Recombinant nucleic acids containing alphaherpesvirus promoter sequences

Abstract

To provide recombinant nucleic acids containing alphaherpesvirus promoter sequences.SOLUTION: The present invention provides a recombinant vector for expression of a target gene in a host cell, comprising: a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for the expression of the target gene in the host cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 801,524, filed February 5, 2019, and U.S. Provisional Application No. 62 / 950,848, filed December 19, 2019, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] background Viruses infect their hosts and introduce their genetic material into the host's cells as part of the viral replication cycle. This genetic material contains the basic "instructions" for producing more copies of these viruses by hijacking the body's normal production mechanisms to serve the viral needs. The host cells execute these instructions to produce more copies of the virus, and more and more of the host's cells become infected. In this way, viruses can be used as vehicles to deliver genes that may provide therapeutic benefits within cells. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ogden, PJ, ED Kelsic, S. Sinai and GM Church (2019). Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design. Science 366(6469): 1139-1143 Summary of the Invention [Problem to be solved by the invention]

[0004] It is an object of the present invention to provide a recombinant nucleic acid comprising an alphaherpesvirus promoter sequence. [Means for solving the problem]

[0005] overview In one aspect, the present invention provides a recombinant vector for expressing a target gene in a host cell, comprising: a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for expression of the target gene in the host cell.

[0006] In another aspect, the present invention provides a recombinant vector for expressing a target gene in a host cell, comprising: a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for expression of the target gene in the host cell.

[0007] In another aspect, the present invention provides a recombinant vector for expressing a target gene in a host cell, comprising: a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for expression of the target gene in the host cell.

[0008] In some embodiments, the at least one non-promoter regulatory element comprises an enhancer element. In some embodiments, the at least one non-promoter regulatory element comprises an inducer element.

[0009] In some embodiments, the recombinant vector further comprises a selectable marker element.

[0010] In some embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0011] In some embodiments, the vector is a non-viral vector.

[0012] In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a neuronal cell. In some embodiments, the host cell is a non-neuronal cell (e.g., a liver cell, a kidney cell, a retinal cell).

[0013] In another aspect, the present invention provides a host cell comprising a recombinant vector described herein.

[0014] In one aspect, the present invention provides a recombinant polynucleotide molecule comprising a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, wherein the nucleic acid is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0015] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, wherein the nucleic acid is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0016] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 9. In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, wherein the nucleic acid is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9.

[0017] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0018] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0019] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9.

[0020] In another aspect, the present invention provides a method for expressing a target gene in a host cell comprising contacting a host cell with a recombinant polynucleotide molecule such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding a target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and wherein expression of the target gene occurs in the host cell.

[0021] In another aspect, the present invention provides a method for expressing a target gene in a host cell comprising contacting a host cell with a recombinant polynucleotide molecule such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding a target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, and wherein expression of the target gene occurs in the host cell.

[0022] In another aspect, the present invention provides a method for expressing a target gene in a host cell, comprising contacting a host cell with a recombinant polynucleotide molecule such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding a target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9, and wherein expression of the target gene occurs in the host cell.

[0023] In some embodiments, the recombinant polynucleotide molecule is introduced into the host cell by transduction upon contacting the host cell with the recombinant polynucleotide, hi other embodiments, the recombinant polynucleotide molecule is introduced into the host cell by transfection upon contacting the host cell with the recombinant polynucleotide.

[0024] The properties of the LAP sequence disclosed herein include: i) similar size when compared with similar promoters, such as EF1α, CAG, TH, CaMKIIa in particular (this is an advantage for AAV vectors with limited gene payload); ii) long-term expression with low susceptibility to transcriptional repression. The LAP sequence disclosed herein achieves long-term chronic transcription of transgenes. This is particularly useful for gene therapy, where therapeutic transgenes may need to be expressed during the lifespan of the patient / host.

[0025] That is, the gist of the present invention relates to the following. Item 1 a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; a cloning site for insertion of a nucleic acid encoding a target gene; and At least one non-promoter regulatory element required for expression of the target gene in a host cell. A recombinant vector for expressing a target gene in a host cell, comprising: Effect of the Invention

[0026] The present invention provides a recombinant nucleic acid comprising an alphaherpesvirus promoter sequence. [Brief description of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] The foregoing will become apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views, and in which the drawings are not necessarily to scale, emphasis instead being placed on illustrating the embodiments. [Figure 1]Figures 1A-1D show the characteristics of the PRV latency-associated transcript promoter (LAP). Figure 1A shows the complete nucleotide sequence of the 902 base pairs (bp) PRV LAP, as well as the 498 bp sub-region LAP1 (bold and underlined), the 404 bp LAP2 (underlined), and the 880 bp LAP1_2. LAP1_2 contains most of the LAP1 and LAP2 sequences but lacks the first 22 nucleotides of LAP1. Black boxes indicate consensus sequences for transcription factors (TFs), including GC box: specificity proteins 1 and 3 (Sp1 and Sp3); CCAAT box: nuclear factor Y (NF-Y); and TATA box: TATA binding protein (TBP). Colored boxes indicate the coordinates for the binding motif sites of TFs: 1, green: SRY-box 10 (SOX10); 2, red: cAMP response element binding protein (CREB); 3, blue: CCCTC-binding factor (CTCF); 4, brown: oligodendrocyte transcription factor 2 (Olig2); 5, pink: signal transducer and activator of transcription (STAT1). Figure 1B shows the plasmid maps of four AAVs designed to transcribe mCherry fluorescent reporters from the LAP1, LAP2, LAP1_2 and EF1α promoters. The 609 bp WPRE is a woodchuck hepatitis virus posttranscriptional enhancer element. All AAVs contain a 479 bp human growth hormone (hGH polyA) polyadenylation sequence, and adjacent AAV2 inverted terminal repeats (ITRs) of 141 bp each. The vectors were packaged into AAV-PhP.eB serotype capsids. The total size of the enhancer-promoter element and promoter sequences is AAV-LAP1: 1.87 Kb; AAV-LAP2: 1.77 Kb; AAV-LAP1_2: 2.25 Kb; and AAV-EF1α: 2.63 Kb. Figure 1C shows the four AAVs used to transduce primary cultures of rat SCG neurons and quantify mCherry expression over a 90-day time-lapse.The relative fluorescence intensity of mCherry expression was measured at 3, 5, 7, 9, 11, 14, 17, 21, 24, 28, 31, 34, 38, 41, 45, 49, 52, 59, 67, 73, 82 and 90 days post-infection (dpi) using 3x1011 AAV genomes. Scale bar = 100 µm. Data are expressed as mean ± SEM; n = 3 SCG culture dishes per group. Figure 1D shows AAV-driven mCherry expression 28 days after AAV transduction with LAP1-mCherry, LAP2-mCherry, LAP1_2-mCherry or EF1α-mCherry in SCG cell bodies. [Diagram 2] Figures 2A-2P show stable and long-term LAP-mediated transgene expression. Figure 2A is a schematic of the systemic route of AAV administration and CNS tissue processing. Intravenous administration of AAV vectors was performed by unilateral injection into the mouse retro-orbital sinuses (4x1011vg / mouse). Brains and spinal cords were harvested at 30 dpi and 190 dpi. The right hemisphere of the brain was processed for iDISCO+ tissue clearing and light sheet microscopy analysis. The left hemisphere of the brain was sectioned sagittally at 50 μm for immunofluorescence and confocal microscopy analysis. The spinal cord was sectioned transversely at 20 μm for immunofluorescence and confocal microscopy analysis. Figures 2B-2P quantify the density of mCherry-positive cells per mm3 across different brain regions in iDISCO+ cleared tissue samples at 30 dpi and 190 dpi. Data are expressed as mean ± SEM; n=2 animals per group, 5-10 500 um sections per animal. Data were normalized to those of vehicle-injected control animals. Significance was determined by Student's t-test (when comparing only two groups) or one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (when comparing more than two groups). p-values ​​of <0.05 were statistically significant (*p<0.033; **p<0.002; ***p<0.001). [Diagram 3]Figures 3A-3J4 show that all three AAV-LAP sequences drive widespread and long-term transgene expression throughput in the brain following retro-orbital injection. Representative immunofluorescence images of sagittal sections show whole-brain distribution of anti-mCherry staining (green) for AAV-LAP1 (Figure 3A), AAV-LAP2 (Figure 3B), AAV-LAP1_2 (Figure 3C) and AAV-EF1α (Figure 3D) at 190 dpi. Cx, cortex; Hip, hippocampus; MRN, midbrain reticular nucleus; Cb, cerebellum; Thal, thalamus; Pn, pons; Hypo, hypothalamus; Str, striatum; OB, olfactory bulb. Scale bar = 1 mm. Representative confocal images show anti-mCherry signal (green) in the cortex (Figure 3E), dentate gyrus (Figure 3F), striatum (Figure 3G) and cerebellum (Figure 3H) at 30 dpi and 190 dpi. All images are stacked confocal sections. Scale bar = 100 μm. Figures 3I1-3I4 quantify the indirect fluorescence intensity of anti-mCherry signals driven by AAV-LAP sequences and AAV-EF1α in the cortex, dentate gyrus, striatum and cerebellum at 30 dpi and 190 dpi. Figures 3J1-3J4 quantify the number of cells expressing mCherry signals per pixel2 by immunohistochemistry (IHC) in the cortex, dentate gyrus, striatum and cerebellum at 190 dpi. Data are presented as mean ± SEM; n = 2 animals and 6 tissue sections per animal. Data were normalized to those of vehicle-injected control animals. Significance was determined by Student's t-test (when comparing only two groups) or one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (when comparing more than two groups). A p-value of <0.05 was statistically significant (*p<0.033; **p<0.002; ***p<0.001). [Figure 4]Figures 4A-4E4 show that LAP2 drives stable and long-term transgene expression in the brain. Representative confocal images show native mCherry fluorescence (red) for AAV-LAP1, AAV-LAP2, AAV-LAP1_2 and AAV-EF1α in the cortex (Figure 4A), dentate gyrus (Figure 4B), striatum (Figure 4C) and cerebellum (Figure 4D) at 30 dpi and 190 dpi. All images are overlapping confocal sections. Scale bar = 100 μm. Figures 4E1-4E4 quantify the direct fluorescence intensity of native mCherry signals driven by AAV-LAP sequences and AAV-EF1α at 30 dpi and 190 dpi shown in the cortex, dentate gyrus, striatum and cerebellum. Data are presented as mean ± SEM; n = 2 animals and 6 tissue sections per animal. Data were normalized to those of vehicle-injected control animals. Significance was determined by Student's t test (when comparing only two groups) or one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (when comparing more than two groups). A p value of <0.05 was considered statistically significant (*p<0.033; **p<0.002; ***p<0.001). [Diagram 5] Figures 5A-5E show that AAV-LAP2 drives long-term transgene transcription in the CNS. The presence of mCherry mRNA was verified by FISH in brain tissue at 190 dpi using a riboprobe specific for mCherry (green). Nuclei were counterstained with DAPI (blue). Figures 5A-5E show 20 μm sagittal brain slices for AAV-LAP2 or AAV-EF1α in the cortex, dentate gyrus, striatum, cerebellum and olfactory bulb, respectively. Panels 2 and 4 show higher magnification images of the indicated areas (squares) in panels 1 and 3, respectively. Images are overlaid confocal sections. Scale bar = 100 μm. [Figure 6]Figures 6A-6F show that AAV-LAP transgene expression is preferential in neurons and not oligodendrocytes. Representative confocal images of AAV-mediated mCherry expression (red) in neurons (Figures 6A and 6B; green labeled for pan-neuronal marker NeuN) or oligodendrocytes (Figures 6C and 6D; green labeled for oligodendrocyte marker Olig2) in the cortex or dentate gyrus at 30 dpi. Cells were counterstained with DAPI (blue). NeuN signal may colocalize with neuronal nuclei and cytoplasm, while staining for Olig2 signal is mostly nuclear. Arrows indicate colabeling between cell markers and mCherry. Scale bar = 100 μm. Figures 6E and 6F quantify the percentage of mCherry-labeled cells corresponding to neurons (NeuN positive) or oligodendrocytes (Olig2 positive) in the cortex (Cx) or dentate gyrus (DG) for the respective promoters. Images are overlaid confocal sections. Data are expressed as mean±SEM; n=2 animals and 6 tissue sections per animal. [Figure 7] Figures 7A-7D show that no AAV-LAP transgene expression is detectable in microglia and astrocytes. Representative confocal immunofluorescence images of anti-mCherry signal (red), microglia (Figures 7A and 7B: green, labeled as Iba1 positive), and astrocytes (Figures 7C and 7D: green, labeled as S100 positive) in the cortex and dentate gyrus at 30 dpi. Absence of co-labeling between mCherry in astrocytes and microglia is indicated by a star (*). Cells were counterstained with DAPI (blue). Images are overlaid confocal sections. Scale bar = 100 µm. [Figure 8]8A-8F show that LAP sequences drive widespread and long-term transgene expression in the spinal cord. Spinal cords (lumbar region) were sectioned in a transverse fashion at 20 μm. Representative confocal images at 190 dpi of native AAV-mediated mCherry expression (red, panels 1 and 4), pan-neuronal marker NeuroTrace (green, panels 2 and 5) and combined signal (yellow, panels 3 and 6) for AAV-LAP1, AAV-LAP2, AAV-LAP1_2 and AAV-EF1α. Images are overlaid confocal sections. DH: dorsal horn; VH ventral horn. Higher magnification images are shown in panels 4-6. Scale bar = 100 μm. [Figure 9] 9A-9C show that LAP sequences drive efficient transgene expression in porcine kidney cells. Representative images of PK15 cells 48 hours after transfection of reporter plasmids containing mCherry driven by LAP1 (FIG. 9A), LAP2 (FIG. 9B) or LAP1_2 (FIG. 9C). Panel 1: bright field; Panel 2: fluorescence; Panel 3: merge. Scale bar=50 μm. [Figure 10] Figures 10A and 10B show that LAP2 drives efficient transgene expression in human kidney cells. Representative images of HEK-293 cells 48 hours after transfection of a reporter plasmid containing mCherry driven by LAP2 (Figure 10A) or the EF1α-promoter (Figure 10B). Panel 1: bright field; Panel 2: fluorescence; Panel 3: merged. Scale bar = 50 μm. [Figure 11] Figures 11A and 11B show that LAP2 drives efficient transgene expression in human liver cells. Representative images of HepG2 cells 48 hours after transfection of a reporter plasmid containing mCherry driven by LAP2 (Figure 11A) or the EF1α-promoter (Figure 11B). Panel 1: bright field; Panel 2: fluorescence; Panel 3: merged. Scale bar = 50 μm. [Figure 12]Figures 12A1-12B4 show that LAP2 drives long-term transgene expression through the liver. Intravenous administration of AAV-LAP2 (Figures 12A1-12A4) or AAV-EF1α-vectors (Figures 12B1-12B4) was performed by unilateral injection into the mouse retro-orbital sinus (4x1011vg / mouse). Livers were harvested at 190 dpi and sectioned longitudinally at 20 μm for IHC and confocal microscopy analysis. Representative confocal images of native mCherry fluorescence (Figures 12A1 and 12B1: red), anti-mCherry immunostaining (Figures 12A2 and 12B2: green), DAPI nuclear counterstaining (Figures 12A3 and 12B3: blue) and combined immunofluorescence and DAPI images (Figures 12A4 and 12B4). CV: central vein. Scale bar = 75 μm. [Figure 13] Figures 13A1-13B4 show that LAP2 drives long-term transgene expression in mouse kidney. Intravenous administration of AAV-LAP2 (Figures 13A1-13A4) or AAV-EF1α-vector (Figures 13B1-13B4) was performed by unilateral injection into the mouse retro-orbital sinus (4x1011vg / mouse). Kidneys were harvested at 190 dpi and sectioned longitudinally at 20 μm for IHC and confocal microscopy analysis. Representative confocal images show native mCherry fluorescence (Figures 13A1 and 13B1: red), anti-mCherry immunostaining (Figures 13A2 and 13B2: green), DAPI nuclear counterstaining (Figures 13A3 and 13B3: blue) and combined immunofluorescence and DAPI images (Figures 13A4 and 13B4). AV: arcuate vasculature. Scale bar = 75 μm. [Figure 14]Figures 14A1-14B4 show that LAP2 drives long-term transgene expression in mouse retina. Intravenous administration of AAV-LAP2 (Figures 14A1-14A4) or AAV-EF1α-vector (Figures 14B1-14B4) was performed by unilateral injection into the mouse retro-orbital sinus (4x1011vg / mouse). Retinas were harvested at 190 dpi and cryosectioned transversely at 20 µm for IHC and confocal microscopy analysis. Representative confocal images show native mCherry fluorescence (Figures 14A1 and 14B1: red), anti-mCherry immunostaining (Figures 14A2 and 14B2: green), DAPI nuclear counterstaining (Figures 14A3 and 14B3: blue) and combined immunofluorescence and DAPI images (Figures 14A4 and 14B4). GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Scale bar = 75 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description A description of exemplary embodiments follows.

[0030] Some aspects of the present invention are described below with reference to examples for illustrative purposes only. It should be understood that many specific details, relationships and methods are described to provide a full understanding of the present invention. However, those skilled in the art will easily understand that the present invention can be practiced without one or more of the specific details, or can be practiced with other methods, protocols, reagents, cell lines and animals. The present invention is not limited to the illustrated order of acts or events, since some acts may occur in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts, steps or events are required to carry out the method according to the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly used using conventional methods by those skilled in the art.

[0031] Unless otherwise defined, all terms, expressions and other scientific or technical terms used in the art are intended to have the meanings that are generally understood by those skilled in the art to which the present invention belongs.In some cases, terms with generally understood meanings are defined herein for clarity and / or easy reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is generally understood in the art.It is further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meanings in the relevant technical field and / or as being defined otherwise herein.

[0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the indefinite articles "a," "an," and "the" are to be understood to include plural references unless the context clearly indicates otherwise.

[0033] As used herein, a "cloning site" refers to a short segment of nucleotides in a vector that contains one or more unique restriction sites that allow for the insertion of a nucleotide "target gene" or "gene of interest" into the vector.

[0034] As used herein, "promoter" refers to a region of DNA to which RNA polymerase binds and initiates transcription (eg, of a gene).

[0035] As used herein, "non-promoter regulatory element" refers to a non-promoter sequence (s) of a nucleic acid molecule that can increase or decrease the expression of a particular gene in a recombinant vector. Such non-promoter regulatory elements include, but are not limited to, enhancer elements, inducer elements, silencer elements, 5' untranslated region (UTR), 3'UTR, terminator elements, CAAT boxes, CCAAT boxes, Pribnow boxes, SECIS elements, polyadenylation signals, A boxes, Z boxes, C boxes, E boxes, G boxes and Cis regulatory elements (CRE).

[0036] As used herein, the phrase "operably linked" means that a nucleic acid is positioned within a recombinant polynucleotide, such as a vector, in a manner that permits expression of the nucleic acid under the control of elements (e.g., a promoter) to which it is linked.

[0037] As used herein, a "selectable marker factor" is a factor that confers a characteristic suitable for artificial selection. Examples of selectable marker factors useful in the present invention include, but are not limited to, β-lactamase, neomycin resistance gene, mutant Fabl genes that confer triclosan resistance, URA3 factors, fluorescent gene products, affinity tags such as GST, His, CBP, MBP, and epitope tags such as Myc HA, FLAG. A selectable marker factor can be a negative or positive selection marker.

[0038] In various aspects, the present invention provides vectors, such as viral vectors (e.g., adeno-associated viral vectors (AAV)), that contain a promoter sequence that is small in size but can provide long-term transcription of a gene of interest. The promoter sequence is smaller than the size of many commonly used promoters and has a size that can drive the transcription of transgenes in most tissues and cell types (e.g., in mammals). The promoter sequences disclosed herein are not susceptible to suppression or inactivation, which makes them particularly useful for gene therapy that requires chronic expression of therapeutic transgenes.

[0039] In one aspect, the present invention provides a gene delivery vector that expresses a gene of interest. In some embodiments, the gene delivery vector comprises a promoter derived from a genomic region of an alphaherpesvirus genome, such as a pseudorabies virus. In some embodiments, the promoter comprises a latency-associated promoter (LAP) region from an alphaherpesvirus genome.

[0040] In certain embodiments, the LAP region used in the vector of the present invention is selected from SEQ ID NO:1 (also referred to herein as LAP1), SEQ ID NO:2 (also referred to herein as LAP2), SEQ ID NO:3 (also referred to herein as LAP1_2) and SEQ ID NO:4 (Table 1). LAP1_2 (880bp) contains the 476bp sequence of LAP1 (498bp) and the complete LAP2 sequence (404bp), i.e., LAP1 and LAP2 in tandem, but lacks the first 22bp of the LAP1 sequence. SEQ ID NO:4 (902bp) contains the complete sequence of SEQ ID NO:1 and SEQ ID NO:2, and is 22bp longer than SEQ ID NO:3.

[0041] In some embodiments, the promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0042] Without wishing to be bound by theory, it is believed that certain active portions or fragments of the LAP region from an alphaherpesvirus genome may drive expression of transgenes in various cell and tissue types. In some embodiments, the promoter used in the present invention comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9 (Table 1). SEQ ID NO:9 consists of the 232 bp "core" sequence of LAP2 and includes a TATA box sequence. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises SEQ ID NO:9.

[0043] Without wishing to be bound by theory, the LAP sequence (Table 1) further comprising SEQ ID NO:10, SEQ ID NO:11 and / or SEQ ID NO:12 may have improved transcriptional performance. Thus, in some embodiments, the promoter further comprises SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or a combination thereof, for example, 5' or 3' of SEQ ID NO:9. SEQ ID NO:10 ("CTCF" motif) has been shown to be associated with epigenetic control of herpesvirus gene expression, chromatin remodeling and recruitment of transcription factors and proteins (Lang et al., 2017, Lee et al., 2018). SEQ ID NO:11 ("alternative CTCF with upstream stabilizing factor" motif) has been shown to stabilize the alternative CTCF motif and further facilitate the binding of four zinc finger domains (Zimmerman et al., 2018). SEQ ID NO:12 ("nuclear transcription factor Y (NF-Y)" motif) is recognized by transcription factors that bind to CAAT boxes located in promoter regions of some eukaryotes (Chikhirzhina et al., 2008; Fleming et al., 2013).

[0044] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

[0045] In some embodiments, the promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 13-58 (Table 1). In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NOs: 13-58.

[0046] Vectors containing the LAP sequences disclosed herein can be used for gene delivery (e.g., in mammals such as humans) and for basic and translational research. The host cell can be a cultured cell (i.e., in vitro) or can be present in a subject (e.g., in an animal model (i.e., in vivo)).

[0047] In some embodiments, vector is lentivirus gene delivery vector, retrovirus gene delivery vector or adenovirus gene delivery vector.One advantage of the LAP sequence described herein is that it has a size much smaller than other commonly used promoters in viral vectors, but can sustain extended transcription in different cell types.

[0048] Considering that the DNA payload capacity is highly restricted in vectors, a relatively small LAP region may be advantageous. For example, the packaging capacity or payload of AAV vectors is restricted to a maximum size of about 5,000bp. In some embodiments, the LAP region used in the vectors of the present invention is about 200bp to about 410bp in length, such as about 220-410bp, about 220-400bp, about 240-400bp, about 240-380bp, about 260-380bp, about 260-360bp, about 280-360bp, about 280-340bp, about 300-340bp, or about 320-340bp in length.

[0049] In certain embodiments, the promoter sequence of the present invention can be used when strong long-term transgene expression is required in cells in culture, in animals or in humans.Promoters are useful in situations where there is genomic space constraint but long-term promoter transcription activity is required.AAV is particularly useful for human gene therapy, and therefore the LAP sequence of the present invention provides gene therapy applications when used with AAV.

[0050] In some embodiments, the gene of interest is expressed in vitro for at least about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, or about 190 days under the control of the LAP sequence provided herein. In some embodiments, the gene of interest is expressed in vivo (e.g., in tissues such as the brain) for at least about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, or about 190 days under the control of the LAP sequence provided herein. In some embodiments, the gene of interest is expressed ex vivo (e.g., in cultured cells such as neurons) for at least about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, or about 190 days under the control of the LAP sequence provided herein.

[0051] In one aspect, the present invention provides a recombinant vector for expressing a target gene in a host cell, comprising: a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for expression of the target gene in the host cell.

[0052] In some embodiments, the promoter comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0053] The LAP sequences described herein can be used in a variety of gene expression vectors, such as plasmids, bacterial artificial chromosomes (BACs), cosmids or other non-viral or viral systems to express a transgene(s).

[0054] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. Non-limiting examples of viral vectors that can be used according to the present invention include DNA or RNA viral vectors, including but not limited to retroviral vectors, herpes viral vectors, adenoviral vectors, lentiviral vectors, rabies viral vectors, lentiviral vectors, VSV vectors, vaccinia viral vectors, reoviral vectors, Semliki forest virus, yellow fever virus and Sindbis viral vectors.

[0055] In some embodiments, the vector is a non-viral vector.The non-viral vector can be plasmid DNA, liposome-DNA complex (lipoplex) and polymer-DNA complex (polyplex).The non-viral vector can be plasmid RNA, liposome-RNA complex (lipoplex) and polymer-RNA complex (polyplex).Oligonucleotides and their analogs, either alone or in complex, are also possible non-viral vector-mediated gene delivery constructs.

[0056] The vectors provided herein (e.g., viral vectors) can be used to express therapeutic transgenes in animal models and human organs or tissues, e.g., in gene therapy to treat acquired or genetic diseases (e.g., of the nervous system).

[0057] The LAP sequence disclosed herein was derived from the genome of pseudorabies virus (PRV). PRV is an alphaherpesvirus that can infect a variety of animals. Although the native host is the adult pig, PRV can infect a wide range of vertebrates, such as cattle, sheep, dogs, birds, cats, goats, raccoons, chickens, skunks, opossums, guinea pigs, horses, rabbits, rats, mice, and non-human primates (Pomeranz et al., 2005). Cultured human cells are also susceptible to PRV infection, and zoonotic PRV infections in humans have been reported (Wong et al., 2019).

[0058] In addition to being able to infect several different animal species, PRV is also a pantropic virus, meaning that most tissues and organs of an infected host can be susceptible and permissive to PRV infection (Blanchard et al., 2006; Boldogkoi et al., 2000; Fan et al., 2019; Gasparini et al., 2019; Pomeranz et al., 2005). Therefore, the LAP sequence can potentially be used for gene therapy in a variety of cell and tissue types.

[0059] In another aspect, the present invention provides a host cell comprising a recombinant vector described herein. In some embodiments, the host cell is a mammalian cell (e.g., a neuronal cell). In other embodiments, the host cell is selected from an avian cell, a fish cell, an amphibian cell, and a reptilian cell. In some embodiments, the host cell comprises an in vitro neuronal cell, an ex vivo neuronal cell, or both. In some embodiments, the host cell is a neuronal cell of a subject. In some embodiments, the neuronal cell is present in the primary motor cortex, the secondary motor cortex, the primary somatosensory cortex, the supplementary somatosensory cortex, the visual cortex, the hippocampal formation, the striatum, the globus pallidus, the thalamus, the hypothalamus, the midbrain region, the hindbrain, the olfactory cortex, or a combination thereof of the subject. In some embodiments, the midbrain region comprises a motor-related cortex, a sensory-related cortex, or both. In some embodiments, the neuronal cell is present in the cortex, the dentate gyrus, the striatum, the cerebellum, the olfactory bulb, or a combination thereof of the subject. In some embodiments, the neuronal cell comprises a neuron, a microglial cell, an astrocyte, or a combination thereof. In some embodiments, the neural cells comprise neurons.

[0060] In some embodiments, the host cell is selected from the group consisting of a bladder cell, a blood cell, a bone cell, an endothelial cell, an epithelial cell, an adipocyte, a cardiac cell, a small intestinal cell, a stomach cell, a kidney cell, a liver cell, a lung cell, a muscle cell, a pancreatic cell, a retinal cell, a germ cell, a skin cell, a spleen cell, a stem cell, and combinations thereof. In some embodiments, the host cell is a cancer cell.

[0061] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, wherein the nucleic acid is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the nucleic acid encoding the target gene comprises greater than about 4 kb.

[0062] In a further aspect, the present invention provides recombinant polynucleotide molecules comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0063] In another aspect, the present invention provides a recombinant vector for expression of a target gene in a host cell, comprising a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for expression of the target gene in the host cell. SEQ ID NO:5 comprises the LAP sequence of Equine Herpesvirus; SEQ ID NO:6 comprises the LAP sequence of Bovine Herpesvirus; SEQ ID NO:7 comprises the LAP sequence of Varicella-Zoster virus; and SEQ ID NO:8 comprises the LAP sequence of Macacine Herpesvirus 1 (Table 1). In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0064] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, wherein the nucleic acid is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the nucleic acid encoding the target gene comprises greater than about 4 kb.

[0065] In a further aspect, the present invention provides recombinant polynucleotide molecules comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0066] In another aspect, the present invention provides a recombinant vector for expression of a target gene in a host cell, comprising a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for expression of the target gene in the host cell. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises SEQ ID NO:9. In some embodiments, the promoter further comprises a nucleic acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or a combination thereof. In some embodiments, the promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs:13-58. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NOs:13-58.

[0067] In some embodiments, the at least one non-promoter regulatory element comprises an enhancer element. In some embodiments, the at least one non-promoter regulatory element comprises an inducer element. In some embodiments, the recombinant vector further comprises a selectable marker element.

[0068] In another aspect, the present invention provides a recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, wherein the nucleic acid is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises SEQ ID NO:9. In some embodiments, the promoter further comprises a nucleic acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof. In some embodiments, the promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs:13-58. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NOs:13-58. In some embodiments, the nucleic acid encoding the target gene comprises greater than about 4 kb.

[0069] In a further aspect, the present invention provides a recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises SEQ ID NO:9. In some embodiments, the promoter further comprises a nucleic acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or a combination thereof. In some embodiments, the promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs:13-58. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NOs:13-58.

[0070] In another aspect, the present invention provides a method of expressing a target gene in a host cell comprising contacting a recombinant polynucleotide molecule with a host cell such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding the target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and expression of the target gene occurs in the host cell. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0071] In another aspect, the present invention provides a method of expressing a target gene in a host cell, comprising contacting a recombinant polynucleotide molecule with a host cell such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding the target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and expression of the target gene occurs in the host cell. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0072] In another aspect, the present invention provides a method for expressing a target gene in a host cell, comprising contacting a recombinant polynucleotide molecule with a host cell such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding the target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9, and expression of the target gene occurs in the host cell. In some embodiments, the promoter comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, the promoter comprises SEQ ID NO:9. In some embodiments, the promoter further comprises a nucleic acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or a combination thereof. In some embodiments, the promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 13-58. In some embodiments, the promoter comprises a nucleic acid sequence selected from SEQ ID NOs: 13-58.

[0073] In some embodiments, the neuronal cells are present in the spinal cord of the subject. In some embodiments, the neuronal cells are present in the dorsal horn of the spinal cord. In some embodiments, the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof.

[0074] In some embodiments, the recombinant polynucleotide molecule is introduced into the host cell by transduction (e.g., infection with a viral vector) when the host cell is contacted with the recombinant polynucleotide. In other embodiments, the recombinant polynucleotide molecule is introduced into the host cell by transfection when the host cell is contacted with the recombinant polynucleotide. In other embodiments, the recombinant polynucleotide molecule is introduced into the host cell by electroporation when the host cell is contacted with the recombinant polynucleotide.

[0075] Recombinant adeno-associated viral vectors (AAVs) are essential neuroscience tools for defining central nervous system (CNS) connectivity and function. In addition, AAVs are used as gene therapy vectors to treat genetic and acquired CNS diseases. Despite their safety and broad tropism, important issues such as limited payload capacity and lack of small gene promoters to provide long-term pan-neuronal transgene expression in the CNS need to be corrected. Commonly used gene promoters are relatively large and can be suppressed several months after viral CNS transduction, compromising the long-term performance of single-dose gene therapy applications.

[0076] Recent improvements to recombinant AAVs, such as capsid engineering and novel gene promoters to optimize transgene expression, have substantially improved gene therapy applications (Mak, Rajapaksha et al. 2017; Hudry and Vandenberghe 2019; Ogden, Kelsic et al. 2019). Given their safety, serotype-dependent broad tropism and transduction efficiency, AAV vectors are widely used in neuroscience and clinical applications (Aschauer, Kreuz et al. 2013; Samulski and Muzyczka 2014; Mak, Rajapaksha et al. 2017). The AAV-9 variant PHP.eB (Chan, Jang et al. 2017), which has an enhanced ability to penetrate the mouse blood-brain barrier (BBB) ​​and to transduce neurons extensively in both the brain and spinal cord after peripheral vascular administration, is one example of recent capsid improvements (Dayton, Grames et al. 2018). The main limitation of recombinant AAVs is their small capsid with a limited payload capacity of only about 4.9 kb (Russell and Hirata 1998). Therefore, the discovery of short promoter sequences that sustain strong and long-lasting transcription is primary to expand the transgene payload and achieve chronic therapeutic effects with a single viral dose.

[0077] Several strong promoters, such as neuron-specific enolase (NSE, 1800bp) (Peel, Zolotukhin et al. 1997; Delzor, Dufour et al. 2012), calcium / calmodulin-dependent protein kinase IIα (CaMKIIa, 1300bp) (Sohal, Zhang et al. 2009) and human elongation factor 1α (EF1α, 1264bp) (Sohal, Zhang et al. 2009; Qin, Zhang et al. 2010), have been used for systemic AAV delivery (Bedbrook, Deverman et al. 2018). However, the considerable size of these promoter sequences (Bedbrook, Deverman et al. 2018) limits the use of large therapeutic transgenes or multiple small transgenes. Furthermore, short promoters such as the human cytomegalovirus immediate early enhancer and promoter (CMV, 600bp) (Gray, Foti et al. 2011) or a truncated version of the human synapsin promoter (hSyn, 468bp) (Jackson, Dayton et al. 2016) are rather weak in driving gene transcription and expression, and in some cases are completely repressed or inactivated just a few weeks after delivery (Brooks, Harkins et al. 2004; Nathanson, Jappelli et al. 2009; Qin, Zhang et al. 2010; Back, Dossat et al. 2019). Similarly, small ubiquitous promoters such as β-glucuronidase (GUSB, 378 bp) (Husain, Passini et al. 2009) or ubiquitin C (UBC, 403 bp) (Qin, Zhang et al. 2010; Powell, Rivera-Soto et al. 2015) showed weak transcription levels.

[0078] Three alphaherpesvirus latency-associated promoters (LAPs), designated LAP1 (498 bp), LAP2 (404 bp) and LAP1_2 (880 bp), derived from the genome of the herpesvirus pseudorabies virus (PRV), are described and validated herein. The Alphaherpesvirinae subfamily of the Herpesviridae family includes bovine herpesvirus-1 (BHV-1), varicella-zoster virus (VZV), herpes simplex virus (HSV) and PRV. These viruses share genome organization and establish latent infections in sensory ganglia of different mammalian hosts (Koyuncu, Hogue et al. 2013).

[0079] The PRV LAP region contains two independent promoters, LAP1 and LAP2 (Cheung 1989; Cheung and Smith 1999; Jin and Scherba 1999; Jin, Schnitzlein et al. 2000) (Figure 1A). PRV LAP1 contains two GC boxes and three CAAT boxes upstream of the first TATA box. PRV LAP2 contains two GC boxes before the second TATA box (Cheung 1989; Jin and Scherba 1999; Taharaguchi, Kobayashi et al. 2002). It has been proposed that binding of different transcription factors (TFs) to consensus promoter elements present in LAPs may facilitate escape from nucleosomal silencing during latent infection (Deshmane and Fraser 1989; Leib, Bogard et al. 1989; Devi-Rao, Goodart et al. 1991; Jin, Schnitzlein et al. 2000; Ono, Tomioka et al. 2007). In transgenic mouse strains, PRV LAP-promoted transcription is neuron-specific in the absence of PRV infection (Taharaguchi, Yoshino et al. 2003). However, in transient expression assays, PRV LAP1 and LAP2 promote transcription in both cultured neurons and non-neuronal cells (Cheung and Smith 1999; Taharaguchi, Kobayashi et al. 2002). Furthermore, the activity of tandem LAP1 and LAP2 sequences is significantly increased compared to LAP1 or LAP2 alone (Cheung and Smith 1999).

[0080] Here, using a whole-CNS screening approach based on retroorbital systemic delivery of AAV-PHP.eB, iDisco+ tissue clearing and light-sheet microscopy, we identified three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp) and LAP1_2 (880 bp). They drive chronic transcription of the virally encoded latency-associated transcript (LAT) during the proliferation and latency phases of the PRV infection cycle. Stable pan-neuronal transgene transcription and translation were observed from AAV-LAPs in the brain and spinal cord for 6 months after AAV transduction. The data suggest that LAP is a suitable candidate for viral vector-based CNS gene therapy that requires chronic transgene expression after a one-time viral vector administration, and that the PRV LAP1, LAP2 and aligned LAP1_2 promoters may be suitable for systemic, less invasive, pan-neuronal gene delivery applications that may require stable, chronic transgene expression after a single administration. EXAMPLES

[0081] Example 1. Materials and Methods Construction of the PRV LAP sequence The PRV latency-associated transcript promoter (LAP) was PCR amplified from equivalents 95106 to 96007 of the PRV Becker strain genome (GenBank: JF797219.1). The LAP1 region (498 bp) was amplified using the primer pair LAP1F (5'- GCA CGC GTA TCT CCG GAA AGA GGA AAT TGA -3') (SEQ ID NO:59) and LAP1R (5'- GCG GAT CCT ATA TAC ACG ATG TGC ATC CAT AAT -3') (SEQ ID NO:60). The LAP2 region (404 bp) was amplified using the primer pair LAP2F (5'- GCA CGC GTA TCC CCG GTC CGC GCT CCG CCC ACC CA -3') (SEQ ID NO:61) and LAP2R (5'- GCG GAT CCG AGC TCC CTC TTC CTC GCC GCG GAC TGG -3') (SEQ ID NO:62). LAP1_2 (902 bp), spanning the entire LAP region, was amplified using LAP1F and LAP2R (Cheung and Smith 1999). The 5' and 3' regions of these PCR sequences contain MluI and BamHI restriction sites, respectively, and were used for directional cloning into the vector pAAV-Ef1α-mCherry, which was a gift from Karl Deisseroth (Addgene plasmid #114470). The three AAV-LAP plasmids were constructed by double digestion of the vector pAAV-Ef1α-mCherry with MluI and BamHI, followed by subcloning of the appropriate LAP fragment upstream of the mCherry reporter gene, flanked by the AAV2 inverted terminal repeats (ITRs), and terminated with the SV40 polyA signal.

[0082] AAV vector construction All expression cassettes were packaged into AAV-PhP.eB capsids (gift from Daniela Gradinaru, Addgene plasmid #103005) at the Princeton Neuroscience Institute Viral Core Facility and purified by iodixanol step gradient and column ultrafiltration as previously described (Zolotukhin, Byrne et al. 1999; Chan, Jang et al. 2017). Capsid-protected viral genomes were measured by TaqMan qPCR and reported as genome copies per milliliter (GC / ml).

[0083] animal Animal studies were performed in accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committee of Princeton University (protocols #1943-16 and 1047). Timed-pregnant Sprague-Dawley rats were obtained from Hilltop Labs Inc. (Scottsdale, PA). E17 rat embryos were harvested for isolation of sympathetic SCG neurons. Adult (4-6 weeks old) wild-type C57BL / 6J male mice were obtained from Jackson Laboratory (The Jackson Laboratory, Bar Harbor, ME). Mice had at least 48 hours of acclimation to the Princeton Neuroscience Institute animal care holding facility before experimental procedures were performed.

[0084] Primary superior cervical ganglion cell culture SCG neurons from rat embryos (E17) were cultured in trichambers as previously described (Curanovic, Ch'ng et al. 2009). Briefly, SCGs were dissociated with trypsin (2.5mg / ml, Sigma-Aldrich, The Woodlands, TX) and plated onto poly-O-ornithine and laminin-coated dishes with medium containing neurobasal medium supplemented with 2% B-27, 100ng / ml nerve growth factor (NGF) and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific, Rockford, IL). Approximately two-thirds of a single ganglion was placed into the soma compartment of the trichamber. Three days after seeding, the culture medium was treated with 0.1 mM cytosine-D-arabinofuranoside, Ara C (Sigma-Aldrich, The Woodlands, TX) for at least 2 days to eliminate dividing non-neuronal cells. The culture medium was changed every 5 days, and neurons were incubated at 37°C, 5% CO2.

[0085] Posterior orbital sinus injection Intravenous administration of AAV vectors was performed in mice by unilateral injection into the retro-orbital sinus (Yardeni, Eckhaus et al. 2011). Prior to the procedure, animals were anesthetized using a ketamine (80mg / kg) / xylazine (10mg / kg) cocktail. Once unresponsive, animals were placed in lateral recumbence for injection into the medial canthus. The injection volume was 100μl, giving a total of 4x10 11 The viral genome was administered via a 29G1 / 2 insulin syringe. Animals were placed on a regulated heating pad and monitored until ambulation.

[0086] Tissue processing and histological procedures Mice were anesthetized with an overdose of ketamine (400 mg / kg) / xylazine (ip) and perfused with 4% paraformaldehyde (PFA) at 30 and 190 days post-infection (dpi). Brains and spinal cords were post-fixed overnight at 4°C in 4% PFA. After rinsing with phosphate-buffered saline (PBS), brains were stored at 4°C in PBS with 0.1% sodium azide. Brains were divided into two parts and the left hemisphere was sectioned sagittally at 50 μm using a Leica VT1200 vibratome and at 20 μm using a Leica CM3050 S cryostat. The right hemisphere was used for the iDISCO+ tissue clearing protocol (below). Fixed brains and spinal cords were successively incubated at 4°C in 10% sucrose, 20% sucrose and 30% sucrose for cryoprotection. Tissues were embedded in OCT (tissue-Tek, Torrance, CA), frozen on dry ice, and stored at -80°C until sectioning. For cryosectioning, spinal cords were placed into embedding molds (Sigma-Aldrich, The Woodlands, TX) and filled with ultrapure low-melting agarose (Thermo Fisher Scientific, Rockford, IL) at 37°C. Semi-solid agarose cubes were removed and glued horizontally for transverse sectioning at 20 μm using a cryostat (Loctite, Rocky Hill, CT).

[0087] immunostaining For immunohistochemistry, free-floating brain sections were washed with PBS and blocked with 3% bovine serum albumin (BSA), 2% donkey serum, and 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 1 h. Samples were incubated with primary antibodies overnight at 4°C and with secondary antibodies diluted in PBS containing 1% BSA, 1% donkey serum, and 0.5% Triton X-100 for 1 h at RT (room temperature). Cell nuclei were counterstained with 0.5 μg / ml DAPI for 5 min (Thermo Fisher Scientific, Rockford, IL). The following primary antibodies were used: rabbit anti-RFP Rockland (1:1000; Limerick, PA), chicken anti-mCherry Abcam (1:500; Cambridge, MA), mouse anti-NeuN (1:500; Millipore Bioscience Research Reagents, Temecula, CA), rabbit anti-Olig2 (1:500, EMD Millipore, Temecula, CA), rabbit anti-Iba1 (1:1000, Wako Chemical, Richmond, VA), and rabbit anti-S100 (1:5000, Dako, Glostrup, Denmark). The following secondary antibodies were used: Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 647 donkey anti-chicken IgG (1:1000, Thermo Fisher Scientific, Rockford, IL). Free-floating spinal cord sections were stained with a 1:300 dilution of NeuroTrace 500 / 525 green fluorescent Nissl stain (Molecular Probes, Eugene, OR) for 1 hour. Sections were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes and washed first with PBS and then with PBS with 0.1% Triton X-100 for 10 minutes. Samples were incubated with 0.5 μg / ml DAPI for 5 minutes and then washed with PBS for 2 hours at room temperature.Fluoromount-G mounting medium (Southern Biotech, Birmingham, Ala.) was applied to brain and spinal cord sections prior to mounting.

[0088] Microscopy Neuronal SCG cultures were imaged with a Nikon Ti-E inverted epifluorescence microscope (Nikon Instruments Inc, Tokyo, Japan) with a Cool Snap ES2 camera (Photometrics, Tucson, AZ) and a 4X objective. Tiled images of all S sections were assembled with Nikon NIS Elements software. To quantify AAV transduction efficiency in different brain regions, brain slices were imaged with a NanoZoomer S60 fluorescence microscope scanner (Hamamatsu, Hamamatsu, Japan). Brain slices were imaged with a Leica STP8000 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) over a 1024x1024 pixel area using 20X and 63X objectives and a Hybrid (HyD) detector for sensitive detection. Corrected total cell fluorescence was calculated using ImageJ software as previously reported (Maturana, Aguirre et al. 2017). Cells were selected and regions of interest (ROIs) were drawn and normalized against background intensity from non-fluorescent cells. Calculation of corrected total cell fluorescence was measured as relative fluorescence intensity (RFI), taking into account the integrated area, density and mean gray value of each cell.

[0089] iDISCO+organizational transparency penetration The right hemisphere was used for iDISCO+ tissue clearing. Brain samples were fixed overnight in 4% PFA followed by tissue clearing as previously described (Renier, Wu et al. 2014). Fixed samples were washed / dehydrated in 20, 40, 60, 80, 100% methanol / water solution for 1 h each, then rehydrated with 5% hydrogen peroxide / methanol overnight washes (Sigma-Aldrich, St. Louis, MO) and reverse gradient methanol / water 100, 80, 60, 40, 20% for 1 h each. Finally, brains were washed with 0.2% Triton X-100 / PBS, followed by 20% DMSO (Thermo Fisher Scientific, Rockford, IL) / 0.3M glycine (Sigma-Aldrich, St. Louis, MO) / 0.2% Triton X-100 / PBS for 2 days at 37°C.

[0090] immunolabeling Samples were incubated for 2-3 days at 37°C in a blocking solution of 10% DMSO / 6% donkey serum (EMD Millipore, Temecula, CA) / 0.2% Triton X-100 / PBS, then washed twice for 1 h in PBS / 0.2% Tween-20 (Sigma-Aldrich, St. Louis, MO) with 10 μg / ml heparin (solution hereafter referred to as PTwH, Sigma-Aldrich, St. Louis, MO). Brains were incubated for 7 days at 37°C with primary rabbit anti-RFP antibody (1:1000; Rockland, Limerick, PA) in 5% DMSO / 3% donkey serum / PTwH. Brains were then washed five times in PTwH (wash intervals: 10 min, 15 min, 30 min, 1 h, 2 h) and incubated with secondary Alexa Fluor 647 donkey anti-rabbit IgG (1:450, Thermo Fisher Scientific, Rockford, IL) in 3% donkey serum / PTwH for 7 days at 37°C, then washed five times in PTwH.

[0091] Tissue transparency Brains were dehydrated sequentially in 20, 40, 60, 80, and 100% methanol / water for 1 h each step, followed by 2:1 dichloromethane (DCM; Sigma-Aldrich, St. Louis, MO) / methanol and 100% DCM washes. Finally, samples were cleared in DBE (Sigma-Aldrich, St. Louis, MO) and stored at room temperature in the dark until imaging.

[0092] Light sheet microscopy and analysis of cleared tissues After immunolabeling and clearing, brain volumes were acquired using a light sheet Ultramicroscope II (LaVision Biotec, Bielefeld, Germany). Half of the brain was glued horizontally on a custom designed and 3D printed holder (Renier, Wu et al. 2014; Renier, Adams et al. 2016) and submerged in DBE. Brains were imaged in the autofluorescence channel with 488 nm laser diode excitation and 525 nm maximum emission filter (FF01-525 / 39-25, Semrock, Rochester, NY) for registration purposes and at 640 nm excitation with 680 nm maximum emission filter (FF01-680 / 42-25, Semrock) for cellular imaging of AAV-infected cells (anti-RFP). Separate left and right illumination autofluorescence images were acquired every 10 micrometers (z-step size) using 0.017 excitation sheet NA and 1.3x magnification. The left and right images were merged in a sigmoid shape at the midline. The autofluorescence volume was registered to the volumetric Allen brain atlas (2015) using affine and b-spline transformations as described by Renier and colleagues (Renier, Wu et al. 2014; Renier, Adams et al. 2016). To account for shifts during acquisition and different imaging parameters between channels, the cell signal volume was registered to the autofluorescence volume by affine transformation. Brain volumes were analyzed with our modified ClearMap software: "ClearMap_cluster" (github.com / PrincetonUniversity / clearmap_cluster), compatible with high-performance computing clusters. For all analyzed samples, detected objects on the brain edge and ventricles were erode 75 μm from the edge of the structure to minimize false positives.

[0093] RNA scope in situ hybridization Brain cryosections were mounted on Superfrost Plus adhesive slides (Thermo Fisher, Waltham, MA) and stored at -80°C. RNA staining was performed using the RNAScope Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics (ACD), Newark, CA) according to the manufacturer's protocol. mCherry probe ACD♯431201-C2 was used. Sections were pretreated with protease IV for 30 min at 40°C, followed by probe incubation for 2 h at 40°C. Different amplifier solutions were then performed for 30, 30 and 15 min at 40°C. Signal was detected by a TSA+fluorescein system (Perkin Elmer, Waltham, MA). Incubation steps were performed in the ACD HybEZ hybridization system. Slides were counterstained with DAPI for 30 s at room temperature and then washed twice with PBS. Finally, slides were mounted with VECTASHIELD Vibrance antifade mounting medium (Vector Laboratories, Burlingame, Calif.).

[0094] statistics Statistical data analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA). Two-tailed Student's test was used to compare between two groups, and nonparametric one-way ANOVA test followed by Bonferroni multiple comparison post-hoc test to compare between multiple groups. A p-value of <0.05 was statistically significant. Data are expressed as mean with SEM.

[0095] Example 2. The small PRV LAP sequence can drive transgene expression in neurons independent of herpesvirus infection. The PRV LAP region contains at least two promoter regions, defined herein as LAP1 and LAP2 (Figure 1A). In the PRV genome, LAP1 and LAP2 are present in tandem as PRV LAP1_2. These sequences, alone or in combination, can efficiently express reporter transgenes in primary sympathetic neurons when used in AAV vectors without PRV infection (Figures 1C and 1D). The LAP sequence was analyzed to identify putative regulators using Jasper (Sandelin, Alkema et al. 2004; Wasserman and Sandelin 2004), RSAT (Nguyen, Contreras-Moreira et al. 2018) and CTCFBSDB 2.0 (Ziebarth, Bhattacharya et al. 2013) software. Three cyclic AMP response element binding protein (CREB) binding motifs were detected upstream of the LAP1 TATA box and one upstream of the LAP2 TATA box. In addition, two CCCTC binding factor (CTCF) binding motifs were detected upstream of the LAP1 TATA box and one downstream of the LAP2 TATA box. A downstream promoter element (DPE) was identified in LAP2 and contained three CG boxes and four signal transducer and activator of transcription 1 (STAT1) binding motifs. In addition, there were lineage-determining TFs (Wang, Pol et al. 2014), such as SRY-Box 10 (SOX10) and oligodendrocyte transcription factor 2 (Olig2), upstream of the LAP1 TATA box and LAP2 TATA box, respectively (Figure 1A).

[0096] The four AAV recombinants were packaged into serotype PHP.eB capsids by standard methods. Three promoter constructs were prepared: LAP1 (498 bp, corresponding to SEQ ID NO:1), LAP2 (404 bp, corresponding to SEQ ID NO:2) and LAP1_2 (880 bp, corresponding to SEQ ID NO:3). The ubiquitous EF-1α promoter (1264 bp) was used as a positive control for transgene expression. All four AAV recombinants expressed the fluorescent reporter mCherry (Figure 1B). To validate the in vitro performance of each promoter, 3x10 of each AAV was injected into rat primary superior cervical ganglion (SCG) neuronal cultures. 11 The genome was transduced and relative mCherry fluorescence intensity (RFI) was quantified over a 90 day period.

[0097] For AAV-LAP1 transduced neurons, mCherry expression suddenly increased at 11 dpi (64220 RFI), albeit at a lower level compared to AAV-LAP2, AAV-LAP1_2 and AAV-EF1α. AAV-EF1α expression further increased approximately 120-fold at 17 dpi (8.70x10 6 ;2.39x10 6 ;8.01x10 6 The highest levels of expression by all four recombinants were at 28 dpi: LAP1 (291,242 RFI); LAP2 (1.36x10 7 RFI;LAP1_2(4.36x10 6 RFI) and EF1α (1.40x10 7 RFI) (Figures 1C and 1D). LAP2 and EF1α had the highest mCherry RFI (LAP2 = EF1α > LAP1_2 > LAP1). Between 38 and 90 dpi, all four AAV recombinants showed a subtle but persistent RFI decline (Figure 1C), most likely due to aging of primary SCG neurons after >100 days in culture. Importantly, all three AAV-LAP recombinants showed mCherry transcription in primary neurons for 90 days in the absence of PRV infection in the context of AAV transduction.

[0098] Example 3. Whole CNS screening reveals pan-neuronal AAV-LAP transgene expression after 6 months. AAV serotype PHP.eB was used in the promoter screening assay given its increased ability to cross the BBB and transduce the C57 / BL6 mouse CNS after systemic intravenous delivery (Chan, Jang et al. 2017). AAV-LAP1-mCherry, AAV-LAP2-mCherry, AAV-LAP1_2-mCherry and AAV-EF1α-mCherry were administered at 4x10 11 AAV was delivered by unilateral retro-orbital sinus injection of 1000 ng / mouse (vg / mouse). As illustrated in Figure 2A, brains and spinal cords were harvested 30 and 190 days after AAV transduction to quantify mCherry transcription and translation. LAP-mCherry expression was determined in intact whole brains after tissues were cleared and immunostained by iDISCO+ (three-dimensional imaging enabled by immunolabeling of solvent-cleared organs) and analyzed by light-sheet microscopy and volumetric registration (Renier, Wu et al. 2014; Renier, Adams et al. 2016).

[0099] As shown in Figures 2B-2P, all four promoters showed stable mCherry expression at both 30 dpi and 190 dpi. 3The number of mCherry positive cells per mCherry cell count (number of mCherry positive cells per mCherry cell count) was higher than that of LAP1 and LAP1_2 and not significantly different from that of EF1α in different brain regions: cortex: primary motor, secondary motor, primary somatosensory and supplementary somatosensory (Figure 2B-2E); hippocampal formation (Figure 2G), globus pallidus (Figure 2I), hypothalamus (Figure 2K) and olfactory cortex (Figure 2P). In the cerebellum, LAP2 showed higher mCherry density than LAP1, LAP1_2 and EF1α (Figure 2O). Furthermore, in the striatum (Figure 2H); thalamus (Figure 2J); midbrain, motor and sensory cortex (Figure 2L and 2M), and rhombencephalon (Figure 2N), LAP2 and LAP1_2 showed significantly higher density than EF1α (p<0.05). Note that although the LAP2 nucleotide sequence is 68% shorter than that of EF1α, it outperforms the capacity of EF1α in some brain regions.

[0100] To further validate LAP transgene expression in the CNS, mCherry protein expression was assessed by immunohistochemistry (IHC) in sagittal brain sections at 30 dpi and 190 dpi (Figure 3A-3H). Confocal microscopy analysis showed abundant mCherry staining in the cortical somatosensory cortex (Figure 3E), dentate gyrus of the hippocampal formation (Figure 3F), caudoputamen of the striatum (Figure 3G) and cerebellar cortex (Figure 3H) at 30 dpi. Importantly, mCherry expression was stable for all three LAP sequences at 190 dpi and similar to that of the larger AAV-EF1α-mCherry promoter (Figure 3E-3H, panels 5-8). mCherry expression was then quantified at 30 dpi and 190 dpi. mCherry RFI was similar for all AAV promoters without significant differences (Figure 3I1-3I4). 1 pixel after 190 days 2The number of mCherry-positive cells per injection was then quantified. In the cortex, the number of LAP2 mCherry-expressing cells was higher than that observed for LAP1-mCherry and LAP1_2-mCherry [LAP2: 297 ± 19.82 vs. LAP1: 149 ± 5.61 vs. LAP1_2: 168 ± 9.22 (n = 6, p < 0.001) (Figure 3J1). In the dentate gyrus, striatum and cerebellum, the number of mCherry-positive cells was similar for all LAP variants and EF1α (Figure 3J2-3J4). Thus, all AAV-LAP variants promote mCherry expression in the brain, further indicating that a single administration of AAV-LAP recombinants may be sufficient to drive long-term pan-neuronal transgene expression in the mouse CNS.

[0101] Example 4. Small LAP2 promoter variants drive strong and stable pan-neuronal transgene expression following systemic AAV administration. We compared the efficacy of mCherry expression under the control of different PRV LAP sequences. Abundant signals were observed in the cortex (Figure 4A and 4E1), dentate gyrus (Figure 4B and 4E2), striatum (Figure 4C and 4E3) and cerebellum (Figure 4D and 4E4) at 30 dpi and 190 dpi. Furthermore, AAV-LAP2 RFI was stable and similar to AAV-EF1α at both 30 dpi and 190 dpi (p<0.05) (Figure 4E1-4E4; Table 2). AAV-LAP1 and AAV-LAP1_2 mCherry RFI levels were stable and did not differ significantly between them at 30 dpi and 190 dpi in the cortex, dentate gyrus, striatum and cerebellum (Figure 4E1-4E4; Table 2), although both promoters showed significantly lower transgene expression compared to LAP2 and EF1α. [Table 2]

[0102] Because mRNA half-life is usually shorter than that of translated proteins (Chan, Mugler et al. 2018), we measured mCherry transcripts in AAV-LAP-transduced mouse brain slices. AAV-LAP2-mCherry mRNA at 30 dpi (data not shown) and 190 dpi was measured using an mCherry-specific riboprobe. Fluorescence in situ hybridization (FISH) demonstrated abundant AAV-LAP2 mCherry RNA in the cortex, dentate gyrus, striatum, cerebellum and olfactory bulb (Figures 5A-5E), further confirming that PRV LAP2 can drive chronic and robust transgene transcription in the CNS.

[0103] Example 5. AAV-LAP transgene expression in the brain is predominant in neurons but not in glial cells. The tropism and specificity of AAV transduction and subsequent transgene expression depend on the AAV serotype (Aschauer, Kreuz et al. 2013, Bedbrook; Deverman et al. 2018) and promoter (von Jonquieres, Frohlich et al. 2016; Hammond, Leek et al. 2017; Dayton, Grames et al. 2018). To characterize which cell types exhibited AAV-LAP-mCherry expression following systemic AAV-PHP.eB delivery, co-immunostaining for mCherry protein with markers for neurons (NeuN), oligodendrocytes (Olig2), microglia (Iba1) and astrocytes (S100) was performed in the cortex and dentate gyrus. Co-staining with NeuN and mCherry revealed that more than 90% of imaged neurons expressed mCherry driven by different AAV-LAP variants in both the cortex and dentate gyrus (Figures 6A, 6B, and 6E). Conversely, fewer than 4% mCherry-positive oligodendrocytes were detected for all LAP variants (Figures 6C, 6D, and 6F). Furthermore, no co-labeling of mCherry with microglial and astrocyte markers was observed for any of the LAP recombinants (Figures 7A-7D). Overall, these results indicate that in the context of systemic brain transduction with AAV-PHP.eB, LAP-mCherry expression is enriched in neurons but not glial cells.

[0104] Example 6. AAV-LAP constructs demonstrate widespread, stable and long-term transgene expression throughout the spinal cord. In addition to the brain, serotype PHP.eB has demonstrated widespread transduction of gray matter (Chan, Jang et al. 2017; Dayton, Grames et al. 2018), so we evaluated AAV-LAP performance in the spinal cord.

[0105] We observed abundant native mCherry expression in both the dorsal and ventral horns of the spinal cord at cervical, thoracic (data not shown) and lumbar levels at 190 dpi (Figures 8A-8D). Quantification of native mCherry fluorescence (RFI) for all promoters was similar with no statistically significant differences (Figure 8E). However, LAP2 and LAP1_2 recombinants showed 1 pixel mCherry expression, with 1 pixel mCherry expression. 2 LAP2 showed the highest density of mCherry-positive cells per sigma, followed by EF1α and LAP1; LAP2 = LAP1_2 > EF1α > LAP1 (Figure 8F). Thus, in the spinal cord, all three PRV LAP sequences effectively mediate pan-neuronal long-term transgene expression.

[0106] Gene therapy has been used to restore gene function in specific target cells in neurological and neurodegenerative disorders (Deverman, Ravina et al. 2018). Gene transfer by systemic vector delivery via peripheral vascular transduction can be challenging for efficient expression in a neuron-specific or pan-neuronal manner in the CNS (Ingusci, Verlengia et al. 2019). Recombinant AAV vectors are among the most efficient vehicles for achieving gene expression in the CNS (Hudry and Vandenberghe 2019, Wang, Tai et al. 2019). Furthermore, genetically engineered AAV capsids have demonstrated improved CNS transduction and enhanced ability to cross the BBB with higher efficiency than naturally occurring serotypes (Chan, Jang et al. 2017; Bedbrook, Deverman et al. 2018; Hordeaux, Yuan et al. 2019; Qin Huang and Alejandro B. Balazs 2019). Despite these advances, AAV gene therapy is hampered by the small payload size limit of 4.9Kb for AAV capsids (Chamberlain, Riyad et al. 2016). For example, CNS therapy for Pompeo disease (Colella, Sellier et al. 2019) and Parkinson's disease (Wang, Muramatsu et al. 2002) is based on the delivery of relatively large genes such as GAA (2.9Kb) and GDNF (2.5Kb). For these and other similar cases, use of larger promoters or even the smaller CMV and hSyn promoters has been shown to be rapidly repressed after delivery (Gray, Foti et al. 2011, Jackson, Dayton et al. 2016).

[0107] As described herein, we identified three small pan-neuronal promoters isolated from the genome of the alphaherpesvirus PRV and demonstrated effective, long-term transgene expression in the mouse CNS following systemic AAV PHP.eB delivery. The results presented herein indicate that these small PRV LAP sequences can drive long-term (>6 months) expression of reporter transgenes in the brain and spinal cord. PRV LAP uniformly transduced neurons in the cortex, striatum, dentate gyrus and cerebellum. The distribution of mCherry-positive cells was not significantly different between LAP sequences in the dentate gyrus, striatum and cerebellum. However, LAP2 transgene expression was significantly higher in the cortex compared to LAP1 and LAP1_2. Whole-brain screening assays showed that the 402 bp LAP2 variant alone could drive stronger mCherry expression than the larger LAP1 and LAP1_2 sequences. Furthermore, abundant mCherry mRNA transcribed from LAP2 was detected in all screened brain regions at 190 dpi. These results demonstrate both efficient transcription and translation driven from the small PRV LAP2 in the CNS following systemic AAV-LAP2 delivery in the absence of PRV infection.

[0108] Although LAP1-mCherry cell density was significantly lower than that for LAP2, mCherry expression remained stable and long-lasting. Therefore, LAP1 could be useful when small amounts of therapeutic protein are needed (e.g. enzyme deficiency) or for cross-correction to non-transduced cells. For example, for lysosomal enzyme deficiency (Husain, Passini et al. 2009) and mucopolysaccharidosis type VII disease (Cearley and Wolfe 2007), the enzyme repaired by AAV therapy could be secreted from the transduced cells and ameliorate the adjacent disease-state cells.

[0109] AAV tropism is primarily determined by the interaction between the capsid and specific receptors in susceptible and permissive cells (Aschauer, Kreuz et al. 2013; Bedbrook, Deverman et al. 2018). Different AAV serotypes have different tissue tropism. However, promoter sequences and other sequences included in the vector, such as inverted terminal repeats (ITRs), can have a substantial effect on tropism (Haberman and McCown 2002). Also, the promoter region that transcribes the transgene is critically important to optimize AAV vector performance. Efficient transgene expression in either a broad-based or cell-type-specific manner requires the binding and action of cell-derived TFs to the promoter region (Dayton, Grames et al. 2018; Andersson and Sandelin 2019). Changes in the neuronal environment, such as aging or differentiation, can also alter the recruitment of cell-specific regulatory proteins and therefore gene expression in the CNS (Herdegen and Leah 1998). Analysis of the PRV LAP sequence presented herein identified a DPE in LAP2 that may control transgene expression initiation, persistence and cell type specificity. Additionally, four STAT1 motifs were identified in HSV-1 LAP that appear to control viral reactivation from latency (Kriesel, Jones et al. 2004). Notably, one of these STAT1 motifs in PRV LAP2 was found to colocalize with a TATA box and an Olig2 motif, a known pleiotropic TF that promotes neuronal and oligodendrocyte fates (Emery and Lu 2015). The effect of proximity associated with these motifs and transcription start sites could explain the different levels of CNS transgene expression between LAP2, LAP1 and LAP1_2.Additionally, one CTCF motif was found downstream of the LAP2 TATA box, which may have a role in resistance to epigenetic silencing during latency as shown for HSV-1 (Lang, Li et al. 2017; Lee, Raja et al. 2018). Indeed, Zimmerman and colleagues (Zimmerman, Patel et al. 2018) found that insertion of a CTCF motif downstream of the EF1α promoter significantly increased transgene expression compared to the native EF1α and CMV promoters. Interestingly, insertion of a second CTCF motif downstream of the CMV TATA box had no effect on luciferase reporter expression, presumably due to the redundant presence of the native CTCF motif (Zimmerman, Patel et al. 2018). Thus, gene expression is susceptible to changes that depend on gene context and sequence-specific DNA-binding proteins. Recruitment of specific TFs from different host cells may regulate transgene transcription by the same mechanisms that control resistance to inactivation during latency. Insulator factors such as CTCF binding factors are independently regulated (Washington, Musarrat et al. 2018) and may protect promoter regions from heterochromatin-mediated repression, maintaining long-term transcription (Lang, Li et al. 2017).

[0110] Histological evaluation of cell-specific transduction by colocalization of LAP-mCherry, glial and neuronal markers revealed that the LAP sequence was expressed more efficiently in neurons than in glia in the cortex and dentate gyrus. LAP-mCherry positive cells colocalized preferentially with neuron-specific markers, but not with microglia or astrocytes, and to a lesser extent with oligodendrocytes. These findings suggest that the PRV LAP sequence has a pan-neuronal promoter profile in the CNS after PHP.eB systemic delivery. This activity has also been found for the HSV LAP sequence due to the presence of a CRE motif upstream of the TATA box (Leib, Nadeau et al. 1991; Kenny, Krebs et al. 1994; Bloom, Stevens et al. 1997). PRV LAP1 and LAP2 are neuron-specific promoters in the absence of other viral proteins, and transgene expression levels vary across different neuronal types (Taharaguchi, Yoshino et al. 2003). Importantly, AAV-PHP.eB preferentially transduces neurons (Chan, Jang et al. 2017), and the combination of this capsid variant with the PRV LAP sequence shows a strong and long-term pan-neuronal expression profile in the CNS. Thus, the PRV LAP sequence can be used in the context of recombinant viral vectors (AAV, adenovirus, lentivirus, herpesvirus) as well as by non-viral gene delivery platforms. Although the native host of PRV is the adult pig, the virus has an extremely broad tropism and can infect many types of mammals, including some birds, fish, and some primates (Baskerville and Lloyd 1977). Furthermore, human cells in culture are susceptible to PRV infection, and there are several reports of zoonotic infections (Wong, Lu et al. 2019). Therefore, the PRV LAP sequence can be optimized for gene therapy applications that require efficient and long-term transgene expression in several different mammals, including humans.

[0111] In summary, the data presented herein demonstrated that PRV LAP promoter activity is independent of PRV infection and that the small AAV-PHP.eB-LAP sequence expresses transgenes in a stable, pan-neuronal manner in the brain and spinal cord (CNS). Long-term transgene transcription and translation are ideal for effective, long-term, single-dose gene therapy applications. Thus, the PRV LAP sequence may be useful for the treatment of genetic CNS diseases.

[0112] Example 7. LAP sequences efficiently drive transgene expression in non-neuronal cells and tissues. In the examples below, the LAP sequence was shown to effectively drive transgene expression of a fluorescent reporter protein (mCherry) in several cell and tissue types, including kidney, liver, and retina. Furthermore, LAP was found to be compatible with viral vectors (e.g., AAV) and non-viral vectors (e.g., direct transfection of plasmid DNA).

[0113] As shown in Figures 9A-9C, the LAP sequence drives efficient transgene expression in porcine kidney cells. Representative images of PK15 cells (porcine kidney epithelial cells, ATCC CCL-33) were obtained 48 hours after transfection of reporter plasmids containing mCherry driven by LAP1 (Figure 9A), LAP2 (Figure 9B), or LAP1_2 (Figure 9C).

[0114] As shown in Figures 10A and 10B, LAP2 drives efficient transgene expression in human kidney cells. Representative images of HEK-293 cells (human embryonic kidney epithelial cells, ATCC CRL-1573) were obtained 48 hours after transfection of a reporter plasmid containing mCherry driven by LAP2 (Figure 10A) or the EF1α promoter (Figure 10B).

[0115] As shown in Figures 11A and 11B, LAP2 drives efficient transgene expression in human liver cells. Representative images of HepG2 cells (human liver epithelial cells, ATCC HB-8065) ​​were obtained 48 hours after transfection of a reporter plasmid containing mCherry driven by LAP2 (Figure 11A) or the EF1α promoter (Figure 11B).

[0116] As shown in Figures 12A1-12B4, LAP2 drives long-term transgene expression throughout the liver. Intravenous administration of AAV-LAP2 (Figures 12A1-12A4) or AAV-EF1α-vector (Figures 12B1-12B4) was performed by unilateral injection into the retro-orbital sinus of mice (4x10 11 vg / mouse). Livers were harvested at 190 dpi and sectioned longitudinally at 20 μm for subsequent IHC and confocal microscopy analysis. Representative confocal images show native mCherry fluorescence (Figures 12A1 and 12B1: red), anti-mCherry immunostaining (Figures 12A2 and 12B2: green), DAPI nuclear counterstaining (Figures 12A3 and 12B3: blue) and combined immunofluorescence and DAPI images (Figures 12A4 and 12B4).

[0117] As shown in Figures 13A1-13B4, LAP2 drives long-term transgene expression in mouse kidney. Intravenous administration of AAV-LAP2 (Figures 13A1-13A4) or AAV-EF1α-vector (Figures 13B1-13B4) was performed by unilateral injection into the retro-orbital sinus of mice (4x10 11 vg / mouse). Kidneys were harvested at 190 dpi and sectioned longitudinally at 20 μm for subsequent IHC and confocal microscopy analysis. Representative confocal images show native mCherry fluorescence (Figures 13A1 and 13B1: red), anti-mCherry immunostaining (Figures 13A2 and 13B2: green), DAPI nuclear counterstaining (Figures 13A3 and 13B3: blue) and combined immunofluorescence and DAPI images (Figures 13A4 and 13B4).

[0118] As shown in Figures 14A1-14B4, LAP2 drives long-term transgene expression in the mouse retina. Intravenous administration of AAV-LAP2 (Figures 14A1-14A4) or AAV-EF1α-vector (Figures 14B1-14B4) was performed by unilateral injection into the mouse retro-orbital sinus (4x10 11 vg / mouse). Retinas were harvested at 190 dpi and cryosectioned transversely at 20 μm for subsequent IHC and confocal microscopy analysis. Representative confocal images show native mCherry fluorescence (Figures 14A1 and 14B1: red), anti-mCherry immunostaining (Figures 14A2 and 14B2: green), DAPI nuclear counterstaining (Figures 14A3 and 14B3: blue) and combined immunofluorescence and DAPI images (Figures 14A4 and 14B4).

[0119] reference [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

[0120] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0121] While example embodiments have been specifically shown and described, it will be understood by those skilled in the art that various changes in form and detail can be made in the present invention without departing from the scope of the embodiments encompassed by the appended claims.

[0122] The aspects of the present invention include the following. Item 1 a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; a cloning site for insertion of a nucleic acid encoding a target gene; and At least one non-promoter regulatory element required for expression of the target gene in a host cell. A recombinant vector for expressing a target gene in a host cell, comprising: Section 2 Item 2. The recombinant vector according to item 1, wherein the vector is a viral vector. Section 3 Item 3. The recombinant vector according to item 2, wherein the viral vector is an adeno-associated viral (AAV) vector. Section 4 Item 2. The recombinant vector of item 1, wherein the vector is a non-viral vector. Section 5 Item 5. The recombinant vector according to any one of Items 1 to 4, wherein the host cell is a mammalian cell. Section 6 6. The recombinant vector according to any one of items 1 to 5, wherein the host cell comprises an in vitro neuronal cell, an ex vivo neuronal cell, or both. Section 7 6. The recombinant vector according to any one of items 1 to 5, wherein the host cell comprises a neuronal cell of a subject. Section 8 8. The recombinant vector of paragraph 7, wherein the neuronal cells are present in the primary motor cortex, secondary motor cortex, primary somatosensory cortex, supplementary somatosensory cortex, visual cortex, hippocampal formation, striatum, globus pallidus, thalamus, hypothalamus, midbrain region, hindbrain, and olfactory cortex, or a combination thereof, of the subject. Section 9 9. The recombinant vector according to item 8, wherein the midbrain region comprises a motor-related cortex, a sensory-related cortex, or both. Item 10 8. The recombinant vector of paragraph 7, wherein the neuronal cell is present in the cortex, dentate gyrus, striatum, cerebellum, olfactory bulb, or a combination thereof of the subject. Section 11 Item 11. The recombinant vector according to any one of items 6 to 10, wherein the neuronal cells include neurons, microglial cells, astrocytes, or a combination thereof. Section 12 12. The recombinant vector of paragraph 11, wherein the neuronal cell comprises a neuron. Section 13 The recombinant vector of paragraph 7, wherein the neuronal cell is present in the spinal cord of the subject. Section 14 14. The recombinant vector of paragraph 13, wherein the neuronal cell is present in the dorsal horn of the spinal cord. Item 15 15. The recombinant vector of paragraph 13 or 14, wherein the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof. Section 16 6. The recombinant vector according to any one of items 1 to 5, wherein the host cell is selected from the group consisting of a bladder cell, a blood cell, a bone cell, an endothelial cell, an epithelial cell, an adipocyte, a cardiac cell, a small intestinal cell, a kidney cell, a liver cell, a lung cell, a muscle cell, a pancreatic cell, a retinal cell, a germ cell, a skin cell, a spleen cell, a stem cell, and a combination thereof. Section 17 Item 17. The recombinant vector according to any one of items 1 to 16, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Section 18 18. The recombinant vector of paragraph 17, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 19 Item 19. The recombinant vector according to any one of items 1 to 18, wherein at least one non-promoter regulatory element comprises an enhancer element. Section 20 20. The recombinant vector according to any one of items 1 to 19, wherein at least one non-promoter regulatory element comprises an inducer element. Section 21 21. The recombinant vector according to any one of items 1 to 20, further comprising a selectable marker element. Section 22 A host cell comprising the recombinant vector according to any one of items 1 to 21. Section 23 A recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 24 24. The recombinant polynucleotide molecule of paragraph 23, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 25 25. The recombinant polynucleotide molecule of paragraph 24, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 26 26. The recombinant polynucleotide molecule according to any one of items 23 to 25, wherein the nucleic acid encoding the target gene is larger than 4 kilobases. Section 27 A recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, the cloning site being operably linked to a promoter comprising a nucleic acid having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 28 28. The recombinant polynucleotide molecule of paragraph 27, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 29 30. The recombinant polynucleotide molecule of paragraph 28, wherein the promoter comprises a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 30 1. A method for expressing a target gene in a host cell, comprising contacting a host cell with a recombinant polynucleotide molecule such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding the target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; A method wherein expression of the target gene occurs in a host cell. Section 31 31. The method of paragraph 30, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Section 32 32. The method of claim 31, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Item 33 33. The method according to any one of items 30 to 32, wherein when contacting the host cell with the recombinant polynucleotide, the recombinant polynucleotide molecule is introduced into the host cell by transduction. Section 34 33. The method according to any one of items 30 to 32, wherein when the host cell is contacted with the recombinant polynucleotide, the recombinant polynucleotide molecule is introduced into the host cell by transfection. Item 35 35. The method according to any one of items 30 to 34, wherein the host cell is a mammalian cell. Section 36 36. The method of any of paragraphs 30 to 35, wherein the host cells comprise in vitro neuronal cells, ex vivo neuronal cells, or both. Section 37 36. The method of any one of items 30 to 35, wherein the host cells comprise neuronal cells of the subject. Section 38 38. The method of paragraph 37, wherein the neuronal cells are present in the primary motor cortex, secondary motor cortex, primary somatosensory cortex, supplementary somatosensory cortex, visual cortex, hippocampal formation, striatum, globus pallidus, thalamus, hypothalamus, midbrain region, hindbrain, olfactory cortex, or a combination thereof of the subject. Item 39 39. The method of paragraph 38, wherein the midbrain region includes motor-related areas, sensory-related areas, or both. Section 40 38. The method of paragraph 37, wherein the neuronal cells are present in the subject's cortex, dentate gyrus, striatum, cerebellum, olfactory bulb, or a combination thereof. Section 41 41. The method of any of items 36 to 40, wherein the neuronal cells comprise neurons, microglial cells, astrocytes, or a combination thereof. Section 42 The method of paragraph 41, wherein the neuronal cell comprises a neuron. Section 43 38. The method of paragraph 37, wherein the neuronal cells are present in the spinal cord of the subject. Section 44 The method of paragraph 43, wherein the neuronal cells are present in the dorsal horn of the spinal cord. Section 45 45. The method of paragraph 43 or 44, wherein the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof. Section 46 36. The method according to any one of items 30 to 35, wherein the host cells are selected from the group consisting of bladder cells, blood cells, bone cells, endothelial cells, epithelial cells, fat cells, cardiac cells, intestinal cells, kidney cells, liver cells, lung cells, muscle cells, pancreatic cells, retinal cells, germ cells, skin cells, spleen cells, stem cells, and combinations thereof. Section 47 a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; a cloning site for insertion of a nucleic acid encoding a target gene; and At least one non-promoter regulatory element required for expression of the target gene in a host cell. A recombinant vector for expressing a target gene in a host cell, comprising: Section 48 48. The recombinant vector of paragraph 47, wherein the vector is a viral vector. Section 49 49. The recombinant vector of paragraph 48, wherein the viral vector is an adeno-associated viral (AAV) vector. Section 50 48. The recombinant vector of paragraph 47, wherein the vector is a non-viral vector. Section 51 51. The recombinant vector according to any one of items 47 to 50, wherein the host cell is a mammalian cell. Section 52 52. The recombinant vector of any one of paragraphs 47 to 51, wherein the host cell comprises an in vitro neuronal cell, an ex vivo neuronal cell, or both. Section 53 52. The recombinant vector according to any one of items 47 to 51, wherein the host cell comprises a neuronal cell of a subject. Section 54 54. The recombinant vector of paragraph 53, wherein the neuronal cells are present in the primary motor cortex, secondary motor cortex, primary somatosensory cortex, supplementary somatosensory cortex, visual cortex, hippocampal formation, striatum, globus pallidus, thalamus, hypothalamus, midbrain region, hindbrain, olfactory cortex, or a combination thereof of the subject. Section 55 55. The recombinant vector of paragraph 54, wherein the midbrain region comprises a motor-related cortex, a sensory-related cortex, or both. Section 56 54. The recombinant vector of paragraph 53, wherein the neuronal cell is present in the cortex, dentate gyrus, striatum, cerebellum, olfactory bulb, or a combination thereof, of the subject. Section 57 57. The recombinant vector according to any one of items 52 to 56, wherein the neuronal cells comprise neurons, microglial cells, astrocytes, or a combination thereof. Section 58 58. The recombinant vector of paragraph 57, wherein the neuronal cell comprises a neuron. Section 59 54. The recombinant vector of paragraph 53, wherein the neuronal cell is present in the spinal cord of the subject. Section 60 60. The recombinant vector of paragraph 59, wherein the neuronal cell is present in the dorsal horn of the spinal cord. Section 61 61. The recombinant vector of paragraph 59 or 60, wherein the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof. Section 62 52. The recombinant vector according to any one of items 47 to 51, wherein the host cell is selected from the group consisting of a bladder cell, a blood cell, a bone cell, an endothelial cell, an epithelial cell, an adipocyte, a cardiac cell, a small intestinal cell, a kidney cell, a liver cell, a lung cell, a muscle cell, a pancreatic cell, a retinal cell, a germ cell, a skin cell, a spleen cell, a stem cell, and a combination thereof. Section 63 63. The recombinant vector according to any one of items 47 to 62, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 64 The recombinant polynucleotide molecule of Paragraph 63, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 65 65. The recombinant vector according to any one of items 47 to 64, wherein at least one non-promoter regulatory element comprises an enhancer element. Section 66 66. The recombinant vector according to any one of items 47 to 65, wherein at least one non-promoter regulatory element comprises an inducer element. Section 67 Item 67. The recombinant vector according to any one of Items 47 to 66, further comprising a selectable marker element. Section 68 Item 68. A host cell comprising the recombinant vector according to any one of Items 47 to 67. Section 69 A recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 70 The recombinant polynucleotide molecule of paragraph 69, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 71 The recombinant polynucleotide molecule of Paragraph 70, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 72 72. The recombinant polynucleotide molecule of any one of claims 69 to 71, wherein the nucleic acid encoding the target gene is greater than 4 kilobases. Section 73 A recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid having at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 74 The recombinant polynucleotide molecule of paragraph 73, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 75 The recombinant polynucleotide molecule of Paragraph 74, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 76 1. A method for expressing a target gene in a host cell, comprising contacting a host cell with a recombinant polynucleotide molecule such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding the target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; A method wherein expression of the target gene occurs in a host cell. Section 77 The method of paragraph 76, wherein the host cell is a mammalian cell. Section 78 78. The method of paragraph 76 or 77, wherein the host cells comprise in vitro neuronal cells, ex vivo neuronal cells, or both. Section 79 78. The method of paragraph 76 or 77, wherein the host cells comprise neuronal cells of the subject. Section 80 80. The method of paragraph 79, wherein the neuronal cells are present in the primary motor cortex, secondary motor cortex, primary somatosensory cortex, supplementary somatosensory cortex, visual cortex, hippocampal formation, striatum, globus pallidus, thalamus, hypothalamus, midbrain region, hindbrain, olfactory cortex, or a combination thereof of the subject. Section 81 81. The method of paragraph 80, wherein the midbrain region includes motor-related areas, sensory-related areas or both. Section 82 80. The method of paragraph 79, wherein the neuronal cells are present in the subject's cortex, dentate gyrus, striatum, cerebellum, olfactory bulb, or a combination thereof. Section 83 83. The method of any of paragraphs 78 to 82, wherein the neuronal cells comprise neurons, microglial cells, astrocytes, or a combination thereof. Section 84 The method of paragraph 83, wherein the neuronal cell comprises a neuron. Section 85 80. The method of paragraph 79, wherein the neuronal cells are present in the spinal cord of the subject. Section 86 The method of paragraph 85, wherein the neuronal cell is present in the dorsal horn of the spinal cord. Section 87 87. The method of paragraph 85 or 86, wherein the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof. Section 88 78. The method of paragraph 76 or 77, wherein the host cells are selected from the group consisting of capsular cells, blood cells, bone cells, endothelial cells, epithelial cells, fat cells, cardiac cells, intestinal cells, kidney cells, liver cells, lung cells, muscle cells, pancreatic cells, retinal cells, germ cells, skin cells, spleen cells, stem cells and combinations thereof. Section 89 89. The method of any of items 76 to 88, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 90 The method of paragraph 89, wherein the promoter comprises a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Section 91 91. The method of any one of paragraphs 76 to 90, wherein when contacting the host cell with the recombinant polynucleotide, the recombinant polynucleotide molecule is introduced into the host cell by transduction. Section 92 91. The method of any one of paragraphs 76 to 90, wherein when contacting the host cell with the recombinant polynucleotide, the recombinant polynucleotide molecule is introduced into the host cell by transfection. Section 93 a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9; a cloning site for insertion of a nucleic acid encoding a target gene; and At least one non-promoter regulatory element required for expression of the target gene in a host cell. A recombinant vector for expressing a target gene in a host cell, comprising: Section 94 The recombinant vector of paragraph 93, wherein the vector is a viral vector. Section 95 95. The recombinant vector of paragraph 94, wherein the viral vector is an adeno-associated viral (AAV) vector. Section 96 The recombinant vector of paragraph 93, wherein the vector is a non-viral vector. Section 97 Item 97. The recombinant vector according to any one of Items 93 to 96, wherein the host cell is a mammalian cell. Section 98 98. The recombinant vector of any one of paragraphs 93 to 97, wherein the host cell comprises an in vitro neuronal cell, an ex vivo neuronal cell, or both. Section 99 98. The recombinant vector of any one of claims 93 to 97, wherein the host cell comprises a neuronal cell of a subject. Item 100 100. The recombinant vector of paragraph 99, wherein the neuronal cells are present in the primary motor cortex, secondary motor cortex, primary somatosensory cortex, supplementary somatosensory cortex, visual cortex, hippocampal formation, striatum, globus pallidus, thalamus, hypothalamus, midbrain region, hindbrain, olfactory cortex, or a combination thereof of the subject. Section 101 101. The recombinant vector of paragraph 100, wherein the midbrain region comprises a motor-related cortex, a sensory-related cortex, or both. Section 102 100. The recombinant vector of paragraph 99, wherein the neuronal cell is present in the cortex, dentate gyrus, striatum, cerebellum, olfactory bulb, or a combination thereof, of the subject. Section 103 103. The recombinant vector of any of paragraphs 98 to 102, wherein the neuronal cells comprise neurons, microglial cells, astrocytes, or a combination thereof. Section 104 The recombinant vector of paragraph 103, wherein the neuronal cell comprises a neuron. Section 105 100. The recombinant vector of paragraph 99, wherein the neuronal cell is present in the spinal cord of the subject. Section 106 The recombinant vector of paragraph 105, wherein the neuronal cell is present in the dorsal horn of the spinal cord. Section 107 107. The recombinant vector of paragraph 105 or 106, wherein the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof. Section 108 98. The recombinant vector according to any one of items 93 to 97, wherein the host cell is selected from the group consisting of a bladder cell, a blood cell, a bone cell, an endothelial cell, an epithelial cell, an adipocyte, a cardiac cell, a small intestinal cell, a kidney cell, a liver cell, a lung cell, a muscle cell, a pancreatic cell, a retinal cell, a germ cell, a skin cell, a spleen cell, a stem cell, and combinations thereof. Section 109 104. The recombinant vector according to any one of items 93 to 103, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9. Section 110 110. The recombinant vector of paragraph 109, wherein the promoter comprises SEQ ID NO:9. Section 111 111. The recombinant vector of any of items 93 to 110, wherein the promoter further comprises the nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or a combination thereof. Section 112 109. The recombinant vector according to any one of items 93 to 108, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13 to 58. Section 113 Item 113. The recombinant vector according to Item 112, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13 to 58. Section 114 114. The recombinant vector of any one of items 93 to 113, wherein at least one non-promoter regulatory element comprises an enhancer element. Section 115 115. The recombinant vector according to any one of items 93 to 114, wherein at least one non-promoter regulatory element comprises an inducer element. Section 116 Item 116. The recombinant vector according to any one of Items 93 to 115, further comprising a selectable marker element. Section 117 A host cell comprising the recombinant vector according to any one of items 93 to 116. Section 118 A recombinant polynucleotide molecule comprising a nucleic acid encoding a target gene, said nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9. Section 119 The recombinant polynucleotide molecule of Paragraph 118, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9. Section 120 The recombinant polynucleotide molecule of Paragraph 119, wherein the promoter comprises SEQ ID NO:9. Section 121 121. The recombinant polynucleotide molecule of any of paragraphs 118 to 120, wherein the promoter further comprises the nucleic acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof. Section 122 19. The recombinant polynucleotide molecule of paragraph 118, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13-58. Section 123 123. The recombinant polynucleotide molecule of any one of paragraphs 118 to 122, wherein the nucleic acid encoding the target gene is greater than 4 kilobases. Section 124 A recombinant polynucleotide molecule comprising a cloning site for insertion of a nucleic acid encoding a target gene, wherein the cloning site is operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9. Section 125 The recombinant polynucleotide molecule of Paragraph 124, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9. Section 126 The recombinant polynucleotide molecule of Paragraph 125, wherein the promoter comprises SEQ ID NO:9. Section 127 127. The recombinant polynucleotide molecule of any of paragraphs 124 to 126, wherein the promoter further comprises the nucleic acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof. Section 128 125. The recombinant polynucleotide molecule of paragraph 124, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13-58. Section 129 129. The recombinant polynucleotide molecule of paragraph 128, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13-58. Section 130 1. A method for expressing a target gene in a host cell, comprising contacting a host cell with a recombinant polynucleotide molecule such that the recombinant polynucleotide molecule is introduced into the host cell, wherein the recombinant polynucleotide molecule comprises a nucleic acid encoding the target gene, the nucleic acid being operably linked to a promoter comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:9; A method wherein expression of the target gene occurs in a host cell. Section 131 The method of paragraph 130, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9. Section 132 The method of paragraph 131, wherein the promoter comprises SEQ ID NO:9. Section 133 133. The method of any of items 130 to 132, wherein the promoter further comprises the nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or a combination thereof. Section 134 131. The method of paragraph 130, wherein the promoter comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 13-58. Section 135 135. The method of claim 134, wherein the promoter comprises a nucleic acid sequence selected from SEQ ID NOs: 13-58. Section 136 136. The method of any of paragraphs 130 to 135, wherein when contacting the host cell with the recombinant polynucleotide, the recombinant polynucleotide molecule is introduced into the host cell by transduction. Section 137 136. The method of any of paragraphs 130 to 135, wherein when contacting the host cell with the recombinant polynucleotide, the recombinant polynucleotide molecule is introduced into the host cell by transfection. Section 138 138. The method of any one of items 130 to 137, wherein the host cell is a mammalian cell. Section 139 139. The method of any of paragraphs 130 to 138, wherein the host cell comprises an in vitro neuronal cell, an ex vivo neuronal cell, or both. Section 140 139. The method of any of paragraphs 130 to 138, wherein the host cells comprise neuronal cells of the subject. Section 141 141. The method of paragraph 140, wherein the neuronal cells are present in the primary motor cortex, secondary motor cortex, primary somatosensory cortex, supplementary somatosensory cortex, visual cortex, hippocampal formation, striatum, globus pallidus, thalamus, hypothalamus, midbrain region, hindbrain, olfactory cortex, or a combination thereof of the subject. Section 142 142. The method of paragraph 141, wherein the midbrain region includes motor-related areas, sensory-related areas or both. Section 143 The method of paragraph 140, wherein the neuronal cells are present in the subject's cortex, dentate gyrus, striatum, cerebellum, olfactory bulb, or a combination thereof. Section 144 144. The method of any of paragraphs 140 to 143, wherein the neuronal cells comprise neurons, microglial cells, astrocytes, or a combination thereof. Section 145 The method of paragraph 144, wherein the neuronal cell comprises a neuron. Section 146 The method of paragraph 140, wherein the neuronal cells are present in the spinal cord of the subject. Section 147 The method of paragraph 146, wherein the neuronal cells are present in the dorsal horn of the spinal cord. Section 148 148. The method of paragraph 146 or 147, wherein the neuronal cells are present in the lumbar spinal cord, the thoracic spinal cord, the cervical spinal cord, or a combination thereof. Section 149 139. The method of any of paragraphs 130 to 138, wherein the host cells are selected from the group consisting of bladder cells, blood cells, bone cells, endothelial cells, epithelial cells, fat cells, cardiac cells, small intestinal cells, kidney cells, liver cells, lung cells, muscle cells, pancreatic cells, retinal cells, germ cells, skin cells, spleen cells, stem cells and combinations thereof.

Claims

[Claim 1] a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; a cloning site for insertion of a nucleic acid encoding a target gene; and At least one non-promoter regulatory element required for expression of the target gene in a host cell. A recombinant vector for expressing a target gene in a host cell, comprising:

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