proc Natl Acad Sci USA, 2003, 100(24):13940-5, which is activated by blue light (470 nm) but not sensitive to orange / red light, ChR2 / H134R (Nagel, G., et al., Curr Biol, 2005, 15(24):2279-84), and ChD / ChEF / ChIEF (Lin, J.Y., et al., Biophys J, 2009, 96(5):1803-14). Additional variants are described in Lin, Experimental Physiology, 2010, 96.1:19-25 and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.

[0071] Certain embodiments include, for functional molecules, DNA and RNA editing tools such as CRISPR / CAS (e.g., guide RNAs and nucleases such as Cas, Cas9 or cpf1). Functional molecules can also include engineered Cpf1s, such as those described in US2018 / 0030425, US2016 / 0208243, WO / 2017 / 184768 and Zetsche et al. (2015) Cell 163:759-771; single gRNAs (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563) or editases, guide RNA molecules or homology recombination donor cassettes.

[0072] Additional information regarding CRISPR-Cas systems and their components is described in US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0073] In certain embodiments, the functional molecule includes a designer receptor exclusively activated by designer drugs (DREADD). Designer receptors exclusively activated by designer drugs (DREADD) can be used to modulate cellular function (Rogan and Roth, Pharmacol. Rev. 2011, 63(2):291-315). This family of evolved muscarinic receptors has been shown to increase (Gs-DREADD; Gq-DREADD) or decrease (Gi / o-DREADD) cellular activity following administration of the inactive synthetic ligand clozapine-N-oxide (Armbruster et al., PNAS, 2007, 104(12):5163-5168). When packaged in viral vectors or expressed in transgenic mouse models, these tools enable the control of cellular activity in a defined spatial and temporal manner. For example, activation of hippocampal neurons by the Gq-DREADD receptor amplifies gamma rhythms and increases spontaneous locomotor activity and social behavior in mice (Alexander et al., Neuron, 2009, 63(1):27-39). DREADDs are formed by point mutations in the third and fifth transmembrane regions of the muscarinic receptor (Y149C and A239G of hM3). Additionally, Gs-coupled DREADDs contain the second and third intracellular loops of β1-AR instead of the loop of the M3 muscarinic receptor. Some exemplary DREADDs include hM3DREADD (hM3D) and hM4DREADD (hM4D). Various plasmids containing DREADD are commercially available. For example, at addgene, AAV plasmids containing DREADD include: pAAV-hSyn-DIO-hM3D(Gq)-mCherry (plasmid number 44361), pAAV-hSyn-DIO-hM4d(Gi)-mCherry) (plasmid number 44362), pAAV-EF1a-DIO-hM4d(Gi)-mCherry) (plasmid number 50461), pAAV-GFAP-HA-hM3D(Gq)-IRES)-mCitrine (plasmid number 50470) and pAAV-CaMKIIa-hM4D(Gi)-mCherry (plasmid number 50477).

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

[0075] Exemplary expressible elements are expression products that do not include effector elements, such as non-functional proteins or defective proteins. In certain embodiments, the expressible elements can provide a method for testing the effects of their functional counterparts. In certain embodiments, the expressible elements are non-functional or defective based on engineered mutations that render them non-functional. In these aspects, the non-expressible elements are as structurally similar as possible to their functional counterparts.

[0076] Exemplary self-cleaving peptides include the 2A peptide that results in the production of two proteins from one mRNA. The 2A sequence is short (e.g., 20 amino acids) and enables more use in size-restricted constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the expression construct includes an internal ribosome entry site (IRES) sequence. The IRES enables ribosomes to initiate translation at a second internal site of the mRNA molecule, resulting in the production of two proteins from one mRNA.

[0077] The coding sequences encoding the molecules described herein (e.g., RNA, protein) can be readily obtained from publicly available databases and publications. The coding sequences can further include various sequence polymorphisms, mutations, and / or sequence variants that do not affect the function of the molecule encoded by such changes. The term "encoding" or "encodes" refers to the property of a nucleic acid sequence such as a vector, plasmid, gene, cDNA, mRNA, etc., that functions as a template for the synthesis of other molecules such as proteins.

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

[0079] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can include strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters. Inducible promoters direct expression in response to specific conditions, signals or cellular events. For example, the promoter can be an inducible promoter that requires a specific ligand, small molecule, transcription factor or hormone protein to affect transcription from the promoter. Specific examples of promoters include minBglobin, CMV, minCMV, mutant minCMV, SV40 early promoter, Hsp68 minimal promoter (proHSP68) and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Minimal promoters have no activity to drive gene expression by themselves, but can be activated to drive gene expression when linked to proximal enhancer elements.

[0080] In certain embodiments, the expression construct is provided within a vector. 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 the vector nucleic acid molecule and, for example, integrated within the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within the cell or sequences that allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0081] Viral vectors are widely used to refer to nucleic acid molecules containing viral-derived nucleic acid elements that facilitate the transfer and expression of non-native nucleic acid molecules within cells. The term "adeno-associated virus vector" refers to a viral vector or plasmid containing structural and functional gene elements or portions thereof mainly derived from AAV. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional gene elements or portions thereof mainly derived from retroviruses, and so on. The term "hybrid vector" refers to a vector containing structural and / or functional gene elements derived from more than one viral type.

[0082] Adenovirus. An "adenovirus vector" refers to a construct that contains sufficient adenovirus sequences to (a) support the packaging of an expression construct and (b) express a coding sequence cloned therein in either the sense or antisense direction. Recombinant adenovirus vectors include genetically engineered forms of adenovirus. The genetic constitution of adenovirus is known to be a 36 kb, linear, double-stranded DNA virus, allowing replacement of a large portion of adenovirus DNA with exogenous sequences up to 7 kb. Since adenovirus DNA can replicate in an episomal manner without potential genotoxicity, in contrast to retroviruses, adenovirus infection of host cells does not result in chromosomal integration. Also, adenoviruses are structurally stable and no genome rearrangement has been detected after extensive amplification.

[0083] Adenovirus is particularly suitable for use as a gene transfer vector due to its medium-sized genome, ease of manipulation, high titer, broad target cell range, and high infectivity. Both ends of this viral genome contain inverted terminal repeats (ITRs) of 100 - 200 base pairs, which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are separated upon initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) leads to the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. The products of late genes, which include most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript made by the major late promoter (MLP). The MLP is particularly efficient during the late stage of infection, and all mRNAs transcribed from this promoter have a 5'-triple leader (TPL) sequence that makes them preferred mRNAs for translation.

[0084] Except for the need for the adenovirus vector to be replication-deficient or at least conditionally deficient, the properties of the adenovirus vector are not considered important for the successful implementation of the specific embodiments disclosed herein. The adenovirus may be any of 42 different known serotypes or subgroups A - F. In certain embodiments, adenovirus type 5 is a human adenovirus for which considerable biochemical and genetic information is known and which has historically been used in most constructs that use adenovirus as a vector. Thus, adenovirus type 5 of subgroup C is a preferred starting material for obtaining a conditionally replication-deficient adenovirus vector for use in certain embodiments.

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

[0086] Adeno-associated virus (AAV) is a parvovirus that was discovered as a contaminant of adenovirus stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) and is not associated with any disease. It is also classified as a dependovirus because its replication is dependent on the presence of a helper virus such as adenovirus. Various serotypes have been isolated, among which AAV-2 is the best characterized. AAV has single-stranded linear DNA encapsulated within a capsid formed by the capsid proteins VP1, VP2, and VP3 into an icosahedral virion 20 - 24 nm in diameter.

[0087] AAV DNA is 4.7 kilobases in length. It contains two open reading frames and is flanked by two ITRs. The AAV genome contains two major genes, rep and cap. The rep gene encodes proteins responsible for viral replication, and cap encodes the capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis-components of AAV for integration into the chromosome. Thus, AAV can be used as a vector in which all viral coding sequences are removed and replaced by a gene cassette for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their map positions. Transcription from p5 and p19 results in the production of rep proteins, and transcription from p40 produces the capsid proteins.

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

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

[0090] Retrovirus. Retroviruses are common tools for gene delivery. "Retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus". The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoding the structural proteins and enzymes necessary to produce new virus particles.

[0091] Examples of retroviruses suitable for use in certain embodiments include: Moloney-mouse leukemia virus (M-MuLV), Moloney-mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV) and lentiviruses.

[0092] "Lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary viruses include: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) can be used.

[0093] Enhanced safety for the use of some vectors can be provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used for this purpose include, for example, the viral Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and Herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters can drive high levels of transcription in a Tat-independent manner. This replacement results in a lack of the complete U3 sequence in the viral production system, reducing the likelihood of recombination resulting in replication-competent virus. In certain embodiments, the heterologous promoter has additional advantages in controlling the way the viral genome is transcribed. For example, the heterologous promoter can be inducible such that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include one or more compounds or physiological conditions such as the temperature or pH at which the host cell is cultured.

[0094] In certain embodiments, the viral vector contains a TAR element. The term "TAR" refers to the "trans-activation response" genetic element located in the R region of the lentiviral LTR. This element interacts with the lentiviral trans-activator (tat) genetic element to increase viral replication. However, this element is not necessary in embodiments where the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0095] The "R region" refers to the region within the retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends immediately prior to the start of the poly(A) tail. The R region is also defined by being flanked by the U3 region and the U5 region. The R region plays a role in moving the initial DNA from one end of the genome to the other during reverse transcription.

[0096] In certain embodiments, the expression of heterologous sequences within a viral vector is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. A variety of post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids. Examples include the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present within hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); etc. (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector includes a post-transcriptional regulatory element such as WPRE or HPRE. In certain embodiments, the vector lacks or does not contain a post-transcriptional regulatory element such as WPRE or HPRE.

[0097] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts can increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector includes a polyadenylation sequence 3' to the polynucleotide encoding the molecule (e.g., protein) to be expressed. The term "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that directs both termination and polyadenylation of the primary RNA transcript by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus can contribute to increased transcriptional efficiency. Certain embodiments can use BGHpA or SV40pA. In certain embodiments, preferred embodiments of the expression construct include a termination element. These elements can contribute to increasing the transcription level and minimizing read-through from this construct to other plasmid sequences.

[0098] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator element can contribute to protecting the viral vector expression array, e.g., an effector element or an expressible element, from the integration site effect (i.e., the position effect; see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001) that can result in deregulated expression of the transduced sequence mediated by cis-acting elements present in the genomic DNA. In certain embodiments, the viral delivery vector comprises one or more insulator elements in the 3' LTR, and when the provirus integrates into the host genome, the provirus will contain one or more insulators in both the 5' LTR and the 3' LTR by replicating the 3' LTR. Insulators suitable for use in certain embodiments include the chicken β-globin insulator (see Chung et al., Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al., Cell 98:387, 1999), the SP10 insulator (see Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer-blocking insulators (see Liu et al., Nature Biotechnology, 33:198, 2015).

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

[0100] Certain embodiments of the vectors disclosed herein include the following:

[0101]

Table 1

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

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

[0104] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can pass through the BBB after intravenous injection. This enables the conversion of large segments of the central nervous system (CNS), thereby allowing for minimally invasive procedures (Naso et al., BioDrugs. 2017;31(4):317) and is described in the context of clinical trials, for example, for the treatment by AveXis (AVXS-101, NCT03505099) of superior mesenteric artery (SMA) syndrome and for the treatment of CLN3 gene-related neuronal ceroid lipofuscinosis (NCT03770572).

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

[0106] AAV1R6 and AAV1R7, two variants isolated from a library of chimeric AAV vectors (with the AAV1 capsid domain replaced within AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing a significant reduction in liver and vascular endothelial transduction.

[0107] rAAVrh.8 was also isolated from rhesus macaques and shows comprehensive transduction of glial and neuronal cell types in clinically important regions after peripheral administration and also shows reduced peripheral tissue tropism compared to other vectors.

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

[0109] AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence QAVRTSL (SEQ ID NO: 92) and transduces neurons in the enteric nervous system and potently transduces the peripheral sensory ascending pathway to invade the spinal cord and brainstem.

[0110] AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 93). It transports genes to the entire CNS with higher efficiency than AAV9 and transduces astrocytes and the majority of neurons across multiple CNS regions.

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

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

[0113] (ii) Compositions for administration. The artificial expression constructs and vectors of the present disclosure (referred to herein as physiologically active components) may be formulated with carriers suitable for administration to cells, tissue slices, animals (e.g., mice, non-human primates), or humans. The physiologically active components in the compositions described herein may be prepared in neutral form, as the free base, or as a pharmaceutically acceptable salt.

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

[0115] Carriers for physiologically active components include solvents, dispersion media, vehicles, coatings, diluents, isotonic agents and absorption delaying agents, buffer solutions, solutions, suspensions, colloids, and the like. The use of such carriers for physiologically active components is well known in the art. Except for conventional media or agents that are incompatible with physiologically active components, they can be used with the compositions described herein.

[0116] The phrase "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or similar adverse reactions when administered to humans and, in certain embodiments, when administered intravenously (e.g., to the posterior orbital plexus).

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

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

[0119] The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulating half-life have been developed (see, for example, U.S. Patent No. 5,741,516). Further, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (see, for example, U.S. Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

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

[0121] Injectable compositions can include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468). For delivery via injection, the form is sterile and fluid to the extent that it can be delivered by a syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage and can optionally contain one or more preservative compounds against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof, and / or vegetable oils. For example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and / or by the use of surfactants, appropriate fluidity can be maintained. Prevention of the action of microorganisms can be brought about by various antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In various embodiments, the preparation contains isotonic agents, such as sugars or sodium chloride. Prolongation of the absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin. Injectable compositions are preferably buffered as required, and the diluent can first be made isotonic with sufficient saline and glucose.

[0122] Dispersions can also be prepared in glycols, liquid polyethylene glycols and their mixtures, and oils. As shown, under normal conditions of storage and use, these preparations can contain preservatives that prevent the growth of microorganisms.

[0123] The sterilized composition can be prepared by incorporating a physiologically active component in an appropriate amount into a solvent together with other optional components (such as the components listed above), and subsequently subjecting it to filtration sterilization. Generally, the dispersion is prepared by incorporating various sterilized physiologically active components into a sterilized vehicle containing a basic dispersion medium and desired other components (such as the components listed above). In the case of sterilized powders for the preparation of sterilized injectable solutions, the preferred methods of preparation may be vacuum drying techniques and lyophilization techniques that yield powders of the physiologically active component and any further desired components from a previously filtration-sterilized solution.

[0124] The oral composition may be in liquid form, such as a solution, syrup or suspension, or presented as a formulation to be reconstituted before use with water or other suitable vehicles. Such liquid preparations can be prepared by conventional means using 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-hydroxybenzoate or sorbic acid). The composition may be in the form of, for example, tablets or capsules, prepared by conventional means using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); diluents (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.

[0125] Inhalable compositions may be delivered in the form of aerosol spray preparations from a pressurized pack or nebulizer by the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound and a suitable powder base, such as lactose or starch.

[0126] Compositions may also include microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0127] An auxiliary active ingredient can also be incorporated into the composition.

[0128] Typically, the composition may contain at least 0.1% or more of a physiologically active component, although the percentage of the physiologically active component may, of course, vary and may conveniently be 1 or 2% to 70% or 80% or more, or 0.5 to 99% of the weight or volume of the total composition. Usually, the amount of the physiologically active component in each of the physiologically useful compositions can be prepared in any given unit dose of the compound in a manner that yields a suitable dosage. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations and, accordingly, various compositions and dosages may be desirable.

[0129] In certain embodiments, for administration to humans, the composition should meet the sterility, pyrogenicity, and general safety and purity standards required by the U.S. Food and Drug Administration (FDA) or other appropriate regulatory authorities in other countries.

[0130] (iii) Cell lines containing artificial expression constructs. The present disclosure includes cells containing the artificial expression constructs described herein. Cells transformed with an artificial expression construct can be used for many purposes, including neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to evaluate the regulatory properties of enhancers.

[0131] A variety of host cell lines can be used. In certain embodiments, the cells are mammalian neurons. In certain embodiments, the enhancer sequence of the artificial expression construct is the combination of SEQ ID NO: 3 and / or 7 and / or the components shown in CN1390, CN1244, CN1389, CN1203, CN1367, CN1498, CN1499, CN1500, CN1838, or FIG. 16, and the cell line is a human, primate, or mouse neuron. Cell lines that can be used for gene transfer in the present disclosure also include primary cell lines derived from biological tissues such as the brains of rats or mice, and organotypic cell cultures including brain slices from animals such as rats or mice. The PC12 cell line (available from the American Type Culture Collection, ATCC, Manassas, VA) has been shown to express several neuronal marker proteins in response to nerve growth factor (NGF). The PC12 cell line is considered a neuronal cell line and is applicable for use in the present disclosure. The JAR cell (available from ATCC) is a platelet-derived cell line that expresses some neuronal genes such as the serotonin transporter gene and can be used in the embodiments described herein.

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

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

[0134] In certain embodiments, "of neuronal cells" describes that which is of, related to, or includes neuronal cells. Neuronal cells are defined by the presence of axons and dendrites. The term "neuron-specific" refers to something or an activity that is found in or occurs in neuronal cells or cells derived from neuronal cells, but not in or occurring in non-neuronal cells or cells not derived from neuronal cells, such as glial cells like astrocytes or oligodendrocytes.

[0135] In certain embodiments, non-neuronal cell lines containing mouse embryonic stem cells can be used. Cultured mouse embryonic stem cells can be used to analyze the expression of gene constructs using transient transfection with plasmid constructs. Mouse embryonic stem cells are pluripotent and undifferentiated. These cells can be maintained in this undifferentiated state by leukemia inhibitory factor (LIF). Withdrawal of LIF induces differentiation of the embryonic stem cells. In culture, the stem cells form various differentiated cell types. Differentiation is caused by the expression of tissue-specific transcription factors and allows for the evaluation of enhancer sequence function (see, for example, Fiskerstrand et al., FEBS Lett 458:171-174, 1999).

[0136] A method for differentiating stem cells into nerve cells involves replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF) heparin, N2 supplement (e.g., transferrin, insulin, progesterone, putrescine, and selenite), laminin, and polyornithine. A method for generating myelinating oligodendrocytes from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. Bibel, et al., 2007, Nat. Protoc. 2:1034-43 describes a protocol for generating glutamatergic neurons from stem cells, and Chatzi, et al., 2009, Exp. Neurol. 217:407-16 describes a procedure for generating GABAergic neurons. This procedure involves exposing the stem cells to all-trans RA for 3 days. Subsequently, after culturing in a serum-free neuron induction medium containing Neurobasal medium supplemented with B27, bFGF, and EGF, 95% of GABA neurons are generated.

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

[0138] In certain embodiments, a yeast one-hybrid system can also be used to identify compounds that inhibit specific protein / DNA interactions, such as the I56i enhancer, its core, and / or the transcription factors of SEQ ID NO: 3 and / or 7.

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

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

[0141] Detailed methods for producing transgenic animals are described in U.S. Patent No. 4,736,866. Transgenic animals can be of any non-human species, but are preferably non-human primates (NHP), sheep, horses, cows, pigs, goats, dogs, cats, rabbits, chickens, and rodents such as guinea pigs, hamsters, shrews, rats, mice, and ferrets.

[0142] In certain embodiments, the construction of transgenic animals results in organisms having engineered constructs present in all cells at the same genomic integration site. Thus, cell lines derived from such transgenic animals are consistent as long as the engineered constructs are at the same genomic integration site in all cells and will thus suffer from the same position effect variegation. In contrast, introduction of a gene into a cell line or primary cell culture can cause heterogeneous expression of the construct. A drawback of this approach is that expression of the introduced DNA can be affected by the specific genetic background of the host animal.

[0143] As indicated above with respect to cell lines, the artificial expression constructs of the present disclosure can be used to genetically modify mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression constructs are introduced into cultured mouse embryonic stem cells. The transformed ES cells are then injected into blastocysts from a host mother and the host embryo is re-implanted into the mother. This results in a chimeric mouse composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Usually, the mouse from which the cultured ES cells used for gene introduction are derived is selected to have a different coat color than the host mouse into which the transformed cells are injected into the embryo. Therefore, the chimeric mouse has a diverse coat color. As long as the germline tissue is at least partially derived from the genetically modified cells, the chimeric mouse is mated with an appropriate strain to produce offspring having the transgene.

[0144] In addition to the above delivery methods, the following techniques are also contemplated as alternative methods for delivering artificial expression constructs to target cells or selected tissues and organs of animals, particularly cells, organs or tissues of mammalian vertebrates: sonophoresis (e.g., ultrasound as described in U.S. Patent No. 5,656,016); intraosseous injection (U.S. Patent No. 5,779,708); microchip devices (U.S. Patent No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrices (U.S. Patent No. 5,770,219 and U.S. Patent No. 5,783,208); and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0145] (v) Method of use. In certain embodiments, a composition comprising a physiologically active component described herein is administered to a subject to provide a physiological effect.

[0146] In certain embodiments, the disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a heterologous gene that is partially or fully encoded at a position downstream of an enhancer in an engineered sequence. Accordingly, provided herein are methods of using the disclosed artificial expression constructs in the research, testing and potential development of medicaments for preventing, treating or ameliorating the symptoms of a disease, dysfunction or disorder.

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

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

[0149] As is well known in the medical field, the dosage for any given subject depends on many factors including the size, surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The dosage of the compounds of the present disclosure will vary, but in certain embodiments, the dosage may be 10 5 ~10 100 copies of the artificial expression construct of the present disclosure. In certain embodiments, patients receiving intravenous, intraspinal, retro-orbital or intrathecal administration may be injected with 10 6 ~10 22 copies of the artificial expression construct.

[0150] "Effective amount" is the amount of the composition required to effect a desired physiological change in a subject. An effective amount is usually administered for research purposes. The effective amounts disclosed herein can cause a statistically significant effect in animal models or in vitro assays.

[0151] In certain embodiments, the constructs disclosed herein can be utilized to treat Dravet syndrome. In certain embodiments, this method reduces or prevents seizures or their symptoms in patients in need thereof. In certain embodiments, the provided method can reduce or prevent one or more different types of seizures. Ideally, the methods of the present disclosure result in complete prevention of seizures. However, the present disclosure also encompasses methods in which the number of seizures is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0152] Generally, seizures can include convulsions, repetitive movements, paresthesia, and combinations thereof. Seizures can be classified into focal seizures (also called partial seizures) and generalized seizures. Focal seizures occur only on one side of the brain, and generalized seizures occur on both sides of the brain. Specific types of focal seizures include simple focal seizures, complex focal seizures, and secondary generalized seizures. Simple focal seizures may be limited to or concentrated in a specific lobe of the brain (e.g., the temporal lobe, frontal lobe, parietal lobe, or occipital lobe). Complex focal seizures generally occur in a larger portion of one hemisphere than simple focal seizures, but generally occur in the temporal lobe or frontal lobe. When a focal seizure spreads from one side (hemisphere) of the brain to both sides, this seizure is called a secondary generalized seizure. Specific types of generalized seizures include absence seizures (also called petit mal seizures), tonic seizures, atonic seizures, myoclonic seizures, tonic-clonic seizures (also called grand mal seizures), and clonic seizures.

[0153] In certain embodiments, the methods described herein can reduce the frequency of seizures, lessen the severity of seizures, change the type of seizures (e.g., from a more severe type to a less severe type), or combinations thereof, in a treated patient as compared to no treatment (e.g., before treatment) or as compared to treatment with an alternative conventional treatment.

[0154] The amount of the expression construct and the administration time of such a composition are within the scope of those skilled in the art having the benefit of this teaching. However, administration of an effective amount of the disclosed composition would appear to be achievable, for example, by a single administration such as a single injection of a sufficient number of infectious particles to effect an outcome in a subject. Alternatively, in some situations, it may be desirable to provide multiple or continuous administrations of the artificial expression construct composition or other gene construct over a relatively short or relatively long period of time, as may be determined by the individual overseeing the administration of such a composition. For example, the number of infectious particles administered to a mammal can be 10 7 、10 8, 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or even more infectious particles / ml. In fact, in certain embodiments, it may be desirable to administer a combination of two or more different expression constructs to achieve the desired effect.

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

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

[0157] Embodiments of the kit or commercial package may also include instructions regarding the use of the components included, for example, in basic research, electrophysiological research, neuroanatomical research, and / or in the research and / or treatment of disorders, diseases or conditions.

[0158] The following exemplary embodiments and experimental examples are included to illustrate specific embodiments of the present disclosure. Those skilled in the art should recognize that many changes can be made to the specific embodiments disclosed herein, and similar or analogous results can still be obtained without departing from the spirit and scope of the present disclosure.

[0159] (vii) Exemplary embodiments.

[0160] 1. The core of the I56i enhancer, the concatenated core of the I56i enhancer, or the concatenated I56i enhancer.

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

[0162] 3. The I56i enhancer core, the concatenated I56i enhancer core, or the concatenated I56i enhancer of Embodiment 1 or 2, wherein the concatenated core contains SEQ ID NO: 2 or 6.

[0163] 4. The I56i enhancer core, the concatenated I56i enhancer core, or the concatenated I56i enhancer of any one of Embodiments 1 to 3, wherein the concatenated core contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the I56i core.

[0164] 5. An I56i enhancer core, concatenated I56i enhancer core, or concatenated I56i enhancer of Embodiment 4, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 2 and / or 6 (e.g., SEQ ID NO: 2 - SEQ ID NO: 2 - SEQ ID NO: 6; SEQ ID NO: 2 - SEQ ID NO: 6 - SEQ ID NO: 6; SEQ ID NO: 2 - SEQ ID NO: 6 - SEQ ID NO: 2; SEQ ID NO: 6 - SEQ ID NO: 6 - SEQ ID NO: 2; SEQ ID NO: 6 - SEQ ID NO: 2 - SEQ ID NO: 2; and SEQ ID NO: 6 - SEQ ID NO: 2 - SEQ ID NO: 6, etc., SEQ ID NO: 2 and SEQ ID NO: 6 within one array).

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

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

[0167] 8. An I56i enhancer core, concatenated I56i enhancer core, or concatenated I56i enhancer of Embodiment 4 or 5, comprising 3 copies of SEQ ID NO: 2.

[0168] 9. An I56i enhancer core, concatenated I56i enhancer core, or concatenated I56i enhancer of Embodiment 4 or 5, comprising 3 copies of SEQ ID NO: 6.

[0169] 10. An I56i enhancer core, concatenated I56i enhancer core, or concatenated I56i enhancer of Embodiment 8, wherein the concatenated core comprises SEQ ID NO: 3.

[0170] 11. The concatenated core of claim 9, the concatenated I56i enhancer core, or the concatenated I56i enhancer, wherein the concatenated core contains SEQ ID NO: 7.

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

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

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

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

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

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

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

[0178] 19. The artificial expression construct according to any one of embodiments 12 to 18, wherein the expression construct is bound to a capsid that passes through the blood-brain barrier.

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

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

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

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

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

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

[0185] 26. A vector comprising a combination of components shown in FIG. 16.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0206] 47. The method according to any one of embodiments 45 or 46, wherein the brain slice is from a mouse, a human or a non-human primate.

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

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

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

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

[0211] 52. An artificial expression construct consisting of, or consisting essentially of, the combination of features shown in FIG. 16.

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

[0213] In the present disclosure, when the context describes a reference to or use of the enhancer in the zebrafish type as described herein, I56i should be construed as I46i.

[0214] (viii) The experimental implementation of Dravet syndrome (DS) is drug-resistant and threatens the life of epilepsy patients. This typically begins in the first year of life, and seizures induced by fever or body temperature develop into generalized tonic-clonic seizures, tonic-clonic seizures, and unilateral seizures. These seizures are usually resistant to current anti-epileptic drugs, which are the first choice for treating this syndrome, and typically complete seizure control is not achieved. As the disease progresses, most affected children also suffer from co-morbidities including developmental delays, intellectual disabilities, impaired motor control and coordination, autistic behaviors, and sleep disorders, and many die early.

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

[0216] Mouse models created by knocking out Scn1a reproduce some of the important phenotypic features of this epilepsy, including infantile (P21)-onset epilepsy, high sensitivity to febrile seizures, ataxia, spontaneous seizures, sleep disorders, autistic behaviors, and early death. Seizures and some co-morbidities result from impaired interneuron function in these mice.

[0217] This mouse model was used to investigate the efficacy of a new viral vector against DS. The virus was delivered by retro-orbital injection after using an insulin syringe, and its ability to suppress seizures was evaluated using a heat seizure test. In this test, a temperature regulator and a heat lamp were used to slowly increase the body temperature at the center of the mouse until a seizure occurred or until 42.5 °C was reached. The body temperature at which seizures developed in treated mice and control mice was compared to determine the efficacy of the intervention. In additional tests, the efficacy of the treatment against spontaneous seizures and early mortality was evaluated using video and electroencephalogram monitoring.

[0218] The viral vector is a new AAV viral vector named CN1500. This viral vector is a recombinant AAV that expresses the transgene SYFP2-P2A-NavSheP-D60N to rescue the deficiency of the voltage-gated sodium channel Nav1.1. NavSheP-D60N is a modified voltage-gated sodium channel derived from bacteria that has been modified to improve its kinetics and expression in mammalian cells. The expression level of the transgene is increased by the addition of the WPRE3 element, and transcription terminates at the bovine growth hormone polyadenylation sequence. The expression of the transgene is high and is limited to inhibitory cells in forebrain structures including the cortex and hippocampus via a 3xhI56iCore synthetic enhancer (SEQ ID NO: 3) immediately 5' of the CMV minimal promoter. Furthermore, the therapeutic transgene NavSheP-D60N is labeled with an HA epitope tag to confirm the correct protein localization.

[0219] To test the efficacy of the therapeutic AAV viral vector, a CN1500 package using the PHP.eB serotype was used. On the 35th day after birth, Scn1a + / - A cohort of mice received 2x10 per animal 11vg was either injected or left standing without injection. AAV was introduced intravenously using the retro-orbital delivery route. Two weeks after virus administration, the susceptibility of animals in the treatment group and the control group to febrile seizures was evaluated. As shown previously, febrile seizures were measured by steadily increasing the body temperature of the mice under a heating lamp at 0.5 degrees Celsius every two minutes and measuring the internal body temperature of the mice with a rectal probe. The body temperature at which the mice had seizures was recorded.

[0220] The new therapeutic vector CN1500 was highly expressed in GABAergic cells in both the mouse cortex and hippocampus, but Scn1a that experienced febrile seizures + / - The average body temperature of the mice also increased from 38.7 °C to 41 °C. These data indicate that CN1500 can substantially rescue the Scn1a deficiency.

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

[0222] (ix) Closing paragraphs. The nucleic acid sequences described herein are shown using standard letter abbreviations for nucleotide bases defined in 37 C.F.R. § 1.822 of the United States Patent Laws. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in the embodiments where appropriate.

[0223] Also included are variants of the sequences disclosed and referred to herein. Guidance on determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs such as DNASTAR™ (Madison, Wisconsin) software, which is well known in the art. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of amino acids that are similarly charged or uncharged. Conservative amino acid changes include substitutions of one amino acid in one family of amino acids that are related in their side chains.

[0224] In peptides or proteins, suitable conservative amino acid substitutions are known to those of skill in the art and generally can be made without altering the biological activity of the resulting molecule. Those of skill in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into the following conservative substitution families: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, nonpolar residues): isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), and cysteine (Cys); Group 7 (uncharged polar): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (nonpolar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Further information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0225] When making such changes, the hydropathic index of the amino acids can be considered. The importance of the hydrophobic amino acid index in conferring an interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has an assigned hydropathic index based on its hydrophobic and charge characteristics (Kyte and Doolittle, 1982). These values are as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamate (-3.5); Gln (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0226] It is known in the art that a particular amino acid can be replaced by other amino acids having similar hydropathic indices and scores, resulting in a protein still having similar biological activity, i.e., still obtaining a biologically functionally equivalent protein. When making such changes, substitutions of amino acids with a hydropathic index of ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitutions of similar amino acids can be effectively made based on hydrophilicity.

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

[0228] As outlined above, amino acid substitutions can be based on the relative similarity of amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc.

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

[0230] Variants of the proteins, nucleic acids, and gene sequences disclosed herein also include sequences having 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 with the proteins, nucleic acids, and gene sequences disclosed herein.

[0231] "Percent sequence identity" refers to the relatedness between two or more sequences, which is determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness determined by the match between strands of such sequences, between proteins, nucleic acids, or gene sequences. "Identity" (often also referred to as "similarity") can be readily calculated by known methods including those described below: 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 for determining identity are designed to give the best match between the sequences to be tested. Methods for determining identity and similarity are embodied in publicly available computer programs. Calculation of sequence alignments and percent identity can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignments of sequences can also be performed using the Clustal alignment method (using the default parameters (gap penalty = 10, gap length penalty = 10) of Higgins and Sharp CABIOS, 5, 151-153 (1989)).Suitable programs also include the following: 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 scope of the context of the present disclosure, when sequence analysis software is used, it will be understood that the results of the analysis are based on the "default values" of the referenced programs. As used herein, "default values" means any set of values or parameters that are initially loaded together with the software when the software is first initialized.

[0232] Variants also include nucleic acids that hybridize to the sequences disclosed herein under stringent hybridization conditions and provide the same function as the reference sequences. Exemplary stringent hybridization conditions include the following: incubation overnight at 42 °C in a solution containing 50% formamide, 5X SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared salmon sperm DNA, followed by washing the filter in 0.1X SSC at 50 °C. Alterations in hybridization stringency and signal detection are achieved initially through manipulation of the formamide concentration (lower percentages of formamide result in lower stringency); salt conditions, or temperature. For example, moderately high stringency conditions include incubation overnight at 37 °C in a solution containing 6X SSPE (20X SSPE = 3 M NaCl; 0.2 M NaH2PO4; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm blocking DNA; followed by washing at 50 °C with 1X SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes at higher salt concentrations (e.g., 5X SSC) may be performed after stringent hybridization. Variations of the above conditions may be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Inclusion of specific blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

[0233] As will be understood by those skilled in the art, each embodiment disclosed herein may include, consist essentially of, or consist of the specific recited elements, steps, components, or ingredients. Accordingly, the terms "include" or "including" should be interpreted to enumerate the following: "comprise, consist of, or consist essentially of". The transition term "comprise" or "comprises" means, but is not limited to, including and enables the inclusion of elements, steps, components, or ingredients not specifically recited, even in large amounts. The transitional phrase "consisting of" excludes all elements, steps, components, or ingredients not specifically recited. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specific recited elements, steps, components, or ingredients and those that do not materially affect the embodiment. The substantial effect would be to cause a statistically significant decrease in selective expression in the targeted cell population determined by scRNA-Seq and the following enhancer / targeted cell population pairing: the concatenated core of the I56i enhancer (e.g., SEQ ID NO: 3) / the targeted cell population mediated by GABAergic neurons.

[0234] Unless otherwise indicated, all numerical values representing amounts of ingredients, properties such as molecular weights, reaction conditions, etc. used in this specification and the 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 this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents of the claims, all numerical parameters should be construed in light of the reported number of significant digits and by applying ordinary rounding techniques. When the term "about" is used in combination with a recited numerical or range, it has the meaning reasonably attributed to such term by one of ordinary skill in the art, i.e., it indicates somewhat more or somewhat less than the recited numerical or range, within a range of ±20%; ±19%; ±18%; ±17%; ±16%; ±15%; ±14%; ±13%; ±12%; ±11%; ±10%; ±9%; ±8%; ±7%; ±6%; ±5%; ±4%; ±3%; ±2%; or ±1% of the recited numerical value.

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

[0236] Unless the context clearly dictates otherwise or is inconsistent with the meaning, the terms "a", "an", "the", and similar designations used in the context of this specification (especially in the context of the following claims) are to be construed to cover both the singular and the plural forms. The recitation of a range 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 herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless the context clearly dictates otherwise or is inconsistent with the meaning. The use of any and all examples, or exemplary language (e.g., "such as") presented herein is intended merely to better illuminate the invention and is not to be construed as limiting the scope of the invention as claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0237] The classification of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Members of each group may be referred to and claimed individually or in any combination with other members of that group or other elements found herein. It is expected that one or more members of a group may be included in or removed from the group for convenience and / or for patentability purposes. If any such inclusion or exclusion occurs, this specification is considered to include the group as modified so as to satisfy all recitations of all Markush groups used in the appended claims.

[0238] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the present invention. Of course, modifications of these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately use such modifications, and the inventors intend for the present invention to be practiced in ways other than those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Further, unless otherwise indicated herein or clearly contradicted by context, any combination of the above elements in all possible variations is included by the present invention.

[0239] Furthermore, numerous references are made throughout this specification to patents, publications, journal articles, and other documents (references herein). Each of the references is hereby incorporated by reference in its entirety into this specification for the teaching it discloses.

[0240] Finally, the embodiments of the invention disclosed herein are understood to illustrate the principles of the invention. Other modifications that may be used are within the scope of the invention. Accordingly, by way of example and not limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to exactly what is shown and described.

[0241] The details shown herein are by way of example and are for purposes of illustrative discussion of the preferred embodiments of the invention only and are presented in order to provide what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the various embodiments of the invention. In this regard, there is no intention to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description, together with the drawings and / or examples, is to make apparent to those skilled in the art how some forms of the invention may be actually implemented.

[0242] The definitions and explanations used in this disclosure are intended to govern any future construction and mean, unless clearly and expressly modified in the following examples or where the application of the meaning gives rise to any structural or essential contradiction, that, where the construction of a term is meaningless or essentially meaningless, the definition should be taken from Webster’s Dictionary, 3rd Edition, or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. (i)A concatemer that is 3, 4, 5, or 6 copies of SEQ ID NO: 2 or 3, 4, 5, or 6 copies of SEQ ID NO: 6; (ii)A promoter; and (iii)An artificial expression construct comprising a coding sequence.

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

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

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

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

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

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

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

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

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

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

12. A composition for use in a method for selectively expressing a coding sequence within a population of nerve cells in vivo or in vitro, the method comprising providing an administrable composition comprising an artificial expression construct to a sample or subject containing the population of nerve cells in a sufficient dosage and for a sufficient time, wherein the artificial expression construct (i) a concatamer that is 3, 4, 5, or 6 copies of SEQ ID NO: 2 or 3, or 4, 5, or 6 copies of SEQ ID NO: 6, (ii) a promoter, and (iii) a coding sequence and that thereby comprises the step of selectively expressing the coding sequence within the population of nerve cells. A composition.

13. The composition according to claim 12, wherein the coding sequence encodes a fluorescent protein or an ion transporter selected from a potassium channel, a calcium channel, or a voltage-gated sodium channel.

14. The composition according to claim 12, wherein the providing comprises pipetting a brain slice containing GABAergic interneurons.

15. The composition according to claim 14, wherein the brain slice contains LAMP5 neurons, VIP neurons, Sst neurons, or Pvalb neurons.

16. The composition according to claim 12, wherein the providing comprises administering to a living subject.

17. The composition according to claim 16, wherein the living subject is a human, a non-human primate, or a mouse.

18. The composition according to claim 16, wherein administering to a living subject is by injection.

19. The composition according to claim 18, wherein the injection comprises intravenous injection, intracerebral parenchymal injection, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection.

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