Targeting damage responsive glial cells with gene delivery vectors

Abstract

The invention relates to isolated nucleic acid molecules and artificial expression constructs comprising selective injury-responsive enhancer elements that activate gene expression in specific cell types following injury. The invention enables the selective expression of heterologous genes in injury-responsive cells and can be used for the treatment or prevention of central nervous system diseases or disorders. The method involves delivering the artificial expression construct to injury-responsive cells, ensuring precise, cell-specific gene activation.

Description

[0001] P7423PC00

[0002] Targeting damage responsive glial cells with gene delivery vectors

[0003] Technical field

[0004] The present disclosure relates to the fields of molecular biology, gene therapy, and central nervous system (CNS) therapeutics. Specifically, it concerns enhancer elements responsive to cellular injury and their use in selectively modulating gene expression in injured cells, particularly within the context of CNS disorders such as spinal cord injury, brain injury, stroke, neuroinflammation, and neurodegeneration.

[0005] Background

[0006] Injuries to the central nervous system (CNS), such as spinal cord injuries, brain injuries, and stroke, represent a significant medical challenge due to the complexity of cellular and molecular responses involved in tissue damage and repair (Silver eta!., 2015). Current therapeutic approaches often aim to promote regeneration or minimise secondary injury but face limitations due to unspecific targeting of multiple cell types, a lack of spatiotemporal control and safety concerns due to the dosage of therapeutic cargo (Van Alstyne eta / ., 2021). A central aspect of CNS injury is the activation of distinct gene regulatory networks that are unique to injured cells, such as astrocytes, which play critical roles in both the progression and resolution of injury (Liddelow et al., 2024). Despite advances in gene therapy and molecular biology which have enabled the targeting of specific cell types (Mich eta!., 2024), the ability to selectively target specific injury-responsive cell populations and modulate their responses remains inadequate.

[0007] Existing gene therapy strategies predominantly rely on general promoters or broadly active enhancers to drive gene expression (High eta / ., 2019, Kuzmin etal., 2021), often lacking the specificity needed to prevent unspecific targeting. This lack of specificity can lead to off-target effects, including unintended gene expression in healthy cells, which may compromise therapeutic efficacy and safety (Van Alstyne et al., 2021 ; Hordeaux etal., 2020). Furthermore, most available gene delivery systems fail to achieve spatiotemporal control, limiting their effectiveness in addressing dynamic injury processes that unfold in specific tissues over defined timeframes. Consequently, there is a pressing need for approaches that can achieve precise, specific targeting of injured cells without affecting the surrounding healthy tissue.

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[0009] Enhancer elements, which are noncoding DNA sequences regulating gene expression, have been increasingly recognised for their ability to confer specificity in cellular and tissue contexts (Long etal., 2016). However, current research on enhancers in the CNS has primarily focused on general or cell-type-specific enhancers without leveraging the unique molecular signatures of injured cells (Mich etal., 2021).

[0010] Summary

[0011] This above-mentioned research gap has hindered the development of highly targeted therapeutic interventions that harness the inherent specificity of injury-induced regulatory elements. It is therefore an objective of the present disclosure to provide an improved means for selectively targeting injury-responsive cells, such as astrocytes, within the CNS. By focusing on the unique molecular responses of these cells to injury, the present disclosure seeks to enable spatiotemporal control of therapeutic gene expression in a manner that is both cell-specific and injury-specific, thereby addressing the limitations of existing therapeutic strategies.

[0012] Accordingly, the present invention provides for isolated nucleic acid molecules and artificial expression constructs comprising selective injury-responsive enhancer elements that are activated in specific cell types which are responsive to injury, as well as uses thereof for the treatment and / or prevention of diseases or disorders related to the central nervous system, and methods for selectively expressing a heterologous gene within a population of selected injury-responsive cells.

[0013] In one aspect, the present invention provides for an isolated nucleic acid construct or an artificial expression construct comprising:

[0014] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0015] b. an encoding sequence operably linked to the enhancer element, wherein the selective injury-responsive enhancer element is configured to drive expression of the encoding sequence in a selected cell type following an injury, and comprises:

[0016] i. at least one injury-responsive motif comprising a consensus sequence recognised by at least one injury-responsive transcription factor activated by injury-induced signalling pathways, which mediates transcriptional activation in response to cellular or tissue stress and / or damage; and

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[0018] ii. at least one lineage-specific motif comprising a consensus sequence recognised by at least one lineage-specific transcription factor that regulates cell-type identity, which restricts expression to a defined lineage, wherein the selective injury-responsive enhancer element provides combinatorial specificity through cooperative activity of said at least one injury-responsive motif and said at least one lineage-specific motif.

[0019] In one aspect, the present invention provides for an isolated nucleic acid molecule comprising:

[0020] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0021] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0022] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0023] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0024] In another aspect, the present invention provides for an artificial expression construct, comprising:

[0025] a. a selective injury-responsive enhancer element that is activated in at least one selected injured cell type; and

[0026] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0027] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

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[0029] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0030] In another aspect, the present invention provides for isolated nucleic acid molecule or artificial expression construct comprising:

[0031] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0032] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0033] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0034] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and

[0035] wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31 -53 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0036] In another aspect, the present invention provides for an isolated nucleic acid molecule or artificial expression construct comprising:

[0037] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0038] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0039] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

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[0041] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and

[0042] wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31 -35 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0043] In another aspect, the present invention provides for use of the isolated nucleic acid molecule, artificial expression construct or pharmaceutical composition of the invention for the treatment and / or prevention of diseases or disorders related to the central nervous system.

[0044] In another aspect, the present invention provides for a method for selectively expressing a heterologous gene within a population of selected injury-responsive cells, comprising contacting said cells with the artificial expression construct or pharmaceutical composition according to the disclosed invention, wherein the heterologous gene is expressed selectively in the injury-responsive cells.

[0045] Description of Figures

[0046] Figure 1. Unbiased single cell transcriptomics and chromatin accessibility profiling in the injured mouse spinal cord reveals cell type specific injury responsive enhancers. A. Schematic workflow of the multiomic experiment. B. UMAP representation of individual cells grouped in cell types according to their molecular similarity (both gene expression and chromatin accessibility). Major cell types are grouped within the dashed lines. C. Heatmap reporting differentially accessible peaks across timepoints for each of the glial cell populations. Shaded bars represent cell type (above) and timepoint (below). Peaks are ordered according to (1) cell type enrichment and (2) activation dynamics and are grouped into modules depending on whether they are expressed in uninjured samples, or during acute, or chronic phases after injury. Astro.: astrocytes; Epend.: ependymal; OPCs: oligodendrocyte progenitors; OLs: oligodendrocytes. D. Feature plots displaying in graysacle the combined chromatin accessibility scores of injury-responsive enhancers (IRENs) for each glial cell type indicating injury- induced gains in accessibility. The cells clusters enriched in cells from injured or uninjured samples are highlighted within the dashed lines. In brackets, peak set size. DPI denotes ‘days post-injury’.

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[0048] Figure 2. Deep learning models predict cell type specific accessibility based on DNA sequence. A, Schematic overview of the architecture of our deep learning models used to predict chromatin accessibility across cell types. B, Coverage plots displaying observed (shaded area) and predicted (black line) accessibility for cell type-enriched genomic regions. Each peak is predicted using a different cell type-specific model (rows).

[0049] Figure 3. Deep learning models identify transcription factor binding site motifs for cell type specific transcription factors. A, Number of peaks used for training the cell type specific models. Bars are shaded based on whether regions carry motifs identified by the models. B, Number of cell type-specific regions carrying one or more motifs detected by the trained models. Cell types ordered according to the legend at the bottom.

[0050] C, Number of unique motifs identified by the trained models in each set of cell typespecific peaks. D, Total number of motifs identified using the traditional motif enrichment analysis compared to our trained models. Bars are split based on whether motifs match expression of the transcription factor in the correct cell type. E, Heatmap displaying the average scaled gene expression levels of transcription factors for which our trained models found enriched motifs in the cell type specific peaks. Columns order by cell type according to the numbers above the heatmap. Cell type order in panels A, B and C are presented.

[0051] Figure 4. AP-1 transcription factors govern injury-induced gene expression changes in glial cells. A, Average gene expression level at each timepoint after spinal cord injury for the genes belonging to the AP-1 transcription factor family in glial cells. Only genes changing in expression level with injury are displayed. B, Violin plots showing the temporal dynamics in the combined chromVAR AP-1 motif activity across glial cells.

[0052] C, Sequence logos recognized by AP-1 transcription factors in damage responsive glial cells.

[0053] Figure 5. Injury induced enhancers harbor active motifs for AP-1 and glial lineage specific factors. A-E, Barplot reporting the most common transcription factors and number of their motif instances recorded by the deep learning models in peaks specific to each cell type and injury condition. AP-1 transcription factors are highlighted in each barplot (*). On the y-axis, multiple transcription factors matching the same motifs are

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[0055] separated by slash ( / ), while co-occurrence of distinct motifs recognized by different genes is reported and genes are separated by comma (,).

[0056] Figure 6. Deep learning identifies motifs for glial lineage specific factors and predicts their activity after injury. A, Barplot reporting the most common transcription factors and number of their motif instances recorded by the deep learning models in peaks specific to each cell type that co-occur with AP-1 sites in injury responsive enhancers. On the y-axis, multiple transcription factors matching the same motifs are separated by slash ( / ), while co-occurrence of distinct motifs recognized by different genes is reported and genes are separated by comma (,). B, Nucleotide contribution scores attributed to an astrocyte-specific, injury-responsive region (SEQ ID NO: 109). Each prediction is performed by a model trained on different sets of cells (astrocytes from uninjured and injured samples -reactive-, as well as microglia). Motifs predicted to be active and found de novo from the contribution scores are highlighted. C, Observed (colored area) and predicted (black line) accessibility of the region in B.

[0057] Figure 7. In vivo screening of injury responsive enhancers containing AP-1 and glial lineage-specific motifs using systemic gene delivery vectors. A, Coverage plots of the regulatory regions driving transgene expression in our AAV-based reporter assay. Tracks are colored by cell type and split by injury condition. B, Overview of a spinal cord section collected from a mouse injected with the AAV library and subjected to an injury. C, Overview of an uninjured spinal cord section collected from a mouse injected with the AAV library (not injured). Scale bar: 250 y.m. D, Magnification of the boxed area from B displaying Sox9+ astrocytes labelled with EYFP. Scale bar: 50 y.m.

[0058] E, UMAP visualization of the scRNA-seq dataset obtained from our reporter assay. UMAP coordinates are calculated based on the gene expression profile and cells are colored according to sample of origin (i.e., uninjured or injured spinal cord). F, Feature plots displaying expression levels of Aldh1l1 (i.e., an astrocyte-specific gene). Astrocytes are highlighted within the dashed line. G, Barplot displaying the number of barcodes delivered and detected (light grey) in the scRNA-seq dataset corresponding to the injury-responsive and the cell type-specific enhancers, respectively. H, Barplot reporting the frequency of detection for each unique barcode in each cell from the injured spinal cord.

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[0060] Figure 8. Additional characterization of the in vivo validated injury responsive enhancers containing AP-1 and glial lineage-specific motifs for cell state targeting. A, Schematic workflow of the AAV-based reporter assay. B, Observed (shaded area) and predicted (black overlay line) accessibility of the enhancer regions selected for our screen. Each profile is predicted by different cell type- and state-specific models (rows).

[0061] C, Nucleotide contribution scores for a 160 bp-long region within the IREN5 enhancer (SEQ ID NO: 110). The region is interpreted by models trained on astrocytes from healthy and injured cords. Motifs with higher contribution scores are highlighted (rectangular shaded grey areas) and the transcription factor binding to such sequences are indicated. The coverage plot displays the observed accessibility of IREN5, along with the region displayed in the contribution plot. D, UMAP visualization of the astrocytes profiled with the AAV-based reporter assay and colored by injury condition. E, Feature plot displaying expression levels of Gfap. F, Feature plots reporting the read counts for each of the IRENs detected in the dataset, along with eGT_380h, an astrocyte-specific human enhancer from Mich et al. (2023).

[0062] Figure 9. Injury responsive enhancers direct expression in damage responsive glial cells after brain and spinal cord injury upon systemic delivery. A, Schematic overview of the experimental design for the in vivo validation of IREN5-driven reporter expression in the spinal cord. B-D, Overviews of spinal cord sections collected from a mouse that had been injected with the IREN5 enhancer virus and injured. The regions correspond to areas that are distal (g, ca 5 mm rostral to the epicenter), proximal (h) to the injury site, and at the epicenter. Scale bars: 250 y.m. E, Magnification of the boxed areas from A and B displaying EYFP-labelled cells. Arrowheads point to a few examples of Gfap+ / Sox9+EYFP-labelled astrocytes. F, Quantification of EYFP+ astrocytes in the spinal cord across the tissue segment spanning the injury. A quadratic function is fit to highlight the spatial distribution. G, Barplot displaying the average proportion of EYFP+ cells identified as astrocytes across the sections of spinal cord. The error bar represents the standard deviation. H, Schematic overview of the experimental design for the in vivo validation of IREN5-driven reporter expression in the brain. The enhancer virus was intravenously (IV) injected in animals that were subsequently subjected to a traumatic brain injury (TBI). I, Overview of cortex from a mouse subjected to a stab-wound injury following enhancer virus injection. The dotted line emphasizes the lesion site. Scale bar: 600 y.m. J, Magnification of the boxed area from g displaying EYFP+ astrocytes. Arrowheads point to a few examples of tdTomato+ / Sox9+EYFP-labelled astrocytes.

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[0064] Scale bar: 150 y.m. K, Barplots reporting the number of EYFP+ astrocytes in the injured spinal cord compared to the contralateral intact side. L, Barplots displaying the proportion of EYFP+ cells identified as astrocytes. M, Schematic representation of an IREN AAV vector. The core region encompassing binding sites for AP1 and lineage specific transcription factors is highlighted. The core region is encoded 1x or 3x in the construct.

[0065] N, Deep learning model predictions of accessibility of IRENs carrying the wild type sequence (IxCORE) or the version with higher activity (3xCORE; SEQ ID NO: 106). O, Overview of cortex from a mouse subjected to a stab wound injury following injection of the wild type (1 x) or boosted enhancer virus (3xCORE; SEQ ID NO: 106) indicating the control of enhancer activity while retaining specificity.

[0066] Figure 10. Injury responsive enhancers, but not cell type specific elements, selectively label damage responsive cells after spinal cord injury. A, Overview of sections spanning the region around the injury site in the spinal cord sampled from a mouse injected with the injury-responsive IREN5 enhancer AAV. B, Plot representing the changes in astrocyte-specificity for the IREN5 enhancer across the spinal cord segment encompassing the injury. Dotted line represents the epicenter of the lesion. C, Overview of sections spanning the region around the injury site in the spinal cord sampled from a mouse injected with the cell type-specific human enhancer AAV from Mich et al. (2023). D, Dotplot displaying the number of EYFP+ astrocytes across the spinal cord segment from c. The number of labelled cells is normalized against the total number of Sox9-expressing cells on each tissue section.

[0067] Figure 11. Sequence determinants of IREN activity and specificity A, Schematic representation of synthetic enhancer validation. IREN5 DNA sequence was mutated in silico to ablate binding sites for AP-1 or astrocyte-specific transcription factors and resulting activity was predicted. Enhancer viruses carrying the synthetic sequences were validated in vivo. B, Bar plots displaying the labeling efficiency of the synthetic sequences. C, Bar plot reporting the labeling specificity of the synthetic sequences. D, Nucleotide contribution scores within a region of the wildtype IREN5 (SEQ ID NO: 111), as interpreted by the reactive astrocyte model. Motifs with higher contribution scores and recognized by astrocyte-specific and AP-1 transcription factors are highlighted in gray.

[0068] E, Mutated IREN5 sequences for which AP-1 (top; SEQ ID NO: 112) or astrocyte-specific sites (middle; SEQ IN NO: 113) were ablated. The bottom plot represents a synthetic sequence (SEQ ID NO: 114) for which all nucleotides had been shuffled, with the

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[0070] exception of the recognized transcription factor binding sites, which remained constant.

[0071] F, Overview of spinal cords from mice subjected to injury following systemic injection of the enhancer viruses.

[0072] Figure 12. Conservation of IREN5 activity in human and across neurological disease. A, Accessibility of the human IREN5 region in human samples of ALS and Alzheimer's disease. B, Top 10 most represented motifs recognized by a human reactive astrocyte model trained on Alzheimer's disease data. The seqlets were identified in a sample of 30,000 regions found to be accessible in human reactive astrocytes from patient samples. C, Nucleotide contribution scores assigned by the human reactive astrocyte model to a region of the human IREN5 sequence (SEQ ID NO:115).

[0073] Detailed description

[0074] Definitions

[0075] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0076] As used in the herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. The phrase “consisting of excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterised by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

[0077] The term “some embodiments” can include one, or more than one embodiment.

[0078] As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of

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[0080] values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0081] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In an aspect, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value unless otherwise stated or otherwise evident from the context.

[0082] As used herein, the term “in vitro” refers to events or experiments that occur in an artificial environment, e.g., in a petri dish, test tube, cell culture, etc., rather than within a multicellular organism. As used herein, the term “in vivo” refers to events or experiments that occur within a multicellular organism.

[0083] The words “treat” or “treating” or “treatment” refer to therapeutic or medical treatment wherein the object is to slow down (lessen), ameliorate, and / or diminish an undesired physiological change, disease, disorder, injury, pathological condition, or disorder in a subject. As used herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment may not necessarily result in the complete clearance of but may reduce or minimize complications, the side effects, and / or the progression of a disease, a disorder, an injury, an infection, a symptom, and / or a complication. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, injury, insult, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or

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[0085] (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease.

[0086] As used herein, “promoter” or “promoters” or “minimal promoter” or “minimal promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. A "minimal promoter" refers to the core promoter region containing the minimal sequence necessary for transcription initiation, such as the TATA box or initiator sequence, but lacking additional regulatory elements or enhancers required for high or tissue-specific transcription.

[0087] As used herein, the term “motif” refers to a short, specific nucleotide sequence within a regulatory DNA element that constitutes a binding site for one or more transcription factors. A motif is characterised by its sequence pattern, which determines the recognition and binding specificity of the corresponding transcription factor(s). Motifs may occur singly or in combination within an enhancer, and their relative arrangement (syntax) influences cooperative or synergistic transcriptional activation. As used herein, the term “injury-responsive motif” encompasses nucleotide sequences that are activated by transcription factors induced by injury signalling pathways. Within this genus, AP-1 motifs represent a specific species, characterised by their consensus sequence and ability to bind AP-1 family transcription factors.

[0088] As used herein, the term "encoding sequence" refers to a DNA sequence that contains the necessary genetic information to direct the production of a specific DNA sequence product, such as a polypeptide, RNA molecule, or other functional element. The encoding sequence is operably linked to regulatory elements, such as a selective injury-responsive enhancer, that regulate its transcription and subsequent expression in a targeted manner. The encoding sequence ensures that the desired DNA sequence product is expressed in a selected cell type under specific conditions, such as after activation of the enhancer by transcription factors in specific cell types (e.g., NFIA, SOX9, JUN, FOS) or in response to injury (e.g., JUN, JUNB, JUND, FOS). This term

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[0090] encompasses naturally occurring, modified, or synthetic DNA sequences designed for therapeutic, diagnostic, or research applications.

[0091] As used herein, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0092] As used herein, an “enhancer” such as a transcription or transcriptional enhancer refers to regulatory DNA segment that is typically found in multicellular eukaryotes. An enhancer can strongly stimulate (“enhance”) the transcription of a linked transcription unit, i.e., it acts in cis. An enhancer can activate transcription over very long distances of many thousand base pairs, and from a position upstream or downstream of the site of transcription initiation. An enhancers can have a modular structure by being composed of multiple binding sites for transcriptional activator proteins. Many enhancers control gene expression in a cell type-specific fashion. Several remote enhancers can control the expression of a singular gene while a singular enhance can stimulate the transcription of one or more genes.

[0093] As used herein, the term “injury-responsive enhancer” or refers to a regulatory DNA segment that specifically activates and / or stimulates transcription in response to cellular or tissue injury, relative to an uninjured state. Similar to traditional enhancers, injury-responsive enhancers act in cis to regulate the transcription of a linked transcription unit, activating transcription over long distances, either upstream or downstream of the transcription initiation site. These enhancers are characterized by their modular structure, comprising multiple motifs, each representing one or more binding sites for transcriptional activator proteins, including both stimulus-induced transcription factors (e.g., AP-1 family), the stimulus being for example injury, and one or more binding sites for lineage-specific transcription factors. Injury-responsive enhancers regulate gene expression in a cell type-specific manner in an injurydependent manner, including but not limited to astrocytes, microglia, oligodendrocytes, or other central nervous system cell types. They are associated with injury- induced

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[0095] transcriptional programs and can direct the expression of one or more genes, often in a spatiotemporally and / or temporally restricted manner. Multiple injury-responsive enhancers may regulate a single gene, and a singular injury-responsive enhancer may influence the transcription of multiple genes.

[0096] As used herein, the term “injury-responsive cells” refers to cells that undergo specific transcriptional, phenotypic, or functional changes in response to cellular or tissue injury. These cells activate distinct injury- induced regulatory programs, including the expression of genes controlled by injury-responsive enhancers. Injury-responsive cells are characterized by their ability to engage in processes such as tissue repair, inflammation, or regeneration, and often display cell type-specific activation of injury-induced pathways. Examples of injury-responsive cells include, but are not limited to, astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells (OPCs), ependymal cells, choroid plexus cells, and border-associated macrophages within the central nervous system. The activation of these cells may be mediated by stimulus-induced transcription factors (e.g., AP-1 family) and lineage-specific factors, which regulate injury-responsive enhancer activity to modulate gene expression in a spatiotemporally restricted and cell type-specific manner.

[0097] As used herein, the term “injury-induced signalling pathways” refers to intracellular molecular cascades that are activated in response to cellular or tissue injury and which mediate transcriptional, phenotypic, or functional changes in affected cells. These pathways typically involve stimulus-responsive kinases and transcription factors that regulate gene expression programmes associated with stress, inflammation, repair, or regeneration. Examples of injury-induced signalling pathways include, but are not limited to, the MAPK / JNK pathway, p38 MAPK pathway, and NF-KB pathway, which activate transcription factors such as AP-1 family members (e.g., JUN, FOS), ATF proteins, and other stress-responsive regulators. Activation of these pathways results in the recruitment of transcription factors to injury-responsive motifs within enhancer elements, thereby initiating selective transcriptional programmes in injury-responsive cells.

[0098] As used herein, the term “injury-responsive transcription factor” refers to a transcription factor that is activated by injury-induced signalling pathways and mediates transcriptional activation of genes involved in stress response, repair, or regeneration.

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[0100] Examples include AP-1 family members such as JUN, JUNB, JUND, FOS, FOSB, FOSL1 , and FOSL2, as well as ATF proteins and related factors.

[0101] As used herein, the term “cellular or tissue stress and / or damage” refers to a physiological state in which cells or tissues experience adverse conditions or insults that disrupt homeostasis and trigger stress-response or injury-response pathways. Such stress or damage may result from mechanical trauma, chemical exposure, oxidative stress, hypoxia, inflammation, infection, or other pathological stimuli.

[0102] This state is characterised by activation of intracellular signalling cascades, including injury- induced pathways such as MAPK / JNK, p38 MAPK, and NF-KB, which lead to transcriptional reprogramming and recruitment of stimulus-responsive transcription factors (e.g., AP-1 family members) to injury-responsive enhancer elements. Cellular or tissue stress and / or damage can occur in various contexts, including central nervous system injury, neuroinflammation, or degenerative conditions, and serves as a trigger for selective enhancer activation described herein.

[0103] As used herein, the term “cooperative syntax” refers to a specific spatial and functional arrangement of two or more transcription factor binding motifs within an enhancer element, wherein the relative positioning, orientation, and spacing of said motifs enables cooperative interaction between the corresponding transcription factors. This arrangement results in synergistic transcriptional activation in target cells, such that the combined activity of the motifs exceeds the additive effect of each motif acting independently. In the present disclosure, the cooperative syntax typically comprises at least one injury-responsive motif and at least one lineage-specific motif configured to promote context-dependent gene expression.

[0104] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms.

[0105] Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more in the range of

[0106] 15-01-2609:01:04P7423PC00

[0107] overlap. Such sequences are also referred to as “variants” herein, e.g., other variants of glycogen branching enzymes and amylases. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and / or 5’-side are 100% identical.

[0108] As used herein, an “isolated” biological component (such as a nucleic acid molecule, protein, or virus) has been substantially separated or purified away from other biological components (e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and / or organelles). Nucleic acids, proteins, and / or viruses that have been “isolated” include nucleic acids, proteins, and viruses purified by standard purification methods. The term also embraces nucleic acids, proteins, and viruses prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” (or purified) does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated or purified nucleic acid, protein, virus, or other active compound is one that is isolated in whole or in part from associated nucleic acids, proteins, and other contaminants. In an aspect, the term “substantially purified” refers to a nucleic acid, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.

[0109] As used herein, the term "motif" refers to a specific, short DNA sequence that functions as a binding site for transcription factors (TFs). Motifs are critical elements within enhancers, enabling the recruitment of transcription factors that regulate the transcription of target genes. These sequences play a key role in gene expression regulation by facilitating the interaction between enhancers and the transcriptional machinery. Motifs can exhibit sequence specificity and are often associated with particular transcription factors that recognize and bind to them.

[0110] As used herein, the term "lineage-specific motif" refers to a DNA sequence within an enhancer that binds transcription factors associated with the identity or function of a specific cell lineage. Lineage-specific motifs enable selective gene regulation in particular cell types by recruiting transcription factors that are predominantly expressed or active in those lineages. For example, lineage-specific motifs may correspond to

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[0112] transcription factor binding sites characterized by position frequency matrices (PFMs) set forth in Table 1 or sequences as set forth in SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88. These motifs can occur as single or multiple instances within an enhancer, contributing to the enhancer's ability to drive gene expression in a cell type-specific manner.

[0113] As used herein, the term “cell-type identity” refers to the distinctive molecular and functional characteristics that define a specific cell type within a tissue or organism. These characteristics include the expression of lineage-specific transcription factors, regulatory elements, and gene expression programmes that maintain the specialised roles of the cell. Cell-type identity is established during development and maintained throughout the life of the cell by transcriptional networks involving lineage-specific factors such as NFIA, SOX9, and others. These factors bind to lineage-specific motifs within enhancer elements to regulate genes essential for the structural and functional properties of the cell. In the context of this disclosure, cell-type identity ensures that selective injury-responsive enhancer elements activate transcription only in the intended cell lineage (e.g., astrocytes, oligodendrocytes) following injury, thereby conferring specificity to therapeutic or experimental constructs.

[0114] As used herein, the term “lineage-specific transcription factor” refers to a transcription factor that regulates the identity, differentiation, and maintenance of a particular cell lineage. These transcription factors bind to lineage-specific motifs within enhancer elements to control gene expression programmes that define the structural and functional characteristics of the cell type. Examples of lineage-specific transcription factors include NFIA, NFIB, NFIC, NFIX, SOX2, SOX4, SOX9, SOX10, RFX family members, and others that are predominantly expressed or active in specific neural cell types such as astrocytes, oligodendrocytes, or progenitor cells. In the context of this disclosure, lineage-specific transcription factors cooperate with injury-responsive transcription factors at selective injury-responsive enhancer elements to achieve combinatorial specificity for gene expression following injury.

[0115] As used herein, the term "injury-specific motif" refers to a DNA sequence within an enhancer that binds transcription factors activated in response to cellular stress or injury. Injury-specific motifs enable the selective activation of gene expression in response to injury signals, such as those mediated by transcription factors in the AP1

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[0117] family (e.g., JUN, FOS). Such motifs are associated with transcription factor binding sites characterized by PFMs, as set forth in Table 2, or sequences as set forth in SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103. Injury-specific motifs may occur as single or multiple instances within an enhancer and are essential for the selective recruitment of transcription factors driving injury-induced gene expression in specific cell types, such as reactive astrocytes or other injury-responsive cells.

[0118] As used herein, the term "DNA sequence product" refers to a nucleic acid-derived molecule encoded by a DNA sequence that is expressed in a selected cell type. This product may include a polypeptide, RNA molecule, or other functional genetic element produced as a result of the activity of a linked regulatory sequence, such as a selective injury-responsive enhancer. A DNA sequence product is expressed in response to specific transcriptional regulation mediated by enhancer elements containing transcription factor binding sites which are activated under particular cellular conditions, such as injury (e.g., JUN, JUNB, JUND, FOS) and / or in specific cell types (e.g., NFIA, SOX9, JUN, FOS). This term encompasses both naturally occurring and synthetically designed sequences that direct the expression of functional products in a targeted manner.

[0119] As used herein, the term "functional homologue" refers to a nucleic acid or protein sequence that exhibits substantial sequence identity to a reference sequence and retains the same or similar biological function or activity. In the context of molecular biology, a functional homologue can include naturally occurring variants, orthologues, paralogues, or synthetic sequences that maintain the ability to perform the same molecular role, such as binding to specific transcription factors, regulating gene expression, or catalysing biochemical reactions. Functional homologues may share at least a specified percentage of sequence identity (e.g., 70%, 80%, 90%, or higher) with the reference sequence and demonstrate equivalent functionality under comparable biological conditions. Modifications, substitutions, deletions, or additions to the sequence are permissible, provided the homologue retains its essential biological activity. This definition encompasses homologues identified across species or engineered for research, therapeutic, or diagnostic applications.

[0120] As used herein, the term “consensus sequence” refers to a nucleotide sequence that represents the most common or preferred bases at each position within a set of

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[0122] transcription factor binding sites identified for a particular transcription factor or motif family. A consensus sequence is typically derived from multiple experimentally validated binding sites and reflects the sequence pattern recognised by the transcription factor. Variability at certain positions may be indicated using standard IUPAC ambiguity codes (e.g., N = any nucleotide, R = A or G, Y = C or T).

[0123] Consensus sequences are used to define motifs functionally rather than by exact nucleotide identity, thereby encompassing sequences that retain binding capability and functional activity even when minor variations occur outside critical positions. In the context of this disclosure, consensus sequences are associated with transcription factor binding sites characterised by position frequency matrices (PFMs) or sequences as set forth in the SEQ ID NO listings provided herein.

[0124] Selective injury-responsive enhancer elements

[0125] In one aspect, the present invention provides for an isolated nucleic acid molecule comprising:

[0126] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0127] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0128] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0129] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0130] Artificial expression constructs

[0131] In one aspect, the present invention provides for an artificial expression construct, comprising:

[0132] a. a selective injury-responsive enhancer element that is activated in at least one selected injured cell type; and

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[0134] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0135] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0136] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0137] In some embodiments, the artificial expression construct further comprises a minimal promoter. In some embodiments, the artificial expression construct further comprises a reporter transgene. In some embodiments, the reporter transgene comprises super yellow fluorescent protein (SYFP) or another fluorescent protein. In some embodiments, the artificial expression construct further comprises a unique barcode, wherein the unique barcode specifically identifies the selective injury-responsive enhancer element. In some embodiments, the unique barcode comprises a nucleic acid sequence as set forth in SEQ ID NO:1-3. In some embodiments, the artificial expression construct further comprises a poly-A tail. In some embodiments, the DNA sequence product is an encoded polypeptide.

[0138] Combinational specificity of specific cell types and stress / injury responses

[0139] In some embodiments, the selective injury-responsive enhancer element comprises at least one lineage-specific motif as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88. In some embodiments, the selective injury-responsive enhancer element comprises at least one lineage-specific motif, as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and wherein the enhancer element comprises multiple instances of the same at least one lineage-specific motif.

[0140] In some embodiments, the selective injury-responsive enhancer element comprises at least one transcription factor binding site characterised by at least one position frequency matrix (PFMs) as set forth in Table 1 (lineage motifs PFM), said binding sites

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[0142] being contained within the DNA sequence product and contribute to the binding specificity for the specific injured cell type.

[0143] In some embodiments, the selective injury-responsive enhancer element comprises at least one AP1 motif as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103. In some embodiments, the selective injury-responsive enhancer element comprises at least one AP1 motif as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103, and wherein the selective injury-responsive enhancer element comprises multiple instances of the same at least one AP1 motif.

[0144] In some embodiments, the selective injury-responsive enhancer element comprises at least one transcription factor binding site characterised by at least one position frequency matrix (PFMs) as set forth in Table 2 (AP1 PFM), said binding sites being contained within the DNA sequence product and contribute to the binding specificity for the specific injured cell type.

[0145] In some embodiments, the selective injury-responsive enhancer element comprises:

[0146] a. at least one lineage-specific motif, such as at least one lineage specific motif comprising a sequence as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and

[0147] b. at least one AP1 motif, such as at least one AP1 motif comprising a sequence as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103.

[0148] In some embodiments, the selective injury-responsive enhancer element comprises:

[0149] a. at least one transcription factor binding site characterised by a position frequency matrix (PFMs) as set forth in Table 1 (lineage motifs), and b. at least one transcription factor binding site characterised by a position frequency matrix (PFMs) as set forth in Table 2 (AP1 motifs),

[0150] said binding sites being contained within the DNA sequence product and contribute to the binding specificity for the specific injured cell type.

[0151] In some embodiments, the selective injury-responsive enhancer element is activated at the onset of injury, during injury pathogenesis and / or post injury. In some

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[0153] embodiments, the selective injury-responsive enhancer element is epigenetically activated. In some embodiments, the selective injury-responsive enhancer element is activated in response to and / or at the onset of and / or during and / or post CNS injury and / or diseases and / or disorders comprising neuroinflammation, including spinal cord injury, brain injury, or stroke and / or diseases and / or disorders comprising neurodegeneration.

[0154] Specific enhancers of the invention - combinations of motifs & specific IRENS

[0155] In some embodiments, the selective injury-responsive enhancer element comprises:

[0156] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0157] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0158] In some embodiments, the selective injury-responsive enhancer element comprises:

[0159] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0160] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0161] wherein the selective injury-responsive enhancer element comprises a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 42 and SEQ ID NO: 46 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0162] In some embodiments, the selective injury-responsive enhancer element comprises:

[0163] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0164] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0165] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 37 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0166] In some embodiments, the selective injury-responsive enhancer element comprises:

[0167] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

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[0169] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0170] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 42 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0171] In some embodiments, the selective injury-responsive enhancer element comprises:

[0172] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0173] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0174] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 46 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0175] In some embodiments, the selective injury-responsive enhancer element comprises:

[0176] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0177] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0178] In some embodiments, the selective injury-responsive enhancer element comprises:

[0179] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0180] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0181] wherein the selective injury-responsive enhancer element comprises a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 41 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0182] In some embodiments, the selective injury-responsive enhancer element comprises:

[0183] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0184] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0185] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 33 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0186] In some embodiments, the selective injury-responsive enhancer element comprises:

[0187] 15-01-2609:01:04P7423PC00

[0188] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0189] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0190] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 34 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0191] In some embodiments, the selective injury-responsive enhancer element comprises:

[0192] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0193] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0194] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 36 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0195] In some embodiments, the selective injury-responsive enhancer element comprises:

[0196] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0197] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0198] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 38 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0199] In some embodiments, the selective injury-responsive enhancer element comprises:

[0200] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0201] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0202] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 39 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0203] In some embodiments, the selective injury-responsive enhancer element comprises:

[0204] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and

[0205] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0206] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 41 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0207] In some embodiments, the selective injury-responsive enhancer element comprises:

[0208] 15-01-2609:01:04P7423PC00

[0209] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 17 and SEQ ID NO: 14, and

[0210] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0211] In some embodiments, the selective injury-responsive enhancer element comprises:

[0212] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 17 and SEQ ID NO: 14, and

[0213] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0214] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 45 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0215] In some embodiments, the selective injury-responsive enhancer element comprises:

[0216] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 17, and

[0217] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0218] In some embodiments, the selective injury-responsive enhancer element comprises:

[0219] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0220] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0221] wherein the selective injury-responsive enhancer element comprises a sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 40, and SEQ ID NO: 44 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0222] In some embodiments, the selective injury-responsive enhancer element comprises:

[0223] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0224] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

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[0226] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 35 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0227] In some embodiments, the selective injury-responsive enhancer element comprises:

[0228] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0229] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0230] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 40 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0231] In some embodiments, the selective injury-responsive enhancer element comprises:

[0232] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0233] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0234] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 44 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0235] In some embodiments, the selective injury-responsive enhancer element comprises:

[0236] a. at least one lineage-specific motif consisting of SEQ ID NO: 10 and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0237] In some embodiments, the selective injury-responsive enhancer element comprises:

[0238] a. at least one lineage-specific motif consisting of SEQ ID NO: 10, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0239] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 31 or SEQ ID NO: 43 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0240] In some embodiments, the selective injury-responsive enhancer element comprises:

[0241] a. at least one lineage-specific motif consisting of SEQ ID NO: 10, and

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[0243] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0244] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 31 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0245] In some embodiments, the selective injury-responsive enhancer element comprises:

[0246] a. at least one lineage-specific motif consisting of SEQ ID NO: 10, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0247] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 43 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0248] In some embodiments, the selective injury-responsive enhancer element comprises:

[0249] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, and

[0250] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0251] In some embodiments, the selective injury-responsive enhancer element comprises:

[0252] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, and

[0253] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0254] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 32 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0255] In some embodiments, the selective injury-responsive enhancer element comprises:

[0256] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13, and

[0257] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0258] In some embodiments, the selective injury-responsive enhancer element comprises:

[0259] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13, and

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[0261] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0262] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 47 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0263] In some embodiments, the selective injury-responsive enhancer element comprises:

[0264] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0265] In some embodiments, the selective injury-responsive enhancer element comprises:

[0266] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0267] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 48 or SEQ ID NO: 49 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0268] In some embodiments, the selective injury-responsive enhancer element comprises:

[0269] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0270] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 48 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0271] In some embodiments, the selective injury-responsive enhancer element comprises:

[0272] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0273] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 49 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0274] 15-01-2609:01:04P7423PC00

[0275] In some embodiments, the selective injury-responsive enhancer element comprises:

[0276] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0277] In some embodiments, the selective injury-responsive enhancer element comprises:

[0278] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0279] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 50 or SEQ ID NO: 51 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0280] In some embodiments, the selective injury-responsive enhancer element comprises:

[0281] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0282] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 50 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0283] In some embodiments, the selective injury-responsive enhancer element comprises:

[0284] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0285] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 51 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0286] In some embodiments, the selective injury-responsive enhancer element comprises:

[0287] 15-01-2609:01:04P7423PC00

[0288] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 19 and SEQ ID NO: 20, and

[0289] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0290] In some embodiments, the selective injury-responsive enhancer element comprises:

[0291] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 19 and SEQ ID NO: 20, and

[0292] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0293] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 52 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0294] In some embodiments, the selective injury-responsive enhancer element comprises:

[0295] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and

[0296] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0297] In some embodiments, the selective injury-responsive enhancer element comprises:

[0298] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and

[0299] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0300] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 53 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0301] In some embodiments, the selective injury-responsive enhancer element comprises multiple instances of the same at least one lineage-specific motif and / or the at least one AP1 motif.

[0302] 15-01-2609:01:04P7423PC00

[0303] Selective injury-responsive enhancer elements

[0304] In one aspect, the invention provides for an isolated nucleic acid molecule or artificial expression construct comprising:

[0305] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0306] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0307] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0308] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and

[0309] wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31 -53 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0310] In some embodiments, the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31 -53 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0311] In one aspect, the invention provides for isolated nucleic acid molecule or artificial expression construct comprising:

[0312] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0313] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury, wherein the selective injury-responsive enhancer element comprises:

[0314] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 ,

[0315] 15-01-2609:01:04P7423PC00

[0316] ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1 , IRF2, IRF3, and IRF4, and

[0317] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and

[0318] wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31 -35 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0319] In some embodiments, the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-35 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0320] Cell types

[0321] In some embodiments, the encoding sequence is selectively expressed in the at least one selected cell type and is not substantially expressed in non-selected other cell types. In some embodiments, the non-selected other cell type is a non-targeted cell type. In some embodiments, the non-selected other cell type is a reference cell type. In some embodiments, the non-selected other cell type or reference cell type is a nontargeted cell type in which the enhancer element is not activated following injury. In some embodiments, the non-selected other cell type or the reference cell type is within the same anatomical structure as the selected cells and / or can project to a common anatomical area as the selected cells. In some embodiments, the non-selected other cell type or the reference cell type is within an anatomical structure that is adjacent to an anatomical structure that comprises the selected cells. In some embodiments, the non-selected other cell type or the reference cell type is a non-targeted cell type with a different gene expression profile than the selected cell type. In some embodiments, the selective expression of the encoding sequence is greater than 50% expression as compared to a reference cell type, such as greater than 60% expression as compared to a reference cell type, such as greater than 70% expression as compared to a reference cell type, such as greater than 80% expression as compared to a reference cell type, such as greater than 90% expression as compared to a reference cell type.

[0322] 15-01-2609:01:04P7423PC00

[0323] In some embodiments, the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type. In some embodiments, the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type, such as at the least 1% of the levels at which the DNA sequence product is expressed in a selected cell type.

[0324] In some embodiments, the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type, such as at the least 1% of the levels at which the DNA sequence product is expressed in a selected cell type, such as at least 2%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20% of the levels at which the DNA sequence product is expressed in a selected cell type.

[0325] In some embodiments, the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type, such as at most 20% of the levels at which the DNA sequence product is expressed in a selected cell type, such as at most 15%, such as at most 10%, such as at most 5%, such as at most 4%, such as at most 3%, such as at most 2%, such as at most 1% of the levels at which the DNA sequence product is expressed in a selected cell type.

[0326] As used herein, the terms “non-neuronal brain cells” and “glial cells” refer to cells within the brain and nervous system that do not primarily function as neurons but are essential for maintaining homeostasis, supporting neuronal function, and responding to injury or disease. Glial cells, a subset of non-neuronal brain cells, include astrocytes, which regulate the extracellular environment and support the blood-brain barrier; microglia, the immune cells of the nervous system; oligodendrocytes, responsible for myelination in the central nervous system; Schwann cells, which provide myelination in the peripheral nervous system; and ependymal cells, which line the ventricles of the brain and spinal cord. Oligodendrocyte precursor cells (OPCs), a progenitor population, are also included within this category. Non-neuronal brain cells also encompass choroid plexus cells, which produce cerebrospinal fluid, and vascular-associated cells, such as pericytes and endothelial cells, which are involved in maintaining the bloodbrain barrier and vascular integrity. These cells play critical roles in structural support, synaptic regulation, immune defence, and repair processes within the nervous system. They are also pivotal in pathological contexts, including neuroinflammation, injury

[0327] 15-01-2609:01:04P7423PC00

[0328] response, and neurodegenerative diseases, often engaging in specialized transcriptional and cellular programs to maintain or restore tissue function.

[0329] In some embodiments, the selected cell type comprises glial cells and / or non-neuronal brain cells. In some embodiments, the selected cell type comprises astrocytes, oligodendrocytes, microglia, oligodendrocyte precursor cells (OPCs), ependymal cells, choroid plexus cells, and / or border-associated macrophages. In some embodiments, the selected cell type is astrocytes. In some embodiments, the DNA sequence product provides therapeutic efficacy in CNS-related disorders. In some embodiments, the reference cell type comprises any cell type distinct from glial cells, including but not limited to neurons or other non-glial cells.

[0330] Vectors

[0331] In some embodiments, the construct is incorporated into a vector. In some embodiments, the construct is incorporated into a vector selected from a viral vector, a chemically synthesized particle, or a lipid nanoparticle. In some embodiments, the vector is a recombinant adeno-associated viral (AAV) vector, lentivirus, retrovirus or adenovirus. In some embodiments, the vector is a recombinant adeno-associated viral (AAV) vector. In some embodiments, the vector is a recombinant adeno-associated viral (AAV) vector which is capable of crossing the blood-brain-barrier. In some embodiments, the recombinant adeno-associated viral (AAV) vector comprises serotype PHP.eB or a capsid variant thereof. In some embodiments, the recombinant adeno-associated viral (AAV) vector comprises serotype PHP.eB or a capsid variant thereof, such as AAV9, AAV.Cap-B10, AAV.Cap-B22, or AAV.CPP.16.

[0332] In some embodiments, the vector is administered systemically. In some embodiments, the vector selectively targets the selected cells in vivo.

[0333] Length of selective injury-responsive enhancer elements

[0334] In some embodiments, the selective injury-responsive enhancer element comprises at least 200 nucleic acids, such as at least 300 nucleic acids, such as at least 400 nucleic acids, at least 500 nucleic acids, such as at least 600 nucleic acids, such as at the least 700 nucleic acids, such as at the least 800 nucleic acids, such as at the least 900 nucleic acids, such as at the least 1000 nucleic acids, such as at the least 1100 nucleic acids, such as at the least 1200 nucleic acids.

[0335] 15-01-2609:01:04P7423PC00

[0336] In some embodiments, the selective injury-responsive enhancer element comprises at the most 1200 nucleic acids, such as at the most 1100 nucleic acids, such as at the most 1000 nucleic acids, such as at the most 900 nucleic acids, such as at the most 800 nucleic acids, such as at the most 700 nucleic acids, such as at the most 600 nucleic acids, such as at the most 500 nucleic acids, such as at most 400 nucleic acids, such as at most 300 nucleic acids, such as at most 200 nucleic acids.

[0337] In some embodiments, the selective injury-responsive enhancer element comprises at least 200 nucleic acids and at the most 1600 nucleic acids. In some embodiments, the selective injury-responsive enhancer element comprises at least 450 nucleic acids and at the most 1100 nucleic acids.

[0338] Compositions, methods and use

[0339] In one aspect, the invention provides for a pharmaceutical composition comprising the isolated nucleic acid construct or artificial expression construct according to any of the preceding sections of the description and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to any excipient, diluent, or vehicle that is compatible with the active ingredient and suitable for administration to a subject without causing undue adverse effects. Such carriers include sterile aqueous solutions such as saline, phosphate-buffered saline or water for injection, buffers including Tris, citrate or acetate buffers, and stabilising agents such as sugars (for example sucrose or trehalose), polyols (for example mannitol or sorbitol) or amino acids such as glycine. Additional carriers may comprise lipids and liposomes for encapsulation or delivery of nucleic acids, polymers such as polyethylene glycol or polyvinylpyrrolidone, oils and emulsions including vegetable oils or oil-in-water emulsions, and controlled-release matrices or biodegradable carriers such as polylactic-co-glycolic acid. Other excipients commonly used in injectable or oral formulations, provided they are recognised as safe and pharmaceutically acceptable, may also be employed.

[0340] In one aspect, the invention provides for use of the isolated nucleic acid molecule, artificial expression construct or pharmaceutical composition as described in any of the above sections of the description, for the treatment and / or prevention of diseases or disorders related to the central nervous system. In some embodiments, the disease or

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[0342] disorder comprises neuroinflammation and / or neurodegeneration. In some embodiments, the disease or disorder is spinal cord injury, brain injury, or stroke.

[0343] As used herein, the term “the disease or disorder comprises neuroinflammation and / or neurodegeneration” refers to any condition characterized by inflammation of nervous tissue and / or the progressive loss of structure or function of neurons, including their death. Neuroinflammation can result from acute injury, autoimmune responses, infection, or chronic neurodegenerative processes. Neurodegeneration refers to pathological processes that lead to the deterioration of the central nervous system, often associated with diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or multiple sclerosis. In the context of the present invention, the term encompasses both acute and chronic conditions affecting the central nervous system. Examples of diseases or disorders involving neuroinflammation and / or neurodegeneration include, but are not limited to, spinal cord injury, brain injury, stroke, multiple sclerosis, Alzheimer's disease, Parkinson's disease, and traumatic brain injury. These conditions may involve neuroinflammatory processes mediated by glial cells, such as microglia and astrocytes, as well as the activation of immune pathways contributing to neuronal damage and degeneration.

[0344] In one aspect, the invention provides for methods for selectively expressing a heterologous gene within a population of selected injury-responsive cells, comprising contacting said cells with the artificial expression construct or pharmaceutical composition as described in any of the above sections of the description, wherein the heterologous gene is expressed selectively in the injury-responsive cells.

[0345] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro.

[0346] Items

[0347] 1. An isolated nucleic acid construct comprising:

[0348] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0349] b. an encoding sequence operably linked to the enhancer element,

[0350] 15-01-2609:01:04P7423PC00

[0351] wherein the selective injury-responsive enhancer element is configured to drive expression of the encoding sequence in a selected cell type following an injury, and comprises:

[0352] i. at least one injury-responsive motif comprising a consensus sequence recognised by at least one injury-responsive transcription factor activated by injury-induced signalling pathways, which mediates transcriptional activation in response to cellular or tissue stress and / or damage; and ii. at least one lineage-specific motif comprising a consensus sequence recognised by at least one lineage-specific transcription factor that regulates cell-type identity, which restricts expression to a defined lineage, wherein the selective injury-responsive enhancer element provides combinatorial specificity through cooperative activity of said at least one injury- responsive motif and said at least one lineage-specific motif.

[0353] 2. An artificial expression construct comprising:

[0354] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0355] b. an encoding sequence operably linked to the enhancer element, wherein the selective injury-responsive enhancer element is configured to drive expression of the encoding sequence in a selected cell type following an injury, and comprises:

[0356] i. at least one injury-responsive motif comprising a consensus sequence recognised by at least one injury-responsive transcription factor activated by injury-induced signalling pathways, which mediates transcriptional activation in response to cellular or tissue stress and / or damage; and

[0357] ii. at least one lineage-specific motif comprising a consensus sequence recognised by at least one lineage-specific transcription factor that regulates cell-type identity, which restricts expression to a defined lineage,

[0358] wherein the selective injury-responsive enhancer element provides combinatorial specificity through cooperative activity of said at least one injury- responsive motif and said at least one lineage-specific motif.

[0359] 15-01-2609:01:04P7423PC00

[0360] 3. The isolated nucleic acid molecule or artificial expression construct according to any of the previous items, wherein the at least one injury-responsive motif comprises at least one binding site for at least one injury-responsive transcription factor activated by injury-induced signalling pathways, wherein said injury-responsive transcription factor is selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0361] 4. The isolated nucleic acid molecule or artificial expression construct according to any of the previous items, wherein the at least one lineage-specific motif comprises at least one binding site for at least one lineage-specific transcription factor that regulates cell-type identity or differentiation, wherein said lineagespecific transcription factor is selected from the group comprising or consisting of; SOX9, SOX2, NFI, NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX4, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4.

[0362] 5. An isolated nucleic acid molecule comprising:

[0363] a. a selective injury-responsive enhancer element that is activated in response to cellular or tissue injury in at least one selected cell type; and b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury,

[0364] wherein the selective injury-responsive enhancer element comprises: at least one binding site for a lineage-specific transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0365] at least one binding site for an injury-responsive transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0366] 15-01-2609:01:04P7423PC00

[0367] 6. An artificial expression construct, comprising:

[0368] a. a selective injury-responsive enhancer element that is activated in response to cellular or tissue injury in at least one selected injured cell type; and

[0369] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury,

[0370] wherein the selective injury-responsive enhancer element comprises: at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0371] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1 , FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

[0372] 7. The artificial expression construct according to any of the preceding items, further comprising a minimal promoter.

[0373] 8. The artificial expression construct according to any of the preceding items, further comprising a reporter transgene.

[0374] 9. The artificial expression construct according to item 8, wherein the reporter transgene comprises super yellow fluorescent protein (SYFP).

[0375] 10. The artificial expression construct according to any of the preceding items, further comprising a unique barcode, wherein the unique barcode specifically identifies the selective injury-responsive enhancer element.

[0376] 11. The artificial expression construct according to item 10, wherein the unique barcode comprises a nucleic acid sequence as set forth in SEQ ID NO:1-3.

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[0378] 12. The artificial expression construct according to any of the preceding items, further comprising a poly-A tail.

[0379] 13. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the DNA sequence product is an encoded polypeptide.

[0380] 14. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one lineage-specific motif is as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88.

[0381] 15. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one lineage-specific motif, is as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and wherein the enhancer element comprises multiple instances of the same at least one lineage-specific motif.

[0382] 16. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one transcription factor binding site characterised by at least one position frequency matrix (PFMs) as set forth in Table 1 (lineage motifs PFM), said binding sites being contained within the DNA sequence product and contribute to the binding specificity for the specific injured cell type.

[0383] 17. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one injury-responsive motif or at least one AP1 motif having the nucleotide sequence TGAXIX2X3X4X5(SEQ ID NO: 104), wherein:

[0384] Xi is G, C or T;

[0385] X2is T or G;

[0386] X3is A, C or T;

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[0388] X4is A, C or G; and;

[0389] X5is absent, A or G.

[0390] 18. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one AP1 motif as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103.

[0391] 19. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one AP1 motif as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103, and wherein the selective injury-responsive enhancer element comprises multiple instances of the same at least one AP1 motif.

[0392] 20. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the at least one injury-responsive motif is as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103, and wherein the selective injury-responsive enhancer element comprises multiple instances of the same at least one injury-responsive motif .

[0393] 21. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one transcription factor binding site characterised by at least one position frequency matrix (PFMs) as set forth in Table 2 (AP1 PFM), said binding sites being contained within the DNA sequence product and contribute to the binding specificity for the specific injured cell type.

[0394] 22. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises:

[0395] a. at least one lineage-specific motif, such as at least one lineage specific motif comprising a sequence as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and

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[0397] b. at least one AP1 motif, such as at least one AP1 motif comprising a sequence as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103.

[0398] 23. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein:

[0399] a. the at least one lineage-specific motif comprises a sequence as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and b. the at least one injury-responsive motif comprises a sequence as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103.

[0400] 24. The isolated nucleic acid molecule or artificial expression construct according to any one of items 22, wherein the selective injury-responsive enhancer element comprises:

[0401] a. at least one transcription factor binding site characterised by a position frequency matrix (PFMs) as set forth in Table 1 (lineage motifs), and b. at least one transcription factor binding site characterised by a position frequency matrix (PFMs) as set forth in Table 2 (AP1 motifs), said binding sites being contained within the DNA sequence product and contribute to the binding specificity for the specific injured cell type.

[0402] 25. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element is activated at the onset of injury, during injury pathogenesis and / or post injury.

[0403] 26. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element is epigenetically activated.

[0404] 27. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element is activated in response to and / or at the onset of and / or during and / or post CNS injury and / or diseases and / or disorders comprising

[0405] 15-01-2609:01:04P7423PC00

[0406] neuroinflammation, including spinal cord injury, brain injury, stroke and / or diseases and / or disorders comprising neurodegeneration.

[0407] 28. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises:

[0408] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0409] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0410] 29. The isolated nucleic acid molecule or artificial expression construct according to item 28, wherein the selective injury-responsive enhancer element comprises:

[0411] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0412] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0413] wherein the selective injury-responsive enhancer element comprises a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 42 and SEQ ID NO: 46 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0414] 30. The isolated nucleic acid molecule or artificial expression construct according to any one of items 28-29, wherein the selective injury-responsive enhancer element comprises:

[0415] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0416] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0417] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 37 or a functional homologue thereof sharing at least 90% sequence identify therewith.

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[0419] 31. The isolated nucleic acid molecule or artificial expression construct according to any one of items 28-29, wherein the selective injury-responsive enhancer element comprises:

[0420] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0421] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0422] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 42 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0423] 32. The isolated nucleic acid molecule or artificial expression construct according to any according to any one of items 28-29, wherein the selective injury- responsive enhancer element comprises:

[0424] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 17, and

[0425] b. at least one AP1 motif selected from the group consisting of SEQ ID NO:

[0426] 4 and SEQ ID NO:5,

[0427] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 46 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0428] 33. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises:

[0429] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0430] 34. The isolated nucleic acid molecule or artificial expression construct according to item 33, wherein the selective injury-responsive enhancer element comprises:

[0431] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0432] 15-01-2609:01:04P7423PC00

[0433] wherein the selective injury-responsive enhancer element comprises a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 41 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0434] 35. The isolated nucleic acid molecule or artificial expression construct according to any one of items 33-34, wherein the selective injury-responsive enhancer element comprises:

[0435] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0436] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 33 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0437] 36. The isolated nucleic acid molecule or artificial expression construct according to any one of items 33-34, wherein the selective injury-responsive enhancer element comprises:

[0438] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0439] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 34 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0440] 37. The isolated nucleic acid molecule or artificial expression construct according to any one of items 33-34, wherein the selective injury-responsive enhancer element comprises:

[0441] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0442] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 36 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0443] 15-01-2609:01:04P7423PC00

[0444] 38. The isolated nucleic acid molecule or artificial expression construct according to any one of items 33-34, wherein the selective injury-responsive enhancer element comprises:

[0445] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0446] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 38 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0447] 39. The isolated nucleic acid molecule or artificial expression construct according to any one of items 33-34, wherein the selective injury-responsive enhancer element comprises:

[0448] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0449] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 39 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0450] 40. The isolated nucleic acid molecule or artificial expression construct according to any one of items 33-34, wherein the selective injury-responsive enhancer element comprises:

[0451] a. at least one lineage-specific motif consisting of SEQ ID NO: 17, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0452] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 41 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0453] 41. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0454] 15-01-2609:01:04P7423PC00

[0455] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 17 and SEQ ID NO: 14, and

[0456] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0457] 42. The isolated nucleic acid molecule or artificial expression construct according to item 41, wherein the selective injury-responsive enhancer element comprises:

[0458] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 17 and SEQ ID NO: 14, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0459] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 45 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0460] 43. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0461] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 17, and

[0462] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0463] 44. The isolated nucleic acid molecule or artificial expression construct according to item 43, wherein the selective injury-responsive enhancer element comprises:

[0464] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0465] b. at least one AP1 motif selected from the group consisting of SEQ ID NO:

[0466] 4 and SEQ ID NO:5,

[0467] wherein the selective injury-responsive enhancer element comprises a sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 44 and SEQ ID NO: 106 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0468] 15-01-2609:01:04P7423PC00

[0469] 45. The isolated nucleic acid molecule or artificial expression construct according to any one of items 43-44, wherein the selective injury-responsive enhancer element comprises:

[0470] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0471] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0472] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 35 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0473] 46. The isolated nucleic acid molecule or artificial expression construct according to any one of items 43-44, wherein the selective injury-responsive enhancer element comprises:

[0474] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0475] b. at least one AP1 motif selected from the group consisting of SEQ ID NO:

[0476] 4 and SEQ ID NO:5,

[0477] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 40 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0478] 47. The isolated nucleic acid molecule or artificial expression construct according to any one of items 43-44, wherein the selective injury-responsive enhancer element comprises:

[0479] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and

[0480] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0481] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 44 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0482] 15-01-2609:01:04P7423PC00

[0483] 48. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0484] a. at least one lineage-specific motif consisting of SEQ ID NO: 10 and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0485] 49. The isolated nucleic acid molecule or artificial expression construct according to item 48, wherein the selective injury-responsive enhancer element comprises:

[0486] a. at least one lineage-specific motif consisting of SEQ ID NO: 10, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0487] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 31 or SEQ ID NO: 43 OR SEQ ID NO: 105 OR SEQ ID NO: 106 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0488] 50. The isolated nucleic acid molecule or artificial expression construct according to any one of items 48-49, wherein the selective injury-responsive enhancer element comprises:

[0489] a. at least one lineage-specific motif consisting of SEQ ID NO: 10, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0490] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 31 OR SEQ ID NO: 105 OR SEQ ID NO: 106 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0491] 51. The isolated nucleic acid molecule or artificial expression according to any one of items 48-49, wherein the selective injury-responsive enhancer element comprises:

[0492] a. at least one lineage-specific motif consisting of SEQ ID NO: 10, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0493] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 43 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0494] 15-01-2609:01:04P7423PC00

[0495] 52. The isolated nucleic acid molecule or artificial expression according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0496] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, and

[0497] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0498] 53. The isolated nucleic acid molecule or artificial expression construct according to item 52, wherein the selective injury-responsive enhancer element comprises: a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, and

[0499] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0500] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 32 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0501] 54. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0502] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13, and

[0503] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0504] 55. The isolated nucleic acid molecule or artificial expression construct according to item 54, wherein the selective injury-responsive enhancer element comprises: a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13, and

[0505] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0506] 15-01-2609:01:04P7423PC00

[0507] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 47 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0508] 56. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0509] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and

[0510] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0511] 57. The isolated nucleic acid molecule or artificial expression construct according to item 56, wherein the selective injury-responsive enhancer element comprises: a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and

[0512] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0513] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 48 or SEQ ID NO: 49 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0514] 58. The isolated nucleic acid molecule or artificial expression construct according to any of items 56-57, wherein the selective injury-responsive enhancer element comprises:

[0515] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and

[0516] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0517] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 48 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0518] 15-01-2609:01:04P7423PC00

[0519] 59. The isolated nucleic acid molecule or artificial expression construct according to any of items 56-57, wherein the selective injury-responsive enhancer element comprises:

[0520] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 and SEQ ID NO: 23, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0521] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 49 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0522] 60. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0523] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and

[0524] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0525] 61. The isolated nucleic acid molecule or artificial expression construct according to item 60, wherein the selective injury-responsive enhancer element comprises: a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and

[0526] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0527] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 50 or SEQ ID NO: 51 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0528] 62. The isolated nucleic acid molecule or artificial expression construct according to any one of items 60-61, wherein the selective injury-responsive enhancer element comprises:

[0529] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and

[0530] 15-01-2609:01:04P7423PC00

[0531] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0532] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 50 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0533] 63. The isolated nucleic acid molecule or artificial expression construct according to any one of items 60-61, wherein the selective injury-responsive enhancer element comprises:

[0534] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and

[0535] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0536] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 51 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0537] 64. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0538] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 19 and SEQ ID NO: 20, and

[0539] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0540] 65. The isolated nucleic acid molecule or artificial expression construct according to item 64, wherein the selective injury-responsive enhancer element comprises: a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 19 and SEQ ID NO: 20, and

[0541] b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0542] 15-01-2609:01:04P7423PC00

[0543] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 52 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0544] 66. The isolated nucleic acid molecule or artificial expression construct according to any one of items 1-27, wherein the selective injury-responsive enhancer element comprises:

[0545] a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

[0546] 67. The isolated nucleic acid molecule or artificial expression construct according to item 66, wherein the selective injury-responsive enhancer element comprises: a. at least one lineage-specific motif selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and b. at least one AP1 motif selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,

[0547] wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 53 or a functional homologue thereof sharing at least 90% sequence identify therewith.

[0548] 68. The isolated nucleic acid molecule or artificial expression construct according to any one of the preceding items, wherein the selective injury-responsive enhancer element comprises multiple instances of the same at least one lineage-specific motif and / or the at least one AP1 motif.

[0549] 69. An isolated nucleic acid molecule or artificial expression construct comprising:

[0550] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0551] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury,

[0552] wherein the selective injury-responsive enhancer element comprises:

[0553] 15-01-2609:01:04P7423PC00

[0554] at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0555] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1 , FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and

[0556] wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-53, SEQ ID NO: 105-106 or functional homologues thereof sharing at least 90% sequence identify therewith.

[0557] 70. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-53, SEQ ID NO: 105-106 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0558] 71. An isolated nucleic acid molecule or artificial expression construct comprising:

[0559] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0560] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury,

[0561] wherein the selective injury-responsive enhancer element comprises: at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4, and

[0562] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1,

[0563] 15-01-2609:01:04P7423PC00

[0564] FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and

[0565] wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-35, SEQ ID NO: 105-106 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0566] 72. An isolated nucleic acid molecule or artificial expression construct comprising:

[0567] a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; and

[0568] b. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury,

[0569] wherein the selective injury-responsive enhancer element comprises: at least one binding site for a transcription factor selected from the group comprising or consisting of; NFIA, NFIB, NFIC, NFIX, RFX2, RFX3, RFX4, RFX7, RORA, RORB, SOX2, SOX9, and

[0570] at least one binding site for a transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1 , FOSL2, ATF1 , ATF3, ATF4, BACH1 , BACH2, JDP2, MAFF, MAFG, and wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-35, SEQ ID NO: 105- 106 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0571] 73. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-35, SEQ ID NO: 105-106 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0572] 74. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the encoding sequence is selectively

[0573] 15-01-2609:01:04P7423PC00

[0574] expressed in the at least one selected cell type and is not substantially expressed in non-selected other cell types.

[0575] 75. The isolated nucleic acid molecule or artificial expression construct according to item 74, wherein the non-selected other cell type is a non-targeted cell type.

[0576] 76. The isolated nucleic acid molecule or artificial expression construct according to any one of items 74-75, wherein the non-selected other cell type is a reference cell type.

[0577] 77. The isolated nucleic acid molecule or artificial expression construct according to any one of items 74-76, wherein the non-selected other cell type or reference cell type is a non-targeted cell type in which the enhancer element is not activated following injury.

[0578] 78. The isolated nucleic acid molecule or artificial expression construct according to any one of items 74-77, wherein the non-selected other cell type or the reference cell type is within the same anatomical structure as the selected cells and / or can project to a common anatomical area as the selected cells.

[0579] 79. The isolated nucleic acid molecule or artificial expression construct according to any one of items 74-77, wherein the non-selected other cell type or the reference cell type is within an anatomical structure that is adjacent to an anatomical structure that comprises the selected cells.

[0580] 80. The isolated nucleic acid molecule or artificial expression construct according to any one of items 74-79, wherein the non-selected other cell type or the reference cell type is a non-targeted cell type with a different gene expression profile than the selected cell type.

[0581] 81. The isolated nucleic acid molecule or artificial expression construct according to any one of items 74-80, wherein the selective expression of the encoding sequence is greater than 50% expression as compared to a reference cell type, such as greater than 60% expression as compared to a reference cell type, such as greater than 70% expression as compared to a reference cell type,

[0582] 15-01-2609:01:04P7423PC00

[0583] such as greater than 80% expression as compared to a reference cell type, such as greater than 90% expression as compared to a reference cell type.

[0584] 82. The isolated nucleic acid molecule or artificial expression construct according to according to any one of the preceding items, wherein the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type.

[0585] 83. The isolated nucleic acid molecule or artificial expression construct according to item 82, wherein the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type, such as at the least 1% of the levels at which the DNA sequence product is expressed in a selected cell type.

[0586] 84. The isolated nucleic acid molecule or artificial expression construct according to any one of items 82-83, wherein the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type, such as at the least 1% of the levels at which the DNA sequence product is expressed in a selected cell type, such as at least 2%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20% of the levels at which the DNA sequence product is expressed in a selected cell type.

[0587] 85. The isolated nucleic acid molecule or artificial expression according to any one of items 82-83, wherein the DNA sequence product may be expressed at low levels in a non-selected other cell type or reference cell type, such as at most 20% of the levels at which the DNA sequence product is expressed in a selected cell type, such as at most 15%, such as at most 10%, such as at most 5%, such as at most 4%, such as at most 3%, such as at most 2%, such as at most 1% of the levels at which the DNA sequence product is expressed in a selected cell type.

[0588] 86. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selected cell type comprises glial cells and / or non-neuronal brain cells.

[0589] 15-01-2609:01:04P7423PC00

[0590] 87. The isolated nucleic acid molecule or artificial expression construct according to item 86, wherein the selected cell type comprises astrocytes, oligodendrocytes, microglia, oligodendrocyte precursor cells (OPCs), ependymal cells, choroid plexus cells, and / or border-associated macrophages.

[0591] 88. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selected cell type is astrocytes.

[0592] 89. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the DNA sequence product provides therapeutic efficacy in CNS-related disorders.

[0593] 90. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the construct is incorporated into a vector.

[0594] 91. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the construct is incorporated into a vector selected from a viral vector, a chemically synthesized particle, or a lipid nanoparticle.

[0595] 92. The isolated nucleic acid molecule or artificial expression construct according to any one of items 90-91 , wherein the vector is a recombinant adeno-associated viral (AAV) vector, lentivirus, retrovirus or adenovirus.

[0596] 93. The isolated nucleic acid molecule or artificial expression construct according to any one of items 90-92, wherein the vector is a recombinant adeno-associated viral (AAV) vector.

[0597] 94. The isolated nucleic acid molecule or artificial expression construct according to item 93, wherein the vector is a recombinant adeno-associated viral (AAV) vector which is capable of crossing the blood-brain-barrier.

[0598] 15-01-2609:01:04P7423PC00

[0599] 95. The isolated nucleic acid molecule or artificial expression construct according to any of items 93-94, wherein the a recombinant adeno-associated viral (AAV) vector comprises serotype PHP.eB or a capsid variant thereof.

[0600] 96. The isolated nucleic acid molecule or artificial expression construct according to any one of items 93-95, wherein the a recombinant adeno-associated viral (AAV) vector comprises serotype PHP.eB or a capsid variant thereof, such as AAV9, AAV.Cap-B10, AAV.Cap-B22, or AAV.CPP.16.

[0601] 97. The isolated nucleic acid molecule or artificial expression construct according to any one of items 90-96, wherein the vector is administered systemically.

[0602] 98. The isolated nucleic acid molecule or artificial expression construct to any one of items 90-97, wherein the vector selectively targets the selected cells in vivo.

[0603] 99. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at least 200 nucleic acids, such as at least 300 nucleic acids, such as at least 400 nucleic acids, at least 500 nucleic acids, such as at least 600 nucleic acids, such as at the least 700 nucleic acids, such as at the least 800 nucleic acids, such as at the least 900 nucleic acids, such as at the least 1000 nucleic acids, such as at the least 1100 nucleic acids, such as at the least 1200 nucleic acids.

[0604] 100. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the selective injury-responsive enhancer element comprises at the most 1200 nucleic acids, such as at the most 1100 nucleic acids, such as at the most 1000 nucleic acids, such as at the most 900 nucleic acids, such as at the most 800 nucleic acids, such as at the most 700 nucleic acids, such as at the most 600 nucleic acids, such as at the most 500 nucleic acids, such as at most 400 nucleic acids, such as at most 300 nucleic acids, such as at most 200 nucleic acids.

[0605] 101. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, the selective injury-responsive

[0606] 15-01-2609:01:04P7423PC00

[0607] enhancer element comprises at least 200 nucleic acids and at the most 1600 nucleic acids.

[0608] 102. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, the selective injury-responsive enhancer element comprises at least 450 nucleic acids and at the most 1100 nucleic acids.

[0609] 103. A pharmaceutical composition comprising the isolated nucleic acid construct or artificial expression construct according to any of the preceding items and a pharmaceutically acceptable carrier.

[0610] 104. Use of the isolated nucleic acid molecule, artificial expression construct or pharmaceutical composition according to any of the preceding items for the treatment and / or prevention of diseases or disorders related to the central nervous system.

[0611] 105. The use according to item 104, wherein the disease or disorder comprises neuroinflammation and / or neurodegeneration.

[0612] 106. The use according to any one of items 104-105, wherein the disease or disorder is spinal cord injury, brain injury, or stroke

[0613] 107. The use according to any one of items 104-106, wherein the disease or disorder is Alzheimer’s disease and amyotrophic lateral sclerosis (ALS).

[0614] 108. A cell comprising the isolated nucleic acid construct or artificial expression construct according to any of the preceding items, wherein the selected cell type comprises astrocytes, oligodendrocytes, microglia, oligodendrocyte precursor cells (OPCs), ependymal cells, choroid plexus cells, and / or border-associated macrophages.

[0615] 109. A method for selectively expressing a heterologous gene within a population of selected injury-responsive cells, comprising:

[0616] 15-01-2609:01:04P7423PC00

[0617] a. contacting said cells with the artificial expression construct or pharmaceutical composition according to any of one of items 6-102, wherein the heterologous gene is expressed selectively in the injury-responsive cells.

[0618] 110. The method according to item 109, wherein the method is performed in vivo.

[0619] 111. The method according to item 109, wherein the method is performed in vitro.

[0620] 112. A method for designing a selective injury-responsive enhancer element comprising a cooperative syntax, comprising:

[0621] a. identifying at least one injury-responsive motif activated by at least one injury-responsive transcription factor, wherein the at least one injury- responsive motif is according to any one of the preceding items and wherein the at least one injury-responsive transcription factor is according to any one of the preceding items,

[0622] b. identifying at least one lineage-specific motif recognised by at least one transcription factor that regulate cell-type identity, wherein the at least one lineage-specific motif is to any one of the preceding items and wherein the at least one lineage-specific transcription factor is according to any one of the preceding items; and

[0623] c. combining said motifs in a configuration that enables cooperative transcriptional activation in selected injury-responsive cells.

[0624] Examples

[0625] Example 1 - Unbiased single cell transcriptomics and chromatin accessibility profiling in the injured mouse spinal cord reveals cell type specific injury responsive enhancers Aims

[0626] The CNS contains the highest degree of cell type specialization, encompassing a large number of neurons and glial subtypes. This specialization in turn leads to a highly heterogenous response to injury, and recent studies have begun to characterize injury or disease associated cell states and the gene expression programs that characterize them (Hasel et al., 2021 ; Matson et al., 2022; Skinnider et al., 2024). How the spatiotemporal regulation of these gene expression programs across the different cell

[0627] 15-01-2609:01:04P7423PC00

[0628] types is encoded within genomic regulatory elements remains largely elusive. Using single-nucleus multiomic approaches, this study aimed to investigate the regulatory mechanisms governing injury-dependent transcriptional programs in the injured mouse spinal cord.

[0629] Materials and Methods

[0630] Animals: Wild-type C57BL6 mice (Janvier) and transgenic mice, generated by crossing Foxj1-CreERT2 (Meletis et al., 2008) or Cx30 (Slezak et al., 2007) with R26R-tdTomato reporter mice (Madisen et al., 2010), were used. Standard housed mice entered the study at 8-12 weeks of age, with balanced assignment to experimental conditions including both sexes. Induction of genetic recombination in the transgenic lines was achieved through oral administration of tamoxifen (Sigma, 20 mg / ml in 1 :9 ethanokcorn oil solution) at 2 mg / injection, on two consecutive days. Subsequent procedures were performed at least two weeks after the last injection to ensure expression of the fluorescent reporter and avoid potential confounders deriving from circulating tamoxifen (Gonzalez et aL, 2016)

[0631] Induction of spinal Cord Injury. Mice were deeply anesthetized with isoflurane (4% induction, 2% maintenance) and kept on a heated pad. Subcutaneous injections of analgesics (Buprenorphine, 0.1 mg / kg; Carprofen, 5 mg / kg) and antibiotics (Sulphadiazine / Trimethoprim, 100 mg / kg) in saline were administered.

[0632] A contusion injury at T9 was performed using the Infinite Horizon Impactor (55 kDyne, 1.3 mm tip) after a laminectomy at T8-T9, leaving the dura mater intact. The vertebral column was stabilized with clamps, and Xylocaine (10 mg / ml) was applied topically. Injury was confirmed by dorsal bruising; animals with displacements <350 were excluded. Muscle and skin were sutured, and animals recovered in heated cages with elevated floor grids. Post-operatively, animals received antibiotics and analgesics (Buprenorphine twice daily; Carprofen once daily) for three days, with diet supplemented by high-energy food (DietGel Boost). Weight was monitored daily for the first week, then weekly. Animals losing >15% of pre-operative weight were euthanized. Bladders were expressed twice daily until regaining control.

[0633] Tissue Preparation for Multiomics: Mice were euthanized at 1 , 3, 7, and 28 days postinjury (dpi) or as uninjured controls. They were deeply anesthetized with intraperitoneal

[0634] 15-01-2609:01:04P7423PC00

[0635] sodium pentobarbital (100 pl) and transcardially perfused with sterile HBSS (without Ca / Mg). Fresh spinal cords were microdissected, cleared of meninges, and 5 mm tissue segments were obtained around the injury epicenter or the equivalent rostrocaudal level in controls. For wild-type C57BL6 mice, spinal cords were dissociated to isolate nuclei. Two samples per timepoint (one per sex) were pooled, and single nuclei suspensions were prepared using the Chromium Nuclei Isolation Kit (10X Genomics). Nuclei were stained with fluorescent antibodies against NeuN (1 :500) or Sox10 (1 :200) for 1 hour on ice, followed by 7AAD (1 :50), and sorted using a BD FACS Influx sorter with a 100 pm nozzle. For ependymal cell enrichment, spinal cords from Foxjl-tdTomato transgenic mice (four cords per condition, two per sex) were enzymatically digested using papain and a GentleMACS Dissociator (Miltenyi) following the Adult Brain Dissociation protocol. Cells were sorted based on fluorescent reporter expression and collected in sorting buffer with 1% BSA for nuclei isolation (10X Genomics protocol CG000124 Rev F). Sequencing-ready libraries were prepared using the Chromium Next GEM Single Cell Multiome protocol (10X Genomics, CG000338 Rev F) and sequenced on an Illumina NextSeq or MGI platform.

[0636] Flaw sequencing data processing: Raw reads from Illumina runs were processed using the Cellranger-ARC pipeline (v2.0.2, 10X Genomics) and mapped to the mm10 reference genome (v2.0.0), supplemented with the tdTomato DNA sequence for the transgenic line. Raw bcl files were converted to fastq files using the mkfastq function with default parameters, and each sample was mapped individually using the count function. For MGI sequencing runs, raw reads in fastq format (R2 and indexes combined) were demultiplexed by first splitting the R2 file, reverse-complementing the index reads (via a custom script), and demultiplexing with deML (v1.1.4). The resulting fastq files were mapped using Cellranger-ARC count as described above. Following alignment, gene expression matrices and fragment files were generated for transcriptomic and epigenomic data, respectively. These datasets were pre-processed and filtered separately before integration for multimodal analysis. The experiment produced 21 high-quality samples.

[0637] Processing of gene expression data'. Gene expression data were processed to remove ambient RNA, low-quality cells, and putative doublets. CellBender (vO.3.2) was applied to raw, unfiltered gene expression matrices using default parameters and an FPR of 0.05 to correct for ambient RNA. Poor-quality nuclei and doublets were identified from

[0638] 15-01-2609:01:04P7423PC00

[0639] raw matrices, while CellBender-corrected data were used for downstream multiomics analysis. Raw data were scaled to a factor of 10,000, normalized for sequencing depth, and log-transformed using the Seurat R package (v4.3.0) (Hao et al., 2021).

[0640] Dimensionality reduction was performed via principal component analysis (PCA), and the top 15 components were used to organize nuclei in UMAP space.

[0641] Processing of chromatin accessibility data'. Fragment files from all samples were merged for peak calling using the ArchR package (Granja et al., 2021 ; v1.0.2).

[0642] Dimensionality reduction was performed with addlterativeLSI on the TileMatrix, followed by harmony integration (addHarmony), clustering (addCluster at resolution 1), and UMAP embedding (addUMAP). Cluster-wise peak calling was conducted with addReproduciblePeakSet using default parameters to generate a consensus peak set. The peak set was imported into Signac (Stuart et al., 2021 ; v1.12.0) to recalculate feature matrices and create new chromatin assay objects using FeatureMatrix and CreateChromatinAssay. This process was repeated for each sample, followed by dimensionality reduction with RunTFIDF, RunSVD, and RunUMAP (dimensions 2-15). Gene annotations from EnsDb.Mmusculus.v79 (v2.99.0) were used to annotate genomic regions and compute TSS enrichment scores.

[0643] Filtering of multiomic data: Low-quality nuclei with fewer than 200 UMIs (RNA assay) or 300 fragments (ATAC assay) were discarded. Data were inspected cluster-wise to account for transcriptional differences between neuronal and non-neuronal cell types. After dimensionality reduction and clustering (FindMultiModalNeighbors and FindClusters at resolution 0.3), cluster-wise interquartile ranges (IQR) were calculated for UMI count, unique genes, fragments, mitochondrial content, TSS enrichment, and nucleosome signal. Nuclei outside 3xlQR below the first quartile or above the third quartile were excluded. Doublets were removed by analyzing RNA and ATAC assays separately. For gene expression data, DoubletFinder (McGinnis et al., 2019; v2.0.3) was applied using principal components 1-15, cluster identities, an optimal pK value, and a sample-adjusted expected doublet rate. For chromatin data, doublets were identified using scDblFinder (Germain et al., 2021 ; v1.16.0) with 1000 features, LSI dimensions 2-15, and an adjusted expected doublet rate. Observations flagged as doublets by either method were excluded. Additional doublets were manually filtered after integration across all samples based on incompatible gene expression profiles.

[0644] 15-01-2609:01:04P7423PC00

[0645] Integration of multiomic data: High-quality data from all samples were merged into a multimodal dataset of -67,000 nuclei (Figure 1). Dimensionality reduction was recomputed for each modality using PCA (RNA, top 2000 variable features) and LSI (ATAC, min. cutoff = 10). The top 30 components (excluding the first for ATAC) were used to calculate multimodal nearest neighbors, generate UMAP visualizations, and cluster data into major cell types at resolution 0.1. Gene-to-region linkages were identified using Signac’s LinkPeaks() function, based on correlations between transcriptional activity and chromatin accessibility, including smaller cell populations.

[0646] Differential analysis: Differentially expressed genes and accessible regions were identified using Seurat’s FindAIIMarkers() or FindMarkers() functions with Wilcoxon’s test (p < 0.05, Iog2-fold change > 0.5). Features differing between uninjured and injured were clustered by dynamic profiles across timepoints using TCseq (v1.28.0) fuzzy clustering, identifying 6 activation patterns to order genes and genomic regions.

[0647] Results

[0648] To study the transcriptional and regulatory response to spinal cord injury, singlenucleus transcriptomics and chromatin accessibility (ATAC) profiling were performed on nuclei isolated from lesion sites of mice subjected to mid-thoracic contusion injury. Samples were collected at acute (1 , 3 dpi), sub-acute (7 dpi), and chronic (28 dpi) phases (Figure 1a).

[0649] High-quality data were obtained for all major resident neuronal, non-neuronal, and immune cell types (Figure 1b). Peripheral immune cells, as expected, were prominent only in the acute and sub-acute phases (Burda & Sofroniew, 2014; Sofroniew, 2018). The dataset, comprising 67,072 high-quality nuclei, aligned with prior studies (Kathe et al., 2022; Skinnider et al., 2024). Enrichment strategies, including fluorescent activated cell sorting, were applied to reduce abundant cell types (e.g., neurons, oligodendrocytes) and enrich rarer populations like astrocytes, ependymal cells, and immune cells, using NeuN, Sox10, and the Foxjl-tdTomato reporter line (Madisen et al., 2010; Meletis et al., 2008).

[0650] Gene regulatory analysis revealed substantial transcriptional changes in glial cells (astrocytes, ependymal cells, OPCs, and microglia), particularly during early phases (1-3 dpi), whereas neurons remained relatively unaffected.

[0651] 15-01-2609:01:04P7423PC00

[0652] Astrocytes upregulated immune and barrier-related genes (Ccl2, Vim, Gfap) while suppressing homeostatic programs like lipid metabolism (Slc27a1) and synapse maintenance (SparcH). Gfap and Stat3 remained elevated in later phases, indicating persistent reactivity. Peak-to-gene linkage analysis linked most injury-induced genes (665 of 1027) to differentially accessible enhancers, highlighting enhancer commissioning as a driver of astrocyte reactivity (Figure 1c).

[0653] Ependymal cells showed similar trends, with downregulation of cilia-related genes (Dnalil) and activation of progenitor, cell cycle, and migratory programs (Nes, Vim, Cd24a) peaking at 3 dpi before returning to baseline. Most gene expression changes (785 of 840) were linked to 1739 injury-responsive enhancers, some of which remained accessible at later stages, suggesting epigenetic memory (Figure 1c).

[0654] OPCs and oligodendrocytes exhibited a milder response. OPCs activated cell cycle genes, while oligodendrocytes upregulated myelin-related genes (Mog, Mag, Plp1 ). Most changes in OPCs (744 of 862) and oligodendrocytes (184 of 308) were linked to newly accessible enhancers.

[0655] Microglia showed a strong, persistent transcriptional response, but only 501 of 1169 gene expression changes were linked to injury-responsive enhancers, indicating a greater reliance on primed regulatory elements (Meijer et al., 2022) (Figure 1c).

[0656] To assess cell type specificity, differential accessibility analyses were performed to identify 27,843 injury-responsive regulatory elements exclusive to specific injury-associated cell states (Figure 1 d).

[0657] Conclusion

[0658] Glial cells initiated a robust injury response driven primarily by the commissioning of thousands of cell type-specific injury-responsive enhancers (IRENs), many of which exhibited high cell state specificity.

[0659] Example 2 - Deep learning models predict cell type specific enhancer accessibility based on DNA sequence

[0660] 15-01-2609:01:04P7423PC00

[0661] Aims

[0662] Deep learning models, particularly interpretable convolutional neural networks (CNNs), effectively decode the regulatory syntax of enhancer elements, predicting accessibility and transcription factor binding based on DNA sequence (Almeida et al., 2021 ; Avsec et al., 2021 ; Janssens et al., 2022; Nair et al., 2023). Leveraging genome-wide chromatin accessibility data and dense sampling of spinal cord cell populations, this study used CNNs to infer the transcription factor motif syntax of lineage-specific regulatory elements.

[0663] Materials and Methods

[0664] Machine Learning Model Architecture: Deep neural network models were trained using the ChromBPNet pipeline (v0.1.7; Nair et al., 2023; code provided at https: / / github.com / kundajelab / chrombpnet) with TensorFlow (v2.8.0) and Keras (v2.8.0) following the BPNet architecture (Avsec et al., 2021). Models used one-hot encoded 2114 bp DNA sequences as input, passed through a convolutional layer (512 filters, kernel size 21 , activation = ‘relu’), followed by 9 dilated convolutions (512 filters, kernel size 3, dilation rate = 2Alayer_number). Outputs generated profile log-likelihoods and count predictions via global average pooling and a dense layer. Models were optimized using Adam (learning rate = 0.001), with early stopping after 5 epochs without validation loss improvement.

[0665] Tn5 Bias Model: To correct Tn5 sequence bias, a bias model was trained on merged fragment files from all experimental conditions. Peaks were called using MACS2 (v2.2.7.1) with parameters -g 1.87e9 -B -p 0.01 -nomodel -extsize 200, followed by filtering of mm10 blacklist regions (Amemiya et al., 2019) and removal of reads mapping to chrM and random sequences. Non-peak background regions matched for GC content were generated with chrombpnet prep nonpeaks. The bias model, trained on specified chromosome subsets, used the same architecture as above to generate de-biased predictions.

[0666] Cell Type Model Training: Chromatin accessibility data and the Tn5 bias model were used to train cell type-specific models. Pseudobulks were generated using Signac’s SplitFragments() function, peaks were prepared as above, and non-peak regions were generated with chrombpnet prep nonpeaks. Sequences were Tn5-adjusted (+4 bp on the +’ strand, -5 bp on the strand) and augmented with 500 bp jittering and reverse

[0667] 15-01-2609:01:04P7423PC00

[0668] complementing of random samples. Five cross-validation sets were prepared using 16 chromosomes for training, and 2 chromosomes each for testing and validation.

[0669] Results

[0670] Convolutional networks were trained for each major cell type to predict accessibility profiles from DNA sequence at base-pair resolution (Figure 2a). The models achieved high predictive power for held-out sequences and showed strong correlations between observed and predicted counts across the genome (Pearson’s R: 0.675 ± 0.013 for ependymal cells; 0.820 ± 0.007 for ventral neurons, Figure 2b).

[0671] Conclusion

[0672] The cell type deep learning models accurately inferred chromatin accessibility from DNA sequence at base-pair resolution, enabling identification of the motif syntax underlying cell type-specific gene regulatory programs.

[0673] Example 3 - Deep learning models identify binding site motifs for cell type specific transcription factors

[0674] Aims

[0675] This study aimed to leverage the interpretability of deep learning models to identify transcription factor motifs associated with cell type-specific regulatory element accessibility.

[0676] Materials and Methods

[0677] Motif Analysis: Nucleotide contribution scores were computed at base-pair resolution using the DeepLIFT algorithm (v0.6.13.0) with the DeepSHAP implementation (Shrikumar et al., 2017). Motifs were extracted using TF-MoDISco (v2.0.7; Shrikumar et al., 2018; https: / / github.com / jmschrei / tfmodisco-lite) with a maximum of 1 million seqlets and matched to a motif database (Vierstra et al., 2014). Enriched motifs were curated by determining transcription factor expression and filtering out motifs with gene expression detected in <1% of cells within the cluster of interest. Model performance was evaluated using conventional motif enrichment analysis on differentially accessible regions (logFC > 0.5, pval_adj < 0.05) with AddMotif () in Signac and the JASPAR2020 CORE vertebrate collection. Over-represented motifs were filtered for expressed transcription factors.

[0678] 15-01-2609:01:04P7423PC00

[0679] Results

[0680] Using DeepLIFT (Shrikumar et al., 2017) and TF-MoDISco (Shrikumar et al., 2018), contribution scores were computed for base-pairs across regulatory elements in each cell type model to infer transcription factor binding sites from enriched motif patterns. Motifs were identified in 52 ± 5.8% of regulatory elements, with 32 ± 11% carrying >1 motif and 3 ± 2% exhibiting >4 motifs (Figure 3a-c).

[0681] The models accurately identified lineage-specific transcription factor motifs and predicted accessibility of elements carrying these motifs (Figure 3d-e). For example, neuronal regions were enriched for Ebf 1 motifs, while glial peaks contained motifs for Rorc and Rfx4 (astrocytes and ependymal cells) and Sox10 (oligodendrocytes).

[0682] Overall, 38 ± 7 consensus motif patterns were identified per cell type, linked to 230 transcription factors, with a total of 1073 motif-transcription factor matches. 82% of matches showed cell type-specific expression of the transcription factor, compared to 57% identified by conventional motif enrichment analysis, which does not consider motif importance (Figure 3d).

[0683] Conclusion

[0684] The cell type-resolved deep learning models accurately identified transcription factor binding motifs underlying cell type-specific gene regulatory programs.

[0685] Example 4 - AP-1 transcription factors govern injury-induced gene expression changes in glial cells

[0686] Aims

[0687] Glial cells coordinate functions like barrier formation and wound repair during injury (Tran et al., 2018). This study aimed to identify transcription factors controlling injury-responsive gene expression in glial cells.

[0688] Materials and Methods

[0689] Differential analysis: Differentially expressed genes and accessible regions were identified using Seurat’s FindAIIMarkers() or FindMarkers() with Wilcoxon’s test (p < 0.05, average Iog2-fold change > 0.5). ChromVar was used to calculate genome-wide motif activity scores.

[0690] 15-01-2609:01:04P7423PC00

[0691] Results

[0692] Transcription factors of the AP-1 family (e.g., Junb, Fosl2, Atf3) were broadly upregulated (Figure 4a), consistent with their role as early response genes activated by growth factors, cytokines, and cellular stressors (Vierbuchen et al., 2017). Upregulation was particularly prominent in astrocytes, ependymal cells, and microglia and correlated with the timing and magnitude of their transcriptional response (Figure 4a). Increased ChromVar motif activity for AP-1 factors was observed across glial cells, especially early after injury, aligning with transcriptional dynamics (Figure 4b-c).

[0693] Conclusion

[0694] AP-1 family transcription factors and their activity were upregulated across glial cells after injury.

[0695] Example 5 - Injury induced enhancers harbor active motifs for AP- 1 and glial lineage specific factors.

[0696] Aims

[0697] Given the broad upregulation of AP-1 transcription factors in glial cells, this study investigated their role in commissioning cell type-specific IRENs.

[0698] Materials and Methods

[0699] The same methods were employed as disclosed in the ‘materials and methods’ section of Example 3.

[0700] Results

[0701] To determine if injury- induced regulatory programs were controlled by AP-1 transcription factors, deep learning models were trained on injured and uninjured cell states to analyze the nucleotide composition of enhancers. The models identified the consensus AP-1 binding site 12-O-tetradecanoyl-phorbol-13-acetate response element (TGA[G / C]TCA; SEQ ID NO: 54 or SEQ ID NO: 103) as highly enriched in IREN sequences across all glial cells (Figure 5a-e).

[0702] 15-01-2609:01:04P7423PC00

[0703] Conclusion

[0704] Deep learning models identified AP-1 transcription factors as drivers of injury-induced enhancer commissioning in glial cells.

[0705] Example 6 - Deep learning identifies motifs for glial lineage specific factors and predicts their activity after injury

[0706] Aims

[0707] To investigate how a common set of injury-responsive transcription factors binding to the same consensus motif (AP-1) could lead to the activation of unique sets of enhancers in different cells.

[0708] Materials and Methods

[0709] The same methods were employed as disclosed in the ‘materials and methods’ section of Example 3.

[0710] Results

[0711] The regulatory syntax of injury-responsive, cell type-specific enhancers was investigated by analyzing motifs predicted to be active near AP-1 binding sites.

[0712] Regulatory elements containing AP-1 transcription factor binding sites, which matched the reference PFMs (Table 2), frequently co-occurred with motifs recognized by lineage-specific transcription factors at injury-responsive enhancers, which also matched their respective PFMs (Figure 6a and Table 1). Lineage-specific motifs included Nfi factors, Rora, and Sox9 for astrocytes, Rfx1 and Rfx2 for ependymal cells, Sox10 and Tcf4 for oligodendrocytes, and Elk1 and Cebpa for microglia (Figure 6a). These findings support that AP-1 factors and lineage-specific transcription factors cooperate to select distinct sets of injury-responsive enhancers in each cell type.

[0713] To further characterise the architecture of injury-responsive elements, the analysis focused on enhancers enriched in reactive astrocytes, a therapeutically relevant cell state common to multiple CNS pathologies (Escartin et al., 2021 ; Liddelow & Sofroniew, 2019). The model trained on chromatin accessibility data from reactive astrocytes correctly predicted state-specific enhancer accessibility and identified Jun / Junb and Sox9 binding sites as key features (Figure 6b-c). In contrast, models

[0714] 15-01-2609:01:04P7423PC00

[0715] trained on non-reactive astrocytes or other reactive cell types identified motifs linked to either lineage-specific or stimulus-induced factors, but not both (Figure 6b-c).

[0716] These results demonstrate that the deep learning models inferred enhancer accessibility across cell states and decoded the regulatory nucleotide composition of state-specific elements. Model interpretation further suggests that AP-1 factors recruit astrocyte-specific transcription factors to injury-responsive enhancers, thereby driving the reactive astrocyte program.

[0717] Conclusion

[0718] The cell type-specificity of IRENs in glial cells was governed by the cooperative activity of stimulus-induced AP-1 factors and lineage-specific transcription factors.

[0719] 15-01-2609:01:04Table 1. Position Frequency Matrix (PFM) for lineage-specific transcription factors. Each column (V1 to V14) corresponds to a specific position in a DNA binding site of 14 base pairs, indicating how frequently each nucleotide base (A, C, G, T) appears at that position.

[0720]

[0721] Table 1 (cont.). Position Frequency Matrix (PFM) for lineage-specific transcription factors.

[0722]

[0723]

[0724] Table 1 (cont.). Position Frequency Matrix (PFM) for lineage-specific transcription factors.

[0725]

[0726]

[0727] Table 1 (cont.). Position Frequency Matrix (PFM) for lineage-specific transcription factors.

[0728]

[0729]

[0730]

[0731]

[0732] Table 1 (cont.). Position Frequency Matrix (PFM) for lineage-specific transcription factors.

[0733]

[0734]

[0735]

[0736] Table 2. Position Frequency Matrix (PFM) for AP-1 transcription factors. Each column (V1 to V14) corresponds to a specific position in a DNA binding site of 14 basepairs, indicating how frequently each nuc eotide base (A, C, G, at that

[0737]

[0738] Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0739]

[0740]

[0741] Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0742]

[0743]

[0744] T 270 20 998 19 1 61 958 1 1 578Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0745]

[0746]

[0747] Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0748]

[0749]

[0750] T 275 14 999 10 0 58 934 0 0 583Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0751]

[0752]

[0753] Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0754]

[0755]

[0756] Table 2 (Cont.). Position Frequency Matrix (PFM) for AP-1 transcription factors

[0757]

[0758]

[0759]

[0760] P7423PC00

[0761] Example 7 - In vivo screening of injury responsive enhancers containing AP- 1 and glial lineage-specific motifs using systemic gene delivery vectors.

[0762] Aims

[0763] This study aimed to achieve selective genetic targeting of reactive astrocytes at the injury site using adeno-associated viruses (AAVs), a therapeutically relevant gene delivery vector.

[0764] Materials and Methods

[0765] In vivo enhancer reporter assay. Astrocyte-specific injury- induced enhancers were tested using a barcoded plasmid library cloned into the CN2249-rAAV-eHGT_451 m-minBglobin-SYFP2-WPRE3-BGHpA backbone (Addgene 164452). First, a library was generated using a 200 bp ultramer containing two 12 bp variable regions flanking a 20 bp constant region, cloned into a Xhol-digested backbone via NEBuilder Hi Fi DNA Assembly (NEB #E2621 L). After transformation into Endura Electrocompetent cells (Biosearch #60242-1), the barcoded plasmid library was extracted (QIAprep Plasmid Plus Midi Prep, QIAGEN).

[0766] Enhancers were then PCR-amplified from mouse genomic DNA with overhangs for assembly into the Mlul / Sacl-digested barcoded library via Gibson assembly (NEB #E2611 L). Successful constructs were transformed into NEB® Stable Competent E. coli cells (NEB #C3040H), and plasmids were recovered using QIAprep Spin Miniprep (QIAGEN).

[0767] The barcoded enhancer library was packaged into AAV-PHP.eB particles (AAVnergene), pooled, and delivered intravenously (2x1011vg) into mice with fluorescently labeled astrocytes (Cx30-Tom). Mice were subjected to spinal cord injury, and astrocytes were sampled at 3 dpi and in uninjured controls.

[0768] Enhancer activity was read out using Chromium Single Cell 3’ v3.1 (1 OX Genomics, PN-1000121). After cDNA amplification, barcoded transcripts were enriched via linear amplification (oEL197) and further amplified with oEL196, oEL195, and Partial R1. Amplicons were barcoded using Dual Index Plate TT SetA and sequenced on Illumina NextSeq 2000.

[0769] 15-01-2609:01:04P7423PC00

[0770] Sequencing reads were processed with CellRanger (v8.0.1) adapted for custom EYFP enhancer barcodes. Count matrices were analyzed with Seurat, filtering cells (500<counts<50,000), computing UMAP on the top 15 PCs, clustering at 0.5 resolution, and identifying astrocytes using canonical markers (Aldhl 11 ) fordownstream analysis, including barcode quantification.

[0771] Results

[0772] Nineteen candidate IRENs (566-1188 bp) enriched in reactive astrocytes were selected based on their association with differentially expressed genes and predicted state-specific accessibility (Figure 7a, 8a-b).

[0773] To test enhancer activity, an AAV-based reporter assay combined with single-cell RNA-sequencing was developed, enabling simultaneous in vivo screening of all candidate sequences. Previously described astrocyte-specific enhancers (Mich et al., 2023) served as controls. Spinal cord tissue was sampled from mice injected with the IREN AAV library and subjected to injury, as well as uninjured controls.

[0774] Consistent with injury-induced accessibility, EYFP expression was higher near the injury site compared to uninjured tissue. EYFP expression was also highly selective for astrocytes, demonstrating strong cell type specificity (Figure 7b-h).

[0775] Conclusion

[0776] This study validated the use of a single-cell-resolved in vivo enhancer reporter assay using systemic AAVs.

[0777] Example 8 - Additional characterisation of the in vivo validated injury responsive enhancers containing AP-1 and glial lineage-specific motifs for cell state targeting

[0778] Aims

[0779] To characterise the in vivo activity of selected IRENs using an in vivo reporter assay with single cell resolution.

[0780] Materials and Methods

[0781] The same methods were employed as disclosed in the ‘materials and methods’ section of Example 7.

[0782] 15-01-2609:01:04P7423PC00

[0783] Results

[0784] After confirming that some enhancers drove injury-responsive reporter expression in astrocytes, astrocytes were isolated, and single-cell RNA-seq with barcode detection was performed (Figure 8a). The model predicted that enhancer accessibility was driven by AP-1 transcription factor binding sites alongside astrocyte-specific factors like Sox9 (Figure 8b-c). Barcodes from 5 of 19 IRENs and 2 of 4 controls were detected, with all IRENs driving reporter expression exclusively in reactive astrocytes (Figure 8d), consistent with their accessibility patterns.

[0785] In contrast, a previously validated astrocyte-specific enhancer (Mich et al., 2023) labeled both reactive and non-reactive astrocytes (Figure 8f). This human-derived enhancer contained Nfib binding sites, identified as important for cell type specificity by the mouse-trained model .However, unlike the IRENs, it lacked AP-1 motifs, explaining its absence of injury-dependent activity. These findings suggest that regulatory principles for enhancer activity are conserved across species (Wray, 2007; Zemke et al., 2023).

[0786] Conclusion

[0787] Together, using a single-cell-resolved in vivo enhancer reporter assay, enhancer elements that can drive expression in a genetically defined cell state were identified.

[0788] Example 9 - Injury responsive enhancers direct expression in damage responsive glial cells after brain and spinal cord injury upon systemic delivery and are tuneable

[0789] Aims

[0790] To further study the capacity and tuneability of individual enhancers to drive different levels of expression in reactive astrocytes in preclinical models of CNS injury.

[0791] Materials and Methods

[0792] Spinal cord injury. The same methods were employed as disclosed in the ‘materials and methods’ section of Example 1.

[0793] 15-01-2609:01:04P7423PC00

[0794] Stab-wound injury: Animals were deeply anesthetized with isoflurane (4% induction, 2% maintenance) and placed on a heated pad. The head was secured in a three-point stereotactic frame, and a 5 mm incision exposed the skull. For unilateral injury, an opening was made at +2.5 mm medio-lateral and +1.0 mm rostral from Bregma. A 26G blunt needle attached to the stereotactic apparatus was lowered 0.7 mm below the dura and moved 1 mm rostro-caudally to extend the lesion. Animals recovered in a heated cage before returning to their home cages.

[0795] Retro-orbital injection: Mice were deeply anesthetized with isoflurane (4% at 400 ml / min) for intravenous injection. Using insulin syringes (30G, 0.5-in), 50 pl of virus diluted in saline was injected unilaterally into the retro-bulbar sinus after applying ophthalmic ointment to the eye. The needle was angled at 30° with the bevel facing upwards. Animals recovered in a heated cage before returning to their home cages.

[0796] Tissue preparation for immunostaining-. Mice were deeply anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) (Sigma). Vertebral columns were isolated, post-fixed overnight in 4% PFA at 4°C, and spinal cords were extracted, cleared of meninges, and cryoprotected in 30% sucrose at 4°C. Tissues were sectioned into 5 mm segments centered on the injury site, embedded in OCT, sliced into 20 pm coronal sections using a Cryostar NX70 (Thermo), and stored at -20°C. Brains from stab-wound-injured animals were post-fixed in 4% PFA, sectioned into 50 pm coronal sections using a vibratome, and processed for further analysis.

[0797] Immunostaining: Cryosections were equilibrated to room temperature, blocked for 1 hour in 10% donkey serum / 0.3% Triton X-100 in DPBS, and incubated overnight at 4°C with primary antibodies: chicken anti-GFP (1 :1000, Aves, GFP1010), rabbit anti-RFP (1 :1000, Rockland, 600-401-379), rabbit anti-Gfap (1 :500, Dako, Z0334), and goat anti-Sox9 (1 :250, R&D, AF3075). Sections were washed (3x10 min, DPBS), incubated with Alexa Fluor-conjugated secondary antibodies (1 :500, Thermo), and counterstained with DAPI (1 pg / ml, BD) during the final wash. Free-floating brain sections were stained similarly and mounted onto glass slides. Images were acquired on a Zeiss LSM 700 confocal microscope and processed using Fiji. Quantification: EYFP+ cells were manually counted in spinal cord sections spanning 5 mm rostral and 10 mm caudal to the injury site. Counts were normalized to the total Sox9+ cells, quantified automatically

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[0799] using Fiji's Analyze Particle tool after binarization. For the stab wound model, quantifications were performed on four sections spanning the injury site, with the contralateral hemisphere as a control.

[0800] Results

[0801] To assess reporter activity, IREN5 AAVs were injected intravenously into mice subjected to spinal cord injury, and reporter expression was analysed three days postinjury (Figure 9a). IREN5, linked to the pan-glial gene Cd44, showed activity specifically in reactive astrocytes near the lesion but not in distal regions (Figure 9b-e).

[0802] 83% of astrocytes at or near the lesion border were labeled (Figure 9f-g) , demonstrating IREN5’s ability to drive spatiotemporally precise gene expression in reactive astrocytes.

[0803] To test generalizability, IREN5 AAVs were injected into mice subjected to stab wound traumatic brain injury (TBI) in the motor cortex (Figure 9h). IREN5 drove EYFP expression in reactive astrocytes surrounding the injured cortex but not in astrocytes of the contralateral hemisphere or other brain regions (Figure 9i-j). Labelling was efficient, with reactive astrocytes along the lesion border showing 98% specificity (Figure 9k-l) . To test the tuneability of IREN5, we tested synthetic sequences that retained the motifcontaining IREN5-core in tandem repeats for predicted activity (Figure 9M & 9N; SEQ ID NO: 106). The 3xCore version (SEQ ID NO: 106), as predicted by the model, had higher activity after TBI than the wild type version as measured by EYFP reporter intensity (Figure 90)

[0804] Conclusion

[0805] Tuneable injury-responsive enhancer elements were identified that specifically targeted reactive astrocytes across the CNS using therapeutically relevant viral vectors delivered systemically.

[0806] Example 10 - Injury responsive enhancers, but not cell type specific elements, selectively label damage responsive cells after spinal cord injury

[0807] Aims

[0808] To compare the specificity of IRENs and cell type specific enhancers to target injury-responsive cells after spinal cord injury.

[0809] 15-01-2609:01:04P7423PC00

[0810] Materials and Methods

[0811] The same methods were employed as disclosed in the ‘materials and methods’ section of Example 9.

[0812] Results

[0813] In contrast to IREN5 (Figure 10A & 10B), consistent with the AAV library screen, the astrocyte-specific human enhancer (Mich et al., 2023) drove EYFP expression in astrocytes across the spinal cord (Figure 10C & 10D). Interestingly, the activity of this enhancer mildly decreased in the proximity of the lesion site (Figure 10C & 10D), contrary to the pattern observed for the injury responsive enhancer element (Figure 10A-B & 9F).

[0814] Conclusion

[0815] Injury responsive enhancers can direct gene expression to specific cell states within damaged tissues even when systemically delivered.

[0816] Example 11 - Identification of the sequence determinants of IREN5 activity and specificity in vivo

[0817] Aims

[0818] To identify the minimal sequence components underlying IREN5 activity and specificity.

[0819] Materials and Methods

[0820] The same methods were employed as disclosed in the ‘materials and methods’ section of Example 7.

[0821] Results

[0822] Using the interpretability of the models to establish sequence to function relationships through in silico mutagenesis (Figure 11 A), an architecture composed of tandem AP-1 motifs along with motifs for the lineage specific factors Sox9 and Nfi was predicted (Figure 11 D) as the minimal functional units of IREN5. Then the accessibility when ablating either AP-1 motifs, lineage specific motifs or of a synthetic sequence that shuffles the original sequence but preserves the learned motif syntax was predicted (Figure 11 A). Ablation of AP-1 led to a drastic reduction in predicted accessibility in reactive astrocytes (despite preserving >97% of the enhancer sequence), while ablation of Sox9 and Nfi motifs led to a more gradual reduction. The shuffled synthetic version had a predicted accessibility that recapitulated the original accessibility better than the

[0823] 15-01-2609:01:04P7423PC00

[0824] individual single motif mutants despite having mutations in >90% of the original base pairs (Figure 11 A & 11 E). Next the model predictions in vivo by delivering synthetic enhancer mutant AAVs systemically prior to injury and measure reporter signal three days post spinal cord injury was validated. In agreement with the model, ablation of the injury-responsive AP1 module led to a drastic reduction in reporter activity, demonstrating a strong AP-1 dependency on IREN5 commissioning (Figure 11B, C & F). In turn, ablation of the Sox9 and Nfi sites led to a milder decrease in reactive astrocyte labeling but was accompanied by a drastic drop in specificity (Figure 11 B, C & F), supporting the role for the cooperative binding with AP-1 in cell type enhancer selection. Interestingly, the synthetic version, best recapitulated reactive astrocyte targeting efficiency and specificity (Figure 11 B, C & F).

[0825] Conclusion

[0826] IREN5 activity and cell type specificity were determined by a cooperative enhancer architecture, in which injury-induced AP-1 transcription factor binding sites provide activation, while lineage-specific transcription factor binding sites, including Sox9 and Nfi, confer astrocyte selectivity. Importantly, enhancer function was governed by motif composition and arrangement rather than primary nucleotide sequence, supporting the use of synthetic and variant enhancer sequences that preserve the cooperative syntax.

[0827] Example 12 - Conservation of damage responsive enhancers in humans and across diseases

[0828] Aims

[0829] To study whether the sequence architecture identified in mouse is conserved in activity and specificity in human cells from Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) samples.

[0830] Materials and Methods

[0831] Mouse IREN5 sequence was used as a candidate to explore conservation. The human sequence matching mouse IREN5 sequence was identified using liftover tool (UCSC genome browser) as hg38dna range=chr11 :35166522-35167340 (SEQ ID NO: 105). Single cell ATAC-seq datasets from public databases were used to explore the accessibility of this sequence in AD and ALS (Gabitto etal.', Li etal. 2023). ATAC-seq tracks were visualized as described for example 2. Machine learning models on

[0832] 15-01-2609:01:04P7423PC00

[0833] astrocytes and reactive astrocytes were generated and interpreted following the methods described for examples 2 and 3.

[0834] Results

[0835] Consistent with mouse I EN5 activity in reactive astrocytes, human I EN5 (SEQ ID NO: 105) showed disease-associated activity in astrocytes in cells from C9 ALS patients as well as in reactive astrocytes from AD patients (Figure 12A). Machine learning models trained on reactive astrocytes from AD patients and subsequent model interpretation identified binding sites for AP-1 factors (Fosb, Junb) as well as binding sites for lineagespecific factors (Nfi, Sox9, Rfx) in this cell state, indicating a high degree of conservation in the regulation of this reactive cell state in humans (Figure 12B). Interpretation of the sequence determinants of human IREN5 activity identify a conserved AP-1 binding site as well a SOX9 binding site at its core (Figure 12C).

[0836] Conclusion

[0837] Human reactive astrocytes across diseases share the same core sequence architecture, including sequence determinants of activity and specificity as identified in mouse, confirming conservation of the injury-responsive and cell type-specific enhancer determinants.

[0838] Sequence overview

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[0924] Gabitto Ml, Travaglini KJ, Rachleff VM, Kaplan ES, Long B, Ariza J, Ding Y, Mahoney JT, Dee N, Goldy J, Melief EJ, Agrawal A, Kana O, Zhen X, Barlow ST, Brouner K, Campos J, Campos J, Carr AJ, Casper T, Chakrabarty R, Clark M, Cool J, Dailey R, Darvas M, Ding SL, Dolbeare T, Egdorf T, Esposito L, Ferrer R, Fleckenstein LE, Gala R, Gary A, Gelfand E, Gloe J, Guilford N, Guzman J, Hirschstein D, Ho W, Hupp M, Jarsky T, Johansen N, Kalmbach BE, Keene LM, Khawand S, Kilgore MD, Kirkland A, Kunst M, Lee BR, Leytze M, Mac Donald CL, Malone J, Maltzer Z, Martin N, McCue R, McMillen D, Mena G, Meyerdierks E, Meyers KP, Mollenkopf T, Montine M, Nolan AL, Nyhus JK, Olsen PA, Pacleb M, Pagan CM, Pena N, Pham T, Pom CA, Postupna N, Rimorin C, Ruiz A, Saldi GA, Schantz AM, Shapovalova NV, Sorensen SA, Staats B, Sullivan M, Sunkin SM, Thompson C, Tieu M, Ting JT, Torkelson A, Tran T, Valera Cuevas NJ, Walling-Bell S, Wang MQ, Waters J, Wilson AM, Xiao M, Haynor D, Gatto NM, Jayadev S, Mufti S, Ng L, Mukherjee S, Crane PK, Latimer CS, Levi BP, Smith KA, Close JL, Miller JA, Hodge RD, Larson EB, Grabowski TJ, Hawrylycz M, Keene CD, Lein ES. Integrated multimodal cell atlas of Alzheimer's disease. Nat Neurosci. 2024 Dec;27(12):2366-2383. doi: 10.1038 / s41593-024-01774-5. Epub 2024 Oct 14. PMID: 39402379; PMCID: PMC11614693.

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[0926] 15-01-2609:01:04

Claims

P7423PC00Claims1. An isolated nucleic acid construct comprising:a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; andb. an encoding sequence operably linked to the enhancer element, wherein the selective injury-responsive enhancer element is configured to drive expression of the encoding sequence in a selected cell type following an injury, and comprises:i. at least one injury-responsive motif comprising a consensus sequence recognised by at least one injury-responsive transcription factor activated by injury-induced signalling pathways, which mediates transcriptional activation in response to cellular or tissue stress and / or damage; andii. at least one lineage-specific motif comprising a consensus sequence recognised by at least one lineage-specific transcription factor that regulates cell-type identity, which restricts expression to a defined lineage,wherein the selective injury-responsive enhancer element provides combinatorial specificity through cooperative activity of said at least one injury- responsive motif and said at least one lineage-specific motif.

2. An artificial expression construct comprising:a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; andb. an encoding sequence operably linked to the enhancer element, wherein the selective injury-responsive enhancer element is configured to drive expression of the encoding sequence in a selected cell type following an injury, and comprises:i. at least one injury-responsive motif comprising a consensus sequence recognised by at least one injury-responsive transcription factor activated by injury-induced signalling pathways, which mediates transcriptional activation in response to cellular or tissue stress and / or damage; andii. at least one lineage-specific motif comprising a consensus sequence recognised by at least one lineage-specific transcription factor that15-01-2609:01:04P7423PC00regulates cell-type identity, which restricts expression to a defined lineage,wherein the selective injury-responsive enhancer element provides combinatorial specificity through cooperative activity of said at least one injury- responsive motif and said at least one lineage-specific motif.

3. The isolated nucleic acid molecule or artificial expression construct according to any of the previous claims, wherein the at least one injury-responsive motif comprises at least one binding site for at least one injury-responsive transcription factor activated by injury-induced signalling pathways, wherein said injury-responsive transcription factor is selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

4. The isolated nucleic acid molecule or artificial expression construct according to any of the previous claims, wherein the at least one lineage-specific motif comprises at least one binding site for at least one lineage-specific transcription factor that regulates cell-type identity or differentiation, wherein said lineagespecific transcription factor is selected from the group comprising or consisting of; SOX9, SOX2, NFI, NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC, SOX4, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1, IRF2, IRF3, and IRF4.

5. The isolated nucleic acid molecule or artificial expression construct according to any of the previous claims, comprising:a. a selective injury-responsive enhancer element that is activated in at least one selected cell type; andb. an encoding sequence operably linked to the enhancer element, wherein the encoding sequence directs the expression of a DNA sequence product in the selected cell type in which the enhancer element is activated following injury,wherein the selective injury-responsive enhancer element comprises: at least one binding site for a lineage-specific transcription factor selected from the group comprising or consisting of; SOX9, SOX2, NFI, NFIA, NFIB, NFIC, NFIX, RFX1, RFX2, RFX3, RFX4, RFX7, RORA, RORB, RORC,15-01-2609:01:04118P7423PC00SOX2, SOX4, SOX9, SOX10, SPI1 , ELK1, ELK4, CEBPA, CEBPB, CEBPC, CEBPG, IRF1 , IRF2, IRF3, and IRF4, andat least one binding site for an injury-responsive transcription factor selected from the group comprising or consisting of; JUN, JUNB, JUND, FOS, FOSB, FOSL1 , FOSL2, ATF1 , ATF3, ATF4, ATF5, BACH1 , BACH2, JDP2, BATF, MAFF, MAFG, and MAFK.

6. The artificial expression construct according to any one of the preceding claims, further comprising a minimal promoter.

7. The artificial expression construct according to any one of the preceding claims, further comprising a reporter transgene.

8. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the DNA sequence product is an encoded polypeptide.

9. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the at least one lineage-specific motif is as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and wherein the enhancer element comprises multiple instances of the same at least one lineage-specific motif.

10. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding items, wherein the at least one injury-responsive motif has the sequence TGAXIX2X3X4X5(SEQ ID NO: 104),wherein:Xi is G, C or T;X2is T or G;X3is A, C or T;X4is A, C or G; and;X5is absent, A or G.

11. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the at least one injury-responsive motif is15-01-2609:01:04119P7423PC00as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103, and wherein the selective injury-responsive enhancer element comprises multiple instances of the same at least one injury-responsive motif .

12. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein:a. the at least one lineage-specific motif comprises a sequence as set forth in any one of SEQ ID NO: 6-30 and / or SEQ ID NO: 60-88, and b. the at least one injury-responsive motif comprises a sequence as set forth in any one of SEQ ID NO: 4-5 and / or SEQ ID NO: 54-59 and / or SEQ ID NO: 103.

13. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-53 or functional homologues thereof sharing at least 90% sequence identity therewith.

14. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the selective injury-responsive enhancer element comprises at least one of sequences as set forth in SEQ ID NO: 31-35 or SEQ ID NO: 105-106 or functional homologues thereof sharing at least 90% sequence identity therewith.

15. The isolated nucleic acid molecule or artificial expression construct according to any one of the preceding claims, wherein the selective injury-responsive enhancer element comprises;a. the at least one binding site for at least one injury-responsive transcription factor selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, BACH1, BACH2, JDP2, MAFF, MAFG, andb. the at least one binding site for the lineage-specific transcription factor SOX9, SOX2, NFIA, NFIB, NFIC, NFIX, RFX2, RFX3, RFX4, RFX7, RORA, RORB.15-01-2609:01:04120P7423PC0016. The isolated nucleic acid molecule or artificial expression construct according to any one of the preceding claims, wherein:a. the at least one lineage-specific motif is selected from the group consisting of SEQ ID NO: 10, and SEQ ID NO: 17, andb. the at least one injury-responsive motif is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5.

17. The isolated nucleic acid molecule or artificial expression construct according to any one of 1-16, wherein the selective injury-responsive enhancer element comprises;a. the at least one binding site for at least one injury-responsive transcription factor selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and b. the at least one binding site for the lineage-specific transcription factor SOX9.

18. The isolated nucleic acid molecule or artificial expression according to any one of claims 1-14, wherein:a. the at least one lineage-specific motif is selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, andb. the at least one injury-responsive motif is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 32 or a functional homologue thereof sharing at least 90% sequence identify therewith.

19. The isolated nucleic acid molecule or artificial expression construct according to any one of 1-14 and 18, wherein the selective injury-responsive enhancer element comprises;a. the at least one binding site for at least one injury-responsive transcription factor selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and15-01-2609:01:04121P7423PC00b. the at least one binding site for the lineage-specific transcription factor SOX2.

20. The isolated nucleic acid molecule or artificial expression construct according to any one of claims 1-14, wherein:a. the at least one lineage-specific motif consists of SEQ ID NO: 17, and b. the at least one injury-responsive motif is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 33 or a functional homologue thereof sharing at least 90% sequence identify therewith.

21. The isolated nucleic acid molecule or artificial expression construct according to any one of claims 1-14, wherein:a. the at least one lineage-specific motif consists of SEQ ID NO: 17, and b. the at least one injury-responsive motif is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 34 or a functional homologue thereof sharing at least 90% sequence identify therewith.

22. The isolated nucleic acid molecule or artificial expression construct according to any one of 1-14 and 20-21 , wherein the selective injury-responsive enhancer element comprises;a. the at least one binding site for at least one injury-responsive transcription factor selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and b. the at least one binding site for the lineage-specific transcription factor NFI.

23. The isolated nucleic acid molecule or artificial expression construct according to any one of claims 1-14, wherein:a. the at least one injury-responsive motif is selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 17, and15-01-2609:01:04122P7423PC00b. the at least one injury-responsive motif is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:5,wherein the selective injury-responsive enhancer element comprises SEQ ID NO: 35 or a functional homologue thereof sharing at least 90% sequence identify therewith.

24. The isolated nucleic acid molecule or artificial expression construct according to any one of 1-14 and 23, wherein the selective injury-responsive enhancer element comprises;a. the at least one binding site for at least one injury-responsive transcription factor selected from the AP-1 family, including JUN, JUNB, JUND, FOS, FOSB, FOSL1, FOSL2, ATF1, ATF3, ATF4, ATF5, BACH1, BACH2, JDP2, BATF, MAFF, MAFG, and MAFK, and b. the at least one binding site for the lineage-specific transcription factor is SOX9 and / or NFI.

25. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the selected cell type comprises astrocytes, oligodendrocytes, microglia, oligodendrocyte precursor cells (OPCs), ependymal cells, choroid plexus cells, and / or border-associated macrophages.

26. The artificial expression construct according to any of claims 2-25, wherein the construct is incorporated into a vector selected from a viral vector, a chemically synthesized particle, or a lipid nanoparticle.

27. The isolated nucleic acid molecule or artificial expression construct according to any of the preceding claims, wherein the selective injury-responsive enhancer element comprises at least 200 nucleic acids and at the most 1600 nucleic acids.

28. A pharmaceutical composition comprising the isolated nucleic acid construct or artificial expression construct according to any of the preceding claims and a pharmaceutically acceptable carrier.15-01-2609:01:04123P7423PC0029. Use of the isolated nucleic acid molecule, artificial expression construct or pharmaceutical composition according to any of the preceding claims for the treatment and / or prevention of diseases or disorders related to the central nervous system.

30. A cell comprising the isolated nucleic acid construct or artificial expression construct according to any of the preceding claims, wherein the selected cell type comprises astrocytes, oligodendrocytes, microglia, oligodendrocyte precursor cells (OPCs), ependymal cells, choroid plexus cells, and / or border- associated macrophages.

31. A method for selectively expressing a heterologous gene within a population of selected injury-responsive cells, comprising:contacting said cells with the artificial expression construct or pharmaceutical composition according to any of one of the preceding claims, wherein the heterologous gene is expressed selectively in the injury-responsive cells.

32. A method for designing a selective injury-responsive enhancer element comprising a cooperative syntax, comprising:a. identifying at least one injury-responsive motif activated by at least one injury-responsive transcription factor, wherein the at least one injury- responsive motif is according to any one of claims 10-12 and 16-24 and wherein the at least one injury-responsive transcription factor is according to any one of claims 1-3 and 5,b. identifying at least one lineage-specific motif recognised by at least one transcription factor that regulate cell-type identity, wherein the at least one lineage-specific motif is according to any one of claims 9, 12, and 16-24 and wherein the at least one lineage-specific transcription factor is according to any one of claims 1-2 and 4-5; andc. combining said motifs in a configuration that enables cooperative transcriptional activation in selected injury-responsive cells.15-01-2609:01:04

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