Genetic regulatory elements and uses thereof

Abstract

Provided herein are regulatory elements capable of defining expression of heterologous nucleotide sequences to specific populations and regions of neurons of the central nervous system (CNS). Also provided herein are vectors comprising such regulatory elements and the use of such regulatory elements and vectors for selective expression in the CNS.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 354,110, filed on June 21, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (REGT_002_01WO_SeqList_ST26.xml; size: 31,745 bytes; and creation date: June 14, 2023) are incorporated herein by reference in their entirety). Technical Field

[0005] The present disclosure relates to genetic regulatory elements and their use for expressing heterologous nucleotide sequences. Background Art

[0006] Gene therapy has the potential to revolutionize the treatment of genetic disorders. Clinical application of this promising technology requires overcoming significant technical limitations. One of these is the need to specifically limit therapeutic intervention to the cell population affected by the disease in order to reduce off-target effects and improve the efficacy of the therapy. Summary of the invention

[0007] Provided herein is a vector comprising (i) a heterologous nucleotide sequence; and (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. or (e) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer, wherein the one or more regions have at least 75% or higher sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In some embodiments, the vector further comprises a promoter.

[0008] In some embodiments, the vector comprises, in 5′ to 3′ order: (i) the promoter; (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:10 and the reverse complementary nucleotide sequence of any one of SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (iii) the heterologous nucleotide sequence;

[0009] In some embodiments, the vector comprises, in 5' to 3' order: (i) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (ii) the promoter; and (iii) the heterologous nucleotide sequence;

[0010] In some embodiments, the vector comprises, in 5′ to 3′ order: (i) the promoter; (ii) the heterologous nucleotide sequence; and (iii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer and having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

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

[0012] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or an adenoviral vector.

[0013] In some embodiments, the AAV vector comprises a 5′ AAV inverted terminal repeat (ITR) and a 3′ AAV ITR flanking the heterologous nucleotide sequence, a promoter, and (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) an inverted nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0014] In some embodiments, the AAV vector comprises, in 5′ to 3′ order: (i) the 5′ AAV ITR; (ii) the promoter; (iii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

[0015] In some embodiments, the AAV vector comprises, in 5′ to 3′ order: (i) the 5′AAVITR; (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:9, the reverse complementary nucleotide sequence of any one of SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer, having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (iii) the promoter; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

[0016] In some embodiments, the AAV vector comprises, in 5′ to 3′ order: (i) the 5′ AAV ITR; (ii) the promoter; (iii) the heterologous nucleotide sequence; (iv) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (v) the 3′ AAV ITR.

[0017] In some embodiments, the promoter comprises an RNA-polymerase II binding site, a TATA box, a TF2B recognition element (BRE), a motif 10 element (MTE), or a downstream promoter element (DPE).

[0018] In some embodiments, the promoter is a minimal promoter. In some embodiments, the minimal promoter is the human β-globin minimal promoter.

[0019] In some embodiments, the vector further comprises an enhancer. In some embodiments, the vector further comprises or encodes a polyadenylation signal sequence. In some embodiments, the vector further comprises or encodes a Kozak sequence. In some embodiments, the vector further comprises or encodes a woodchuck hepatitis virus post-transcriptional element (WPRE), a hepatitis B virus post-transcriptional regulatory element, an RNA transport element (RTE), a WPRE3 or a wsl3 regulatory element. In some embodiments, the vector further comprises an artificial intron. In some embodiments, the vector further encodes a 2A self-cleaving peptide. In some embodiments, the vector further comprises or encodes an internal ribosome entry site (IRES).

[0020] In some embodiments, the heterologous nucleotide sequence encodes a protein. In some embodiments, the heterologous nucleotide sequence encodes a gene silencing nucleic acid. In some embodiments, the heterologous nucleotide sequence encodes a CRISPR-associated protein. In some embodiments, the heterologous nucleotide sequence also encodes a guide RNA (gRNA).

[0021] Provided herein is a viral particle comprising a vector disclosed herein. In some embodiments, the viral particle is a recombinant adenoviral particle. In some embodiments, the viral particle is a recombinant AAV (rAAV) particle. In some embodiments, the rAAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV26, AAV27, AAV28, AAV29, AAV30, AAV31, AAV32, AAV33, AAV34, AAV35, AAV36, AAV37, AAV38, AAV39, AAV40, AAV41, AAV42, AAV43, AAV44, AAV45, AAV46, AAV47, AAV48, AAV49, AAV50, AAV510, AAV52, AAV53, AAV54, AAV55, AAV56, AAV57, AAV58, AAV59, AAV6, AAV6, AAV6, AAV7, AAV7, AAV8, AAV9, AAV110, AAV1112, AAV13 3. AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAVAnc80, TM-AAV6, AAV-PHP.A, AAV-PHP.B, AAV-PHR.S, AAV-PHP.eB, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV2-TLH, or AAV2-GMN serotype particles.

[0022] Provided herein is a population of viral particles comprising a plurality of viral particles disclosed herein.

[0023] Provided herein is a pharmaceutical composition comprising a vector, viral particle or population disclosed herein, and a pharmaceutically acceptable carrier, excipient or diluent.

[0024] Provided herein is a nanoparticle comprising a carrier disclosed herein.

[0025] Provided herein is a cell comprising a vector or viral particle disclosed herein. In some embodiments, the cell is a mammalian cell or an insect cell.

[0026] Provided herein is a method for producing rAAV particles, the method comprising: (i) culturing cells disclosed herein under conditions that allow packaging of the rAAV particles; and (ii) harvesting the cultured host cells or culture medium for collecting the rAAV particles.

[0027] Provided herein is a method for selectively expressing a heterologous nucleotide sequence in neurons of the central nervous system (CNS) of a subject in need thereof, the method comprising administering to the subject a vector, viral particle, population, pharmaceutical composition or nanoparticle disclosed herein.

[0028] In some embodiments of the methods provided herein, the heterologous nucleotide sequence is expressed and enriched in a specific region of the CNS. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of one or more of the following: forebrain, cerebellum, and spinal cord. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of the forebrain and cerebellum. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of the forebrain. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of the cerebellum. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of the cerebellum. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of the cerebellum and hippocampus. In some embodiments, the heterologous nucleotide sequence is expressed and enriched in neurons of the spinal cord.

[0029] In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10, and expression of the heterologous nucleotide sequence is enriched in neurons of the CNS.

[0030] In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 9, and expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain and the cerebellum.

[0031] In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 11, and expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain.

[0032] In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 6, and expression of the heterologous nucleotide sequence is enriched in neurons of the cerebellum and hippocampus.

[0033] In some embodiments of the methods provided herein, the vector, the viral particle, the colony, the pharmaceutical composition, or the nanoparticle is administered intravenously to the subject. In some embodiments of the methods provided herein, the vector, the viral particle, the colony, the pharmaceutical composition, or the nanoparticle is administered to the subject via injection into the CNS. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A Schematic diagram and representative images depicting transgene expression profiles across organs. Recombinant adeno-associated virus (rAAV) particles (with AAV-PHP.eB capsid) containing regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V3 (SEQ ID NO: 2), CNS2_V7 (SEQ ID NO: 4) and CNS2_V10 (SEQ ID NO: 6) (from top to bottom) were systemically injected into adult mice after 3 weeks of incubation, and the expression profiles of the regulatory elements in the indicated organs are shown. Representative images show the expression of viral reporter genes. n = 3 mice per construct. Organs shown from left to right are: sagittal IHC (immunohistochemistry); cortex; hippocampus; cerebellum; spinal cord; DRG (dorsal root ganglion); liver; heart; muscle; lung.

[0035] Figure 1B Schematic diagram and representative images depicting transgene expression profiles across organs. RAAV particles (with AAV-PHP.eB capsid) containing regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V4.1 (SEQ ID NO: 10), CNS2_V3 (SEQ ID NO: 2), CNS2_V3.3 (SEQ ID NO: 9), CNS2_V7 (SEQ ID NO: 4), CNS2_V7.1 (SEQ ID NO: 11), CNS2_V11 (SEQ ID NO: 5) and CNS2_V10 (SEQ ID NO: 6) (from top to bottom) were injected systemically into adult mice after 3 weeks of incubation, and the expression profiles of the regulatory elements in the indicated organs are shown. Representative images show the expression of viral reporter genes. N = at least 3 mice per construct. The organs shown from left to right are: sagittal IHC (immunohistochemistry); cortex; hippocampus; striatum; cerebellum; spinal cord; DRG (dorsal root ganglion); liver; heart; muscle; lung.

[0036] Figure 2A-2C Depicted are the results of quantitative analysis of the intensity, specificity and sensitivity of transgene expression using regulatory elements CNS2_V3 (V3) (SEQ ID NO: 2), CNS2_V4 (V4) (SEQ ID NO: 1) and CNS2_V7 (V7) (SEQ ID NO: 4). Results are shown for neurons in the cortex (expressing the pan-neuronal marker NeuN), corresponding to Figure 1A The data presented in . Figure 2A Results of analyses of neuronal specificity are depicted. Figure 2B Results of the analysis of neuronal sensitivity are depicted. Figure 2C Results of the analysis of cortical layer sensitivity are depicted.

[0037] Figure 3A-3D Depicted are the results of quantitative analysis of the intensity, specificity and sensitivity of transgene expression using regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V4.1 (SEQ ID NO: 10), CNS2_V3 (SEQ ID NO: 2), CNS2_V3.3 (SEQ ID NO: 9), CNS2_V7 (SEQ ID NO: 4) and CNS2_V7.1 (SEQ ID NO: 11). Results are shown for neurons in the relevant region (expressing the pan-neuronal marker NeuN), corresponding to Figure 1B The data presented in . Figure 3A Depicted are results of an analysis of the specificity of neurons within the somatosensory cortex (ssctx). Figure 3B Depicted are results of an analysis of the sensitivity of neurons within the somatosensory cortex (ssctx). Figure 3C Results of the analysis of cortical layer sensitivity are depicted. Figure 3D Depicted are the results of an analysis of the sensitivity of neurons within the striatum.

[0038] Figure 4A-4B Depicted are the results of a study of transgene expression following intracerebroventricular (ICV) injection of rAAV particles (with AAV9 capsid) containing the regulatory element CNS2_V4.1 (SEQ ID NO: 10) into postnatal day 1 (PND1) mice after 4 weeks of incubation. Figure 4A Images depicting the expression of a fluorescent reporter gene (dTomato) driven by CNS2_V4.1 in the brain. Figure 4B Depicted are the results of analysis of dCas9 transgene expression driven by CNS2_V4.1 detected by qPCR. DETAILED DESCRIPTION

[0039] The present disclosure provides regulatory elements capable of selectively expressing heterologous nucleotide sequences in specific neuronal populations and regions of the central nervous system (CNS), while reducing or preventing the heterologous nucleotide sequences from being expressed in other organs. The regulatory elements enrich the expression of heterologous nucleotide sequences in neurons and reduce or prevent expression in other cell populations (e.g., glial cells, microglia, and endothelial cells). When integrated into a DNA-based expression vector, the regulatory elements can be used to reduce off-target effects and increase the efficacy of gene therapy. The regulatory elements show evidence of broad applicability, because the same regulatory elements with different rAAV serotypes are delivered in mice of different ages, and different transgenes are expressed, showing the same expression profile.

[0040] Regulatory elements and vectors

[0041] Provided herein are genetic control elements for selectively expressing heterologous nucleotide sequences in neurons of CNS.When integrated into an expression vector based on DNA, these control elements can limit the expression of heterologous nucleotide sequences to neurons of CNS.In some embodiments, the control element limits the expression of heterologous nucleotide sequences to neurons of CNS, with at least about 75%, about 80%, about 85%, about 90%, about 95% or about 99% specificity to the cell type.In some embodiments, the control element limits the expression of heterologous nucleotide sequences to neurons of CNS, with at least about 75%, about 80%, about 85%, about 90%, about 95% or about 99% selectivity to the cell type.

[0042] In some embodiments, the regulatory element restricts the expression of the heterologous nucleotide sequence to neurons of the forebrain and cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for this cell type. In some embodiments, the regulatory element restricts the expression of the heterologous nucleotide sequence to neurons of the forebrain and cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for this cell type.

[0043] In some embodiments, the regulatory element restricts expression of the heterologous nucleotide sequence to neurons of the forebrain with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for the cell type. In some embodiments, the regulatory element restricts expression of the heterologous nucleotide sequence to neurons of the forebrain with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for the cell type.

[0044] In some embodiments, the regulatory element restricts expression of the heterologous nucleotide sequence to neurons of the cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for the cell type. In some embodiments, the regulatory element restricts expression of the heterologous nucleotide sequence to neurons of the cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for the cell type.

[0045] In some embodiments, the regulatory element restricts the expression of the heterologous nucleotide sequence to neurons of the cerebellum and neurogenic niches with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for the cell type. In some embodiments, the regulatory element restricts the expression of the heterologous nucleotide sequence to neurons of the cerebellum and neurogenic niches with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for the cell type.

[0046] In some embodiments, the regulatory element expresses a heterologous nucleotide sequence in a non-neuronal cell of the CNS (e.g., a glial cell, a microglia cell, or an endothelial cell) with up to about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% specificity for the cell type. In some embodiments, the regulatory element expresses a heterologous nucleotide sequence in a non-neuronal cell of the CNS (e.g., a glial cell, a microglia cell, or an endothelial cell) with up to about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% selectivity for the cell type.

[0047] In some embodiments, the regulatory element does not express a heterologous nucleotide sequence in one or more of liver, heart, muscle, and lung.

[0048] Provided herein is a genetic regulatory element comprising a nucleotide sequence of any one of SEQ ID NOs: 1-13, a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or a reverse complementary sequence of any one of SEQ ID NOs: 1-13.

[0049] Provided herein is a genetic control element comprising a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; or a reverse complementary sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

[0050] Provided herein is a vector (e.g., a DNA vector) comprising (i) a heterologous nucleotide sequence; and (ii) a nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or a reverse complementary sequence of any one of SEQ ID NOs: 1-13.

[0051] Provided herein is a vector (e.g., a DNA vector) comprising (i) a heterologous nucleotide sequence; and (ii) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; The reverse complementary sequence of any one of NO:11.

[0052] In some embodiments, the vector comprises (i) a heterologous nucleotide sequence; and (ii) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer having at least 75%, 80%, 85%, 90%, 95%, 97% or 99% or more sequence identity to any one of SEQ ID NOs: 1-13.

[0053] In some embodiments, the vector comprises (i) a heterologous nucleotide sequence; and (ii) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer, wherein the one or more regions have at least 75%, 80%, 85%, 90%, 95%, 97% or 99% or more sequence identity to any one of SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

[0054] In some embodiments, the vector comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 regulatory elements disclosed herein. In some embodiments, the vector comprises 2, 3, 4, 5, 6, 7 or 8 regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V4.1 (SEQ ID NO: 10), CNS2_V3 (SEQ ID NO: 2), CNS2_V3.3 (SEQ ID NO: 9), CNS2_V7 (SEQ ID NO: 4), CNS2_V7.1 (SEQ ID NO: 11), CNS2_V11 (SEQ ID NO: 5) and CNS2_V10 (SEQ ID NO: 6).

[0055] In some embodiments, the vectors provided herein further comprise a promoter. In some embodiments, the promoter is a minimal promoter. In some embodiments, the minimal promoter comprises one or more short sequences capable of promoting transcription. In some embodiments, the promoter comprises an RNA-polymerase II binding site, a TATA box, a TF2B recognition element (BRE), a motif 10 element (MTE), or a downstream promoter element (DPE). In, for example, et al., (2005) J. Virol. 79(17): 11082-11094 describe the TATA transcriptional regulatory activation site.

[0056] In some embodiments, the promoter is the human β-globin minimal promoter.

[0057] In some embodiments, the promoter is recognized by RNA polymerase II. In some embodiments, the promoter is recognized by RNA polymerase II. In some embodiments, the promoter is human U6 (hU6) promoter, mouse U6 promoter or human H1 promoter.

[0058] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter, a GUSB240 promoter, a GUSB379 promoter, a HSVTK promoter, a CMV promoter, an SV40 early promoter, a SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus (MMTV) promoter, a Rous sarcoma virus (RSV) promoter, a polyhedrin promoter, an EF-1α promoter, a dihydrofolate reductase (DHFR) promoter, or a phosphoglycerol kinase (PGK) promoter.

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] In some embodiments, the vector provided herein comprises (a) a promoter; (b) a nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse complementary sequence of any one of SEQ ID NOs: 1-13; or a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13; and (c) a heterologous nucleotide sequence.

[0065] In some embodiments, the vectors provided herein comprise (a) a promoter; (b) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:11; or a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (c) a heterologous nucleotide sequence.

[0066] In some embodiments, the vector provided herein comprises, in 5′ to 3′ order: (i) a promoter; (ii) (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-1 3; and (iii) the heterologous nucleotide sequence;

[0067] In some embodiments, the vectors provided herein comprise, in 5′ to 3′ order: (i) a promoter; (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:10 and the reverse complementary nucleotide sequence of any one of SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (iii) the heterologous nucleotide sequence;

[0068] In some embodiments, the vector provided herein comprises, in 5′ to 3′ order: (i) (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-1 3; and (ii) the promoter; and (iii) the heterologous nucleotide sequence;

[0069] In some embodiments, the vectors provided herein comprise, in 5' to 3' order: (i) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (ii) the promoter; and (iii) the heterologous nucleotide sequence;

[0070] In some embodiments, the vector provided herein comprises, in 5′ to 3′ order: (i) a promoter; (ii) the heterologous nucleotide sequence; and (iii) (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13.

[0071] In some embodiments, the vectors provided herein comprise, in 5′ to 3′ order: (i) a promoter; (ii) the heterologous nucleotide sequence; and (iii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:6, SEQ ID NO:9, SEQ ID NO:1θ and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer and having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0072] In some embodiments, the vectors provided herein further comprise an enhancer.

[0073] In some embodiments, the vectors provided herein further comprise or encode a polyadenylation (polyA) signal sequence. As used herein, a "polyadenylation signal sequence" refers to a DNA sequence that regulates the addition of a polyA tail to an mRNA transcript when transcribed. In some embodiments, the polyA signal sequence is an SV40, human, cattle, or rabbit polyA signal sequence. In some embodiments, the polyA signal sequence is an SV40 polyA signal sequence. In some embodiments, the polyA signal sequence is a β-globin polyA signal sequence. In some embodiments, the polyA signal sequence is a human growth hormone polyA signal sequence or a bovine growth hormone polyA signal sequence.

[0074] In some embodiments, the SV40 polyA signal sequence comprises or consists of:

[0075]

[0076] In some embodiments, the human growth hormone polyadenylation signal (bGHpA) sequence comprises or consists of the following sequence:

[0077]

[0078] In some embodiments, the human β-globin polyadenylation signal (hBGpA) sequence comprises or consists of the following sequence:

[0079]

[0080] In some embodiments, the vectors provided herein further comprise or encode a Kozak sequence (e.g., a DNA sequence that is transcribed into an RNA Kozak sequence). In some embodiments, the vector comprises a Kozak sequence upstream of the transgene. In some embodiments, the Kozak sequence is encoded by GCCACC (SEQ ID NO: 19). In some embodiments, the Kozak sequence (e.g., RNA Kozak sequence) comprises or consists of: ACCAUGG (SEQ ID NO: 20), GCCGCCACCAUGG (SEQ ID NO: 21), CCACCAUG (SEQ ID NO: 22), or CCACCAUGG (SEQ ID NO: 23).

[0081] In some embodiments, the vectors provided herein further comprise or encode a woodchuck hepatitis virus post-transcriptional element (WPRE). See, e.g., Wang and Verma, Proc. Natl. Acad. Sci., USA, 96: 3906-3910 (1999). In some embodiments, the vectors comprise or encode a hepatitis B virus post-transcriptional regulatory element (HBVPRE) and / or an RNA transport element (RTE). In some embodiments, the WPRE or HBVPRE sequence is any of the WPRE or HBVPRE sequences disclosed in US 6,136,597 or US 6,287,814. In some embodiments, the vectors provided herein further comprise or encode a WPRE3 or wsl3 regulatory element.

[0082] In some embodiments, WPRE3 comprises or is encoded by the following sequence:

[0083]

[0084] In some embodiments, WPRE comprises or is encoded by the following sequence:

[0085]

[0086] In some embodiments, the vectors provided herein further comprise an artificial intron.In some embodiments, the vectors provided herein further comprise a chimeric intron.

[0087] In some embodiments, the vectors provided herein further encode a 2A self-cleaving peptide.

[0088] In some embodiments, the vectors provided herein also contain or encode an internal ribosome entry site (IRES). In some embodiments, the IRES is located at the 5' end of the gene of interest and can translate RNA in a cap-independent manner. The transcribed IRES can bind directly to the ribosomal subunit so that the start codon of the mRNA is properly oriented in the ribosome for translation. The IRES sequence is usually located in the 5'UTR of the mRNA (directly upstream of the start codon). IRES functionally replaces the need for various protein factors that interact with the eukaryotic translation machinery.

[0089] Provided herein is a vector comprising (a) a promoter; (b) a nucleotide sequence of any one of SEQ ID NOs: 1-13, or a reverse complementary sequence of any one of SEQ ID NOs: 1-13; and (c) a heterologous nucleotide sequence; and comprising or encoding one or more of the following: an enhancer, a polyadenylation signal sequence, a Kozak sequence, a WPRE, a hepatitis B virus post-transcriptional regulatory element, an RTE, a WPRE3, a wsl3 regulatory element, an artificial intron, a chimeric intron, a 2A self-cleaving peptide, and an IRES.

[0090] Provided herein is a vector comprising (a) a promoter; (b) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, or a reverse complementary sequence of any one of SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; and (c) a heterologous nucleotide sequence; and comprising or encoding one or more of the following: an enhancer, a polyadenylation signal sequence, a Kozak sequence, a WPRE, a hepatitis B virus post-transcriptional regulatory element, an RTE, a WPRE3, a wsl3 regulatory element, an artificial intron, a chimeric intron, a 2A self-cleaving peptide and an IRES.

[0091] In some embodiments, the vectors provided herein further encode a nuclear localization signal (NLS). In some embodiments, the NLS is a Simian Virus 40 (SV40) NLS. In some embodiments, the NLS is a nucleoplasmin NLS.

[0092] The genetic regulatory elements provided herein can be used to limit gene therapy interventions (e.g., gene replacement, gene regulation, gene editing, and / or cell activity modulation) to distinct neuronal populations in the CNS while preventing or reducing the effects of the gene therapy interventions in any other organs.

[0093] In some embodiments, the heterologous nucleotide sequence encodes a protein.

[0094] In some embodiments, the heterologous nucleotide sequence encodes a gene silencing nucleic acid. In some embodiments, the gene silencing nucleic acid is a short hairpin RNA (shRNA). In some embodiments, the gene silencing nucleic acid is a short interfering RNA (siRNA).

[0095] In some embodiments, the heterologous nucleotide sequence encodes a CRISPR-associated protein. In some embodiments, the heterologous nucleotide sequence encodes a nuclease-deficient CRISPR-associated protein (also referred to as a catalytically inactive CRISPR nuclease, a dead Cas protein, or a dCas protein). In some embodiments, the heterologous nucleotide sequence encodes a CRISPR-associated protein and a guide RNA (gRNA).

[0096] In some embodiments, the heterologous nucleotide sequence encodes a transcription activator-like effector (TALE) protein.

[0097] In some embodiments, the vector provided herein is a plasmid or viral expression cassette comprising another nucleic acid sequence. In some embodiments, the vector provided herein can be used to produce recombinant virus particles to be used as a viral vector for gene delivery. In some embodiments, the vector provided herein is formulated for use via a non-viral delivery system. Further provided herein is a plasmid comprising any vector nucleic acid sequence disclosed herein.

[0098] In some embodiments, the vector provided herein is a non-viral vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a bacterial plasmid vector. In some embodiments, the vector is a yeast plasmid vector. In some embodiments, the vector is a bacterial artificial chromosome vector, a yeast plasmid vector or a yeast minichromosome vector.

[0099] In some embodiments, the vectors provided herein are viral vectors.

[0100] In some embodiments, the viral vector is an adenoviral vector. Adenoviral vectors and recombinant adenoviruses are described, for example, in U.S. Pat. No. 10,781,427. In some embodiments, the adenoviral vector is a helper virus-dependent adenoviral vector that lacks all viral genes except for the ψ packaging and inverted terminal repeat (ITR) sequences.

[0101] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0102] In some embodiments, the AAV vectors provided herein are self-complementary. In some embodiments, the AAV vectors provided herein are single-stranded.

[0103] In some embodiments, the AAV vector provided herein comprises a first AAV ITR located upstream of a heterologous nucleotide sequence, a promoter, and a nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse complementary sequence of any one of SEQ ID NOs: 1-13; or a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13; and a second AAV ITR located downstream of a heterologous nucleotide sequence, a promoter, and a nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; a reverse complementary sequence of any one of SEQ ID NOs: 1-13; or a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13. ITRs are sequences that mediate AAV proviral integration and packaging of AAV DNA into virions.

[0104] In some embodiments, the AAV vectors provided herein comprise a first AAV ITR located upstream of a heterologous nucleotide sequence, a promoter, and a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a complementary nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; NO:11; or a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; and a second AAV ITR located downstream of the heterologous nucleotide sequence, the promoter, and the nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; the reverse complementary sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; the complementary nucleotide sequence of any one of SEQ ID NO:1-13; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. the reverse complementary sequence of any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11;or a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. ;

[0105] In some embodiments, the AAV vector provided herein comprises a 5′ AAV inverted terminal repeat (ITR) and a 3′ AAV ITR flanked by a heterologous nucleotide sequence, a promoter and (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13.

[0106] In some embodiments, the AAV vectors provided herein comprise a 5′ AAV inverted terminal repeat (ITR) and a 3′ AAV ITR flanked by a heterologous nucleotide sequence, a promoter, and (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (b) an inverted nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1. NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:1; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:1.

[0107] In some embodiments, the AAV vector provided herein comprises, in 5′ to 3′ order: (i) the 5′AAVI TR; (ii) the promoter; (iii) (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13; (iv) the heterologous nucleotide sequence; and (v) the 3′AAV ITR.

[0108] In some embodiments, the AAV vector provided herein comprises, in 5′ to 3′ order: (i) the 5′AAVITR; (ii) the promoter; (iii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 1; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 1; (c) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 1; (d) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 1; NO:6, SEQ ID NO:9, SEQ ID NO:10 and the reverse complementary nucleotide sequence of any one of SEQ ID NO:1; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer, having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:1; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

[0109] In some embodiments, the AAV vector provided herein comprises, in 5′ to 3′ order: (i) the 5′AAVI TR; (ii) (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-13; (iii) the promoter; (iv) the heterologous nucleotide sequence; and (v) the 3′AAV ITR.

[0110] In some embodiments, the AAV vector provided herein comprises, in 5′ to 3′ order: (i) the 5′AAVITR; (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1. NO:9, SEQ ID NO:10 and the reverse complementary nucleotide sequence of any one of SEQ ID NO:1; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer, having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:1; (iii) the promoter; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

[0111] In some embodiments, the AAV vector provided herein comprises, in 5′ to 3′ order: (i) the 5′AAV ITR; (ii) the promoter; (iii) the heterologous nucleotide sequence; (iv) (a) a nucleotide sequence of any one of SEQ ID NOs: 1-13; (b) a reverse nucleotide sequence of any one of SEQ ID NOs: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; (d) a reverse complementary nucleotide sequence of any one of SEQ ID NOs: 1-13; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NOs: 1-1 3; and (v) the 3′AAV ITR.

[0112] In some embodiments, the AAV vector provided herein comprises, in 5′ to 3′ order: (i) the 5′AAVITR; (ii) the promoter; (iii) the heterologous nucleotide sequence; (iv) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1-13; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1. NO:1; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:1; and (v) the 3′ AAV ITR.

[0113] The viral vectors and plasmids provided herein can be used to produce recombinant viral particles.

[0114] Provided herein is a viral particle (also referred to as a virion) comprising a vector disclosed herein. In some embodiments, the viral particle is a recombinant adenovirus particle. In some embodiments, the viral particle is a recombinant AAV (rAAV) particle. In some embodiments, the rAAV particle is an AAV-PHP.eB serotype particle. In some embodiments, the rAAV particle is an AAV9 serotype particle. In some embodiments, the rAAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i 8. AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHP.A, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV2-TLH or AAV2-GMN serotype particles, or any other AAV serotype particles now known or later discovered.

[0115] Provided herein is a population of viral particles comprising a plurality of viral particles disclosed herein. Provided herein is a population of recombinant adenoviral particles comprising a plurality of recombinant adenoviral particles disclosed herein. Provided herein is a population of rAAV particles comprising a plurality of rAAV particles disclosed herein.

[0116] In some embodiments, the carrier provided herein is suitable for delivery via a non-viral delivery system. In some embodiments, the carrier provided herein is formulated for delivery via a non-viral delivery system. In some embodiments, the non-viral delivery system is a lipid nanoparticle or an exosome. In some embodiments, the non-viral system for gene delivery can be lipid-based, polymer-based or other nanomaterial-based. Cationic lipids or cationic polymers can be compounded with nucleic acid molecules to produce a synthetic vehicle for gene delivery.

[0117] Provided herein is a cell comprising any of the vectors or viral particles disclosed herein.

[0118] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell can be a mammalian cell, an insect cell, a plant cell, a fungal cell, a eukaryotic algae cell, a nematode cell, a protozoan cell, or a fish cell.

[0119] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell, a COS cell, a Vero cell, a SP2 / 0 cell, a NS / O myeloma cell, a human embryonic kidney cell, an immature hamster kidney cell, a HeLa cell, a human B cell, a cv-1 / EBNA cell, a L cell, a 3T3 cell, a HEPG2 cell, a PerC6 cell, a 293 cell or a MDCK cell. In some embodiments, the mammalian cell is a HEK293 cell. In some embodiments, the mammalian cell is a CHO cell.

[0120] In some embodiments, the cell is an insect cell. In some embodiments, the insect cell is a Spodoptera frugiperda cell (e.g., Sf9 or ExpiSf9T M The Sf9 insect cell line (ThermoFisher Scientific, Waltham, MA) is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE. TM The cells (Thermo Fisher Scientific, Waltham, MA) are a non-engineered derivative of Sf9 insect cells that have been adapted for high-density suspension growth.

[0121] Pharmaceutical composition

[0122] Provided herein is a pharmaceutical composition comprising any carrier, viral particle, nucleic acid molecule or viral particle colony disclosed herein, and a pharmaceutically acceptable carrier, vehicle or diluent. "Pharmaceutically acceptable" refers to nontoxic or otherwise undesirable materials, that is, the material can be applied to a subject without causing any undesirable biological effects. Typically, a pharmaceutically acceptable material has one or more benefits that exceed any undesirable biological effects that the material may have. Undesirable biological effects may include, for example, excessive toxicity, irritation, allergic reactions and other problems and complications.

[0123] For injection, the carrier is typically a liquid. For other methods of administration, the carrier can be either a solid or a liquid.

[0124] In some embodiments, the pharmaceutical composition may include other medicinal agents, pharmaceutical agents, stabilizers, buffers, adjuvants, and / or diluents.

[0125] In some embodiments, the pharmaceutical composition includes at least one pharmaceutically acceptable carrier, excipient and / or vehicle, such as solvent, buffer, solution, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic agent and absorption delay agent. In some embodiments, pharmaceutically acceptable carrier, excipient and / or vehicle include saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer or their combination. In some embodiments, pharmaceutically acceptable carrier, excipient and / or vehicle include phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) or its suitable mixture. In some embodiments, the composition disclosed herein further includes an emulsifier or wetting agent, or a pH buffer. Such materials may be present in minor amounts (eg, less than 10% by weight of the composition, such as less than 5% by weight of the composition, 2% by weight of the composition, 1% by weight of the composition, or less).

[0126] In some embodiments, the pharmaceutical composition further comprises one or more other pharmaceutical ingredients, such as one or more preservatives or chemical stabilizers. Examples of preservatives and chemical stabilizers include, but are not limited to, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, p-chlorophenol, and albumin. In some embodiments, the compositions disclosed herein further comprise antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal; isotonic agents, such as sugars and sodium chloride; and / or agents that delay absorption, such as aluminum monostearate and gelatin.

[0127] In some embodiments, the pharmaceutical composition is in the form of an injectable solution or dispersion, such as an aqueous solution or dispersion. In some embodiments, the pharmaceutical composition is a sterile powder for the temporary preparation of a sterile injectable solution or dispersion. The dispersion can be prepared in water, glycerol, liquid polyethylene glycol, oil or any combination thereof. Delivery vehicles (such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce pharmaceutical compositions provided herein.

[0128] In some embodiments, the pharmaceutical composition comprises a sterile saline (e.g., pharmaceutical grade saline) solution and a carrier or consists of a sterile saline solution and a carrier. In some embodiments, the pharmaceutical composition comprises a carrier and sterile water (e.g., pharmaceutical grade water) or consists of a carrier and sterile water. In some embodiments, the pharmaceutical composition comprises a carrier and phosphate buffered saline (PBS) (e.g., pharmaceutical grade PBS) or consists of a carrier and phosphate buffered saline.

[0129] In some embodiments, the pharmaceutical compositions provided herein comprise a carrier and one or more excipients. In some embodiments, the excipient is water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose or polyvinyl pyrrolidone.

[0130] In some embodiments, the pharmaceutical compositions provided herein include lipid moieties. For example, the carrier can be introduced into a preformed liposome or lipid complex made from a mixture of a cationic lipid and a neutral lipid. In some embodiments, a carrier complex with a monocationic lipid or a polycationic lipid is formed in the absence of a neutral lipid. In some embodiments, the lipid moiety is selected to increase the distribution of the agent to a specific cell or tissue. In some embodiments, the lipid moiety is selected to increase the distribution of the agent to neurons.

[0131] In some embodiments, the pharmaceutical composition comprises a delivery system. The example of a delivery system includes but is not limited to liposomes and emulsions. Some delivery systems can be used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In some embodiments, an organic solvent is used, such as dimethyl sulfoxide. In some embodiments, the pharmaceutical composition comprises a delivery system based on nanoparticles. In some embodiments, nanoparticles are prepared using polylactic-co-glycolic acid (PLGA), poly(β-amino ester) (PBAE) and / or polyethyleneimine (PEI).

[0132] In some embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver the vector to a specific tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies or cell-specific antibodies.

[0133] In some embodiments, the pharmaceutical composition comprises a co-solvent system. Some co-solvent systems comprise, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is a VPD co-solvent system, which is a solution of anhydrous ethanol comprising 3% w / v benzyl alcohol, 8% w / v non-polar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300. The proportions of such co-solvent systems can vary greatly without significantly changing their solubility and toxicity characteristics. In addition, the types of co-solvent components can be changed: for example, other surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be changed; other biocompatible polymers can replace polyethylene glycol, such as polyvinyl pyrrolidone; and other sugars or polysaccharides can replace dextrose.

[0134] Provided herein is a nanoparticle comprising a carrier disclosed herein. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a solid lipid nanoparticle (SLN).

[0135] In some embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution (such as water) or a physiologically compatible buffer (such as Hanks solution, Ringer's solution or saline buffer). In some embodiments, other ingredients (e.g., ingredients that help dissolve or serve as preservatives) are included. In some embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. In some embodiments, pharmaceutical compositions for injection are prepared in unit dosage forms, such as ampoules or multi-dose containers. Some pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulations such as suspending agents, stabilizers and / or dispersants. Exemplary solvents suitable for pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, and liposomes.

[0136] In some embodiments, the carrier can be mixed with pharmaceutically acceptable active substances and / or inert substances for the preparation of pharmaceutical compositions or formulations. The composition and the method for formulating the pharmaceutical composition depend on many criteria, including but not limited to the route of administration, the extent of the disease or the dose to be administered.

[0137] In some embodiments, the pharmaceutical composition is suitable or formulated for systemic administration. In some embodiments, the pharmaceutical composition is suitable or formulated for intravenous administration.

[0138] In some embodiments, the pharmaceutical composition is suitable for or formulated for intraventricular, intrathecal, intracarotid, or intraparenchymal injection.

[0139] Methods for producing rAAV particles

[0140] Provided herein are methods for producing rAAV particles by using any of the vectors and cells disclosed herein.

[0141] Provided herein is a method for producing rAAV particles, the method comprising: (i) culturing cells comprising a vector disclosed herein under conditions that allow packaging of the rAAV particles; and (ii) harvesting the cultured host cells or culture medium for collecting the rAAV particles.

[0142] In some embodiments, the method of producing rAAV particles comprises providing to a cell: (a) a vector comprising two AAV ITRs located 5′ and 3′ of a polynucleotide sequence desired to be packaged into rAAV particles; and (b) AAV sequences sufficient to replicate the nucleic acid template and encapsidate into an AAV protein capsid (e.g., AAV rep sequences encoding AAV capsid subunits and AAV cap sequences, also referred to as “helper functions”). Typically, the AAV rep and cap sequences will not be flanked by AAV ITRs to prevent rescue and / or packaging of these sequences.

[0143] Any suitable genetic elements can be used to deliver the vector (nucleic acid template), rep sequence, cap sequence, and any other auxiliary functions required for producing the rAAV particles disclosed herein to the packaging host cells. Further details on methods for preparing rAAV particles are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; Fisher et al., J. Virol., 70: 520-532 (1993) and US 5,478,745.

[0144] The nucleic acid template and the AAV rep and cap sequences are provided under conditions that produce a viral vector comprising the nucleic acid template packaged in an AAV protein capsid in the cell. The method may further comprise the step of collecting the packaged viral vector from the cell. The packaged viral vector may be collected from the culture medium and / or by lysing the cells.

[0145] The cell may be a cell that allows AAV virus replication. Any suitable cell known in the art may be used. In some embodiments, the cell is a mammalian cell (e.g., HEK293 cell). In some embodiments, the cell may be a trans-complementation packaging cell line that provides a function deleted from a replication-deficient helper virus, such as HEK293 cells or other E1a trans-complementation cells. The auxiliary sequence may be embedded in the chromosome or maintained as a stable extrachromosomal element.

[0146] In some embodiments, rAAV particles are produced using a triple transfection method as described in US 6,001,650. In some embodiments, rAAV is produced by transfecting host cells with an AAV vector (i.e., an AAV expression cassette) to be packaged into rAAV particles, an AAV auxiliary (helper) function vector, and an accessory (accessory) function vector. The AAV auxiliary function vector encodes "AAV auxiliary function" sequences (i.e., rep and cap), which act in a trans-directed manner for productive AAV replication and encapsidation. Non-limiting examples of AAV auxiliary function vectors include pHLP19 and pRep6cap6 vectors described in US 6,001,650 and US 6,156,303, respectively. The auxiliary function vector encodes the nucleotide sequence of the non-AAV derived virus and / or cell function (i.e., "accessory function") that AAV relies on for replication. Auxiliary functions include those required for AAV replication, including but not limited to those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based auxiliary functions can be derived from any known helper virus, such as adenovirus, herpes virus (e.g., except herpes simplex virus type 1), and vaccinia virus.

[0147] In some embodiments, rAAV is produced using a recombinant baculovirus vector. rAAV production using a baculovirus vector is described in, for example, Urabe et al. (2002) Hum Gene Ther 13 (16): 1935-1943; Smith et al. (2009) Mol Ther 17 (11): 1888-1896; US 8,945,918; US 9,879,282; and US2018 / 0371495. In some embodiments, the baculovirus vector genome is derived from Autographa calfornica multicapsid nuclear polyhedrosis virus (AcMNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV), Helicoverpa armigera (HearNPV) or Spodoptera exigua MNPV. Baculovirus vectors are used to produce recombinant AAV in insect cells (e.g., Spodoptera frugiperda cells). In some embodiments, Sf9 or ExpiSf9 TM The Spodoptera frugiperda cell line is used to produce rAAV. In some embodiments, the methods of the present disclosure include co-infecting insect cells with a recombinant baculovirus (rBV) population to produce rAAV disclosed herein. At least two rBV populations can be used in the methods of the present disclosure. Methods for generating recombinant baculovirus are known in the art (see, e.g., Baculovirus Expression System (Thermo Fisher Scientific, Waltham, MA)).

[0148] In some embodiments, the rAAV particles produced by the methods provided herein include AAV-PHP.eB capsid protein. In some embodiments, the rAAV particles produced by the methods provided herein include AAV9 capsid protein. In some embodiments, the rAAV particles produced by the methods provided herein include AAV1, AAV2, AAV3 (including type 3A and type 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh.8, AAVrh.10, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHP.A, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV2-TLH or AAV2-GMN capsid protein, or any other AAV capsid protein now known or later discovered.

[0149] Methods of using vectors

[0150] Provided herein is a method for selectively expressing a heterologous nucleotide sequence in neurons of the CNS of a subject in need thereof, the method comprising administering to the subject a vector, viral particle, population, pharmaceutical composition or nanoparticle disclosed herein.

[0151] In some embodiments of the methods disclosed herein, the regulatory elements restrict expression of the heterologous nucleotide sequence to neurons of the CNS with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for the cell type. In some embodiments of the methods disclosed herein, the regulatory elements restrict expression of the heterologous nucleotide sequence to neurons of the CNS with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for the cell type.

[0152] In some embodiments of the methods disclosed herein, the regulatory element expresses a heterologous nucleotide sequence in a non-neuronal cell of the CNS (e.g., a glial cell or an endothelial cell) with up to about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% specificity for the cell type. In some embodiments of the methods disclosed herein, the regulatory element expresses a heterologous nucleotide sequence in a non-neuronal cell of the CNS (e.g., a glial cell or an endothelial cell) with up to about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% selectivity for the cell type.

[0153] In some embodiments of the methods disclosed herein, the heterologous nucleotide sequence expression is enriched in a specific region of the CNS. The terms "enrichment" and "sensitivity" can be used interchangeably. The regulatory elements provided herein, when integrated into a DNA-based expression vector, can drive the functional expression of a heterologous nucleotide sequence in a specific region of the brain, with a sensitivity of more than 10% for neurons in a given target region (i.e., forebrain), while preventing the heterologous nucleotide sequence from being expressed in other regions of the brain (e.g., less than 5% sensitivity for cells, or less than 2% sensitivity for cells).

[0154] In some embodiments of the methods disclosed herein, the regulatory element limits the expression of the heterologous nucleotide sequence to neurons of the CNS, with at least about 70% specificity for the cell type; and enriches the expression of the heterologous nucleotide sequence in a specific area of ​​the brain, with more than 10% sensitivity to neurons in a given target area.

[0155] In some embodiments of the methods disclosed herein, the regulatory element limits the expression of the heterologous nucleotide sequence to neurons of the CNS, with at least about 80% specificity for the cell type; and enriches the expression of the heterologous nucleotide sequence in a specific area of ​​the brain, with more than 10% sensitivity to neurons in a given target area.

[0156] In some embodiments of the methods disclosed herein, the regulatory element limits the expression of the heterologous nucleotide sequence to neurons of the CNS, with at least about 90% specificity for the cell type; and enriches the expression of the heterologous nucleotide sequence in a specific area of ​​the brain, with greater than 10% sensitivity to neurons in a given target area.

[0157] In some embodiments of the methods disclosed herein, the heterologous nucleotide sequence expression is enriched in neurons of one or more of the following: forebrain, cerebellum, and spinal cord. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the forebrain and the cerebellum. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the forebrain. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the cerebellum. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the spinal cord. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of a neurogenic niche.

[0158] In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10, and expression of the heterologous nucleotide sequence is enriched in neurons of the CNS (eg, pan-neuronal expression across all brain regions).

[0159] In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 9, and expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain and the cerebellum.

[0160] In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 11, and expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain.

[0161] In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 6, and the heterologous nucleotide sequence expression is enriched in neurons of the cerebellum and hippocampus.

[0162] In some embodiments, the vector, the viral particle, the colony, the pharmaceutical composition, or the nanoparticle is administered to the subject via systemic administration. In some embodiments, the vector, the viral particle, the colony, the pharmaceutical composition, or the nanoparticle is administered to the subject intravenously. In some embodiments, the vector, the viral particle, the colony, the pharmaceutical composition, or the nanoparticle is administered to the subject via injection into the CNS. In some embodiments, the vector, the viral particle, the colony, the pharmaceutical composition, or the nanoparticle is administered to the subject via ICV injection.

[0163] In some embodiments, the subject suffers from or is suspected of having a disease or disorder associated with improper expression of a protein or nucleic acid in a CNS neuron. In some embodiments, the subject suffers from or is suspected of having a disease or disorder associated with improper expression of a protein or nucleic acid in neurons of one or more of the forebrain, cerebellum, and spinal cord. In some embodiments, the subject suffers from or is suspected of having a disease or disorder associated with improper expression of a protein or nucleic acid in neurons of a neurogenic niche.

[0164] In some embodiments, the subject has or is suspected of having a neurodegenerative, neurological, neurodevelopmental, or neuropsychiatric disease or disorder.

[0165] In some embodiments, the vectors, viral particles, colonies, pharmaceutical compositions or nanoparticles disclosed herein are administered to a subject for the treatment or prevention of a disease or condition associated with inappropriate expression of a protein or nucleic acid in a CNS neuron. In some embodiments, the vectors, viral particles, colonies, pharmaceutical compositions or nanoparticles disclosed herein are administered to a subject for the alleviation or amelioration of one or more symptoms of a disease or condition associated with inappropriate expression of a protein or nucleic acid in a CNS neuron.

[0166] In some embodiments, the subject is a human. In some embodiments, the subject is less than 2 years old. In some embodiments, the subject is between about 2 years old and about 18 years old. In some embodiments, the subject is greater than 18 years old.

[0167] definition

[0168] Unless otherwise indicated, the terms used herein have the definitions commonly used in the art.Some terms are defined below, and additional definitions can be found in the remainder of the detailed description.

[0169] The term "a" or "an" refers to one or more of the entity, i.e., it may refer to plural referents. Therefore, the terms "a", "one", "one or more", and "at least one" are used interchangeably herein. In addition, an "element" referred to by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires the presence of one and only one element.

[0170] Unless otherwise indicated or apparent from the context, the term "about" means within 10% above or below the reported value (unless the number is more than 100% or less than 0% of the possible value). When used in conjunction with a range of values ​​or a series of values, the term "about" applies to the endpoints of the range or each value in the recited values ​​in the series unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0171] As used herein, the terms "specificity" and "sensitivity" are defined as follows. For the quantification of colocalization analysis, only the corresponding color channel is used to count the cells expressing the specified reporter gene, and only the corresponding color channel is used to independently identify the cells (i.e., neurons) expressing the pan-neuronal marker NeuN. The ratio of the cells co-expressing two kinds of markers to the total number of cells expressing only the reporter gene is then calculated, and reported as mean ± sem (mean standard error) (i.e., specificity). The ratio of the cells co-expressing two kinds of markers to the total number of cells expressing the pan-neuronal marker is calculated, and reported as mean ± sem (i.e., sensitivity).

[0172] As used herein, the term "vector" refers to a delivery vehicle for a polynucleotide (e.g., a heterologous nucleotide sequence). A vector can be used to deliver a heterologous nucleotide sequence to a host cell. In some embodiments, the vector comprises a heterologous nucleotide sequence encoding a protein or a gene silencing nucleic acid operably inserted therein, and can express the protein or gene silencing nucleic acid in genetic engineering recombinant technology. The vector can be used for transformation, transduction or transfection of a host cell, and enables the genetic material elements carried by the vector to be expressed in a host cell." vector " can be any suitable vector, including chromosome, non-chromosome and synthetic nucleic acid vector.

[0173] As used herein, the term "heterologous" refers to a substance from some source other than its natural source. For example, the term "heterologous nucleotide sequence" refers to a nucleotide sequence from a non-natural source or location that has been artificially provided to a biological system.

[0174] As used herein, the term "gRNA molecule" or "gRNA" refers to a guide RNA that is capable of targeting a CRISPR nuclease or a nuclease-deficient CRISPR-associated protein to a target nucleic acid. Depending on the context, the term "gRNA molecule" refers to a guide ribonucleic acid or a nucleic acid encoding a gRNA.

[0175] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotides or polypeptide sequences are unchanged over the entire alignment window of residues (e.g., nucleotides or amino acids). The "identity score" of the aligned segments of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). "Identity percentage" is the identity score multiplied by 100. The alignment program Clustal Omega available at ebi.ac.uk / Tools / msa / clustalo can be used to calculate the identity percentage using default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (2011October 11) Molecular Systems Biology 7: 539. For the purpose of calculating identity to a sequence, extensions such as tags are not included.

[0176] As used herein, a regulatory sequence (e.g., a promoter or genetic regulatory element provided herein) is considered "operably linked" when it is in a functional position and orientation relative to a nucleic acid sequence it regulates to control transcription initiation and / or expression of that sequence.

[0177] As used herein, when referring to AAV vectors, the term "self-complementary" refers to an AAV vector that contains nucleic acid (i.e., DNA) that forms spontaneously annealed dimeric inverted repeat molecules, resulting in earlier and more robust transgene expression compared to conventional single-stranded (ss) AAV genomes. See, e.g., McCarty, Molecular Therapy 16(10): 1648-1656 (2008). Unlike conventional ssAAV, self-complementary AAV (scAAV) can bypass second-strand synthesis, which is the rate-limiting step for gene expression. In addition, double-stranded scAAV is less susceptible to DNA degradation after viral transduction, thereby increasing the copy number of stable episomes.

[0178] As used herein, a "therapeutically effective amount" is an amount of a vector, viral particle, viral particle population, pharmaceutical composition, or nanoparticle provided herein that is effective in treating or preventing a disease in a subject or in ameliorating its signs or symptoms. A "therapeutically effective amount" may vary depending on, for example, the disease and / or disease symptoms, the severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the patient to be treated, and the judgment of the prescribing physician.

[0179] In the present specification, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and include fractions thereof (such as one tenth and one hundredth of an integer) where appropriate.

[0180] Use of alternatives (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

[0181] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the reference to any reference, article, publication, patent, patent publication and patent application cited herein does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0182] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0183] Numbered implementation plan

[0184] Notwithstanding the appended claims, the present disclosure sets forth the following numbered embodiments:

[0185] Embodiment 1. A vector comprising (i) a heterologous nucleotide sequence; and (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) an inverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. or (e) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer, wherein the one or more regions have at least 75% or higher sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0186] Embodiment 2. The vector of embodiment 1, wherein the vector further comprises a promoter.

[0187] Embodiment 3. The vector of embodiment 2, which comprises, in order from 5′ to 3′: (i) the promoter; (ii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. NO:9, the reverse complementary nucleotide sequence of any one of SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (iii) the heterologous nucleotide sequence;

[0188] Embodiment 4. The vector of embodiment 2, comprising, in 5′ to 3′ order: (i) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11;

[0189] (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (ii) the promoter; and (iii) the heterologous nucleotide sequence.

[0190] Embodiment 5. The vector of embodiment 2, which comprises, in 5′ to 3′ order: (i) the promoter; (ii) the heterologous nucleotide sequence; and (iii) (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0191] Embodiment 6. The vector of any one of Embodiments 1-5, wherein the vector is a non-viral vector.

[0192] Embodiment 7. The vector of embodiment 6, wherein the non-viral vector is a plasmid vector.

[0193] Embodiment 8. The vector of any one of Embodiments 1-5, wherein the vector is a viral vector.

[0194] Embodiment 9. The vector of embodiment 8, wherein the viral vector is an adeno-associated virus (AAV) vector or an adenoviral vector.

[0195] Embodiment 10. The vector of embodiment 9, wherein the AAV vector comprises a 5′ AAV inverted terminal repeat (ITR) and a 3′ AAV ITR flanking the heterologous nucleotide sequence, a promoter, and (a) a nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) an inverted nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer and having at least 75% or higher sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0196] Embodiment 11. The vector as described in embodiment 10, which comprises, in order from 5' to 3', (i) the 5'AAVITR; (ii) the promoter; (iii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (d) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

[0197] Embodiment 12. The vector of embodiment 10, which comprises, in order from 5′ to 3′: (i) the 5′AAVITR; (ii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (d) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (iii) the promoter; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

[0198] Embodiment 13. The vector of embodiment 10, which comprises, in order from 5′ to 3′: (i) the 5′AAVITR; (ii) the promoter; (iii) the heterologous nucleotide sequence; (iv) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (d) the complementary nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and (v) the 3′ AAV ITR.

[0199] Embodiment 14. A vector as described in any of embodiments 2-13, wherein the promoter comprises an RNA-polymerase II binding site, a TATA box, a TF2B recognition element (BRE), a motif 10 element (MTE), or a downstream promoter element (DPE).

[0200] Embodiment 15. The vector of any one of Embodiments 2-13, wherein the promoter is a minimal promoter.

[0201] Embodiment 16. The vector of embodiment 15, wherein the minimal promoter is the human β-globin minimal promoter.

[0202] Embodiment 17. The vector of any one of Embodiments 1-16, wherein the vector further comprises an enhancer.

[0203] Embodiment 18. The vector of any one of embodiments 1-17, wherein the vector further comprises or encodes a polyadenylation signal sequence.

[0204] Embodiment 19. The vector of any one of Embodiments 1-18, wherein the vector further comprises or encodes a Kozak sequence.

[0205] Embodiment 20. The vector of any one of embodiments 1-19, wherein the vector further comprises or encodes a woodchuck hepatitis virus post-transcriptional element (WPRE), a hepatitis B virus post-transcriptional regulatory element, an RNA transport element (RTE), a WPRE3, or a wsl3 regulatory element.

[0206] Embodiment 21. The vector of any one of Embodiments 1-20, wherein the vector further comprises an artificial intron.

[0207] Embodiment 22. The vector of any one of embodiments 1-21, wherein the vector further encodes a 2A self-cleaving peptide.

[0208] Embodiment 23. The vector of any one of embodiments 1-22, wherein the vector further comprises or encodes an internal ribosome entry site (IRES).

[0209] Embodiment 24. The vector of any one of embodiments 1-23, wherein the heterologous nucleotide sequence encodes a protein.

[0210] Embodiment 25. The vector of any one of embodiments 1-23, wherein the heterologous nucleotide sequence encodes a gene silencing nucleic acid.

[0211] Embodiment 26. A vector as described in any of Embodiments 1-23, wherein the heterologous nucleotide sequence encodes a CRISPR-associated protein.

[0212] Embodiment 27. A vector as described in Embodiment 26, wherein the heterologous nucleotide sequence also encodes a guide RNA (gRNA).

[0213] Embodiment 28. A viral particle comprising the vector of any one of embodiments 1-5 and 8-27.

[0214] Embodiment 29. The viral particle of Embodiment 28, wherein the viral particle is a recombinant adenoviral particle.

[0215] Embodiment 30. The viral particle of embodiment 28, wherein the viral particle is a recombinant AAV (rAAV) particle.

[0216] Embodiment 31. The viral particle of embodiment 30, wherein the rAAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHRA, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, AAV-CAP.B 1 0, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV2-TLH, or AAV2-GMN serotype particles.

[0217] Embodiment 32. A population of viral particles comprising a plurality of viral particles according to any one of embodiments 28-32.

[0218] Embodiment 33. A pharmaceutical composition comprising the vector of any one of embodiments 1-27, the viral particle of any one of embodiments 28-32, or the population of embodiment 32, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0219] Embodiment 34. A nanoparticle comprising the carrier of any one of embodiments 1-7 and 14-27.

[0220] Embodiment 35. A cell comprising the vector of any one of embodiments 1-27 or the viral particle of any one of embodiments 28-32.

[0221] Embodiment 36. The cell of embodiment 35, wherein the cell is a mammalian cell or an insect cell.

[0222] Embodiment 37. A method for producing rAAV particles, the method comprising: (i) culturing the cells described in embodiment 35 or 36 under conditions that allow packaging of the rAAV particles; and (ii) harvesting the cultured host cells or culture medium for collecting the rAAV particles.

[0223] Embodiment 38. A method for selectively expressing a heterologous nucleotide sequence in neurons of the central nervous system (CNS) of a subject in need thereof, the method comprising administering to the subject the vector of any one of embodiments 1-27, the viral particle of any one of embodiments 28-32, the population of embodiment 32, the pharmaceutical composition of embodiment 33, or the nanoparticle of embodiment 34.

[0224] Embodiment 39. A method as described in Embodiment 38, wherein the expression of the heterologous nucleotide sequence is enriched in a specific region of the CNS.

[0225] Embodiment 40. The method of embodiment 38, wherein the heterologous nucleotide sequence expression is enriched in neurons of one or more of: forebrain, cerebellum, and spinal cord.

[0226] Embodiment 41. A method as described in Embodiment 38, wherein the heterologous nucleotide sequence expression is enriched in neurons of the forebrain and the cerebellum.

[0227] Embodiment 42. A method as described in Embodiment 38, wherein the heterologous nucleotide sequence expression is enriched in neurons of the forebrain.

[0228] Embodiment 43. A method as described in Embodiment 38, wherein the heterologous nucleotide sequence expression is enriched in neurons of the cerebellum.

[0229] Embodiment 44. A method as described in Embodiment 38, wherein the heterologous nucleotide sequence expression is enriched in neurons in the spinal cord.

[0230] Embodiment 45. The method of embodiment 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10, and wherein expression of the heterologous nucleotide sequence is enriched in neurons of the CNS.

[0231] Embodiment 46. A method as described in embodiment 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 9, and wherein the heterologous nucleotide sequence expression is enriched in neurons of the forebrain and the cerebellum.

[0232] Embodiment 47. A method as described in Embodiment 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 11, and wherein the heterologous nucleotide sequence expression is enriched in neurons of the forebrain.

[0233] Embodiment 48. The method of embodiment 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 6, and wherein expression of the heterologous nucleotide sequence is enriched in neurons of the cerebellum and hippocampus.

[0234] Embodiment 49. The method of any one of Embodiments 38-48, wherein the vector, the viral particle, the population, the pharmaceutical composition, or the nanoparticle is administered to the subject intravenously.

[0235] Embodiment 50. The method of any one of Embodiments 38-48, wherein the vector, the viral particle, the population, the pharmaceutical composition, or the nanoparticle is administered to the subject via injection into the CNS.

[0236] The present disclosure will be further clarified by the following examples, which are intended to be merely illustrative of the present disclosure and not limiting.

[0237] Example

[0238] Generation and analysis of regulatory elements

[0239] Identification of regulatory elements

[0240] The sequences of 13 synthetic regulatory elements were engineered using a proprietary method. The Regulatory Element High Throughput Screening (RE-HTS) platform combines comprehensive analysis of genomic and epigenomic next-generation sequencing data relevant to the target cell type to identify short DNA sequences predicted to be able to restrict viral transgene expression to the target cell type.

[0241] rAAV cloning and production

[0242] The regulatory elements are then synthesized from scratch and cloned individually into custom vectors. The custom vector contains (in the order of 5' to 3') 5'AAV ITR, cloning site, coding sequence of red fluorescent reporter gene tDTomato or coding sequence of dCas9, WPRE, polyadenylation sequence and 3'AAV ITR. The vector is synthesized from scratch and used as a common skeleton for all constructs. "dCas" or "dead Cas" protein is a nuclease-deficient CRISPR-associated protein (also known as catalytically inactive CRISPR nuclease). The construct includes a regulatory element, followed by a minimal promoter sequence, immediately downstream of 5'AAV ITR. Full plasmid Sanger sequencing is then performed to ensure the integrity of each construct. The corresponding rAAV is produced in-house using standard production methods. PEI is used for transfection and OptiPrep gradient (Sigma, USA) is used for viral particle purification. All batches produced are within the range of 10E+11 to 10E+1 3 viral genomes / ml.

[0243] Systemic viral injection

[0244] For systemic injections in adult mice, 10E+11 produced viral particles with AAV capsid PHP.eB (Chan et al., Nat. Neurosci. 20, 1172-1179 (2017)) were injected into the tail vein of the animals. Five days post-intervention monitoring was performed after injection.

[0245] Immunohistochemistry

[0246] All animals injected with the virus were perfused with 4% paraformaldehyde (PFA) through the heart. The organs were placed in 4% PFA for 3 hours and then sliced ​​using a Leica VTS1200 vibrator. Floating slices were permeabilized with 0.3% Triton X 100, 5% normal donkey serum and PBS for 30 minutes. The slices were then incubated overnight at 4°C in 0.1% Triton X 100 with the specified combination of the following primary antibodies: 1:1000 rabbit anti-Ds-red (Clontech, 632496) and 1:500 mouse anti-NeuN (Invitrogen, MA5-33103). The slices were then washed three times with PBS, incubated with 1:1,000 Alexa Fluor-conjugated secondary antibodies (Invitrogen, USA), counterstained with DAPI (Sigma, USA), and mounted on slides using Fluoromount-G (Sigma, USA).

[0247] Quantification, Statistics, and Reproducibility

[0248] For expression analysis, fluorescent images were taken at standard magnification and exposure time for each organ. Figure 1A and Figure 1B Representative images equivalent between constructs are presented in . Brain heatmaps and representative images of transgene expression driven by the indicated engineered regulatory elements across indicated brain regions and organs are shown. Note that the heatmap has 5 levels, with darker shades corresponding to a higher percentage of cells expressing the transgene (i.e., the darkest shade is the strongest expression observed, and the lightest shade corresponds to almost no expression).

[0249] The expression profiles of regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V3 (SEQ ID NO: 2), CNS2_V7 (SEQ ID NO: 4) and CNS2_V10 (SEQ ID NO: 6) are shown in FIG. Figure 1A As shown. CNS2_V4 shows neuron-specific expression throughout the CNS. CNS2_V3 shows neuron-specific expression that is highly enriched in the forebrain and cerebellum. CNS2_V7 shows neuron-specific expression that is highly enriched in the forebrain. CNS2_V10 shows neuron-specific expression that is highly enriched in the cerebellum and neurogenic niches.

[0250] The expression profiles of the regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V4.1 (SEQ ID NO: 10), CNS2_V3 (SEQ ID NO: 2), CNS2_V3.3 (SEQ ID NO: 9), CNS2_V7 (SEQ ID NO: 4), CNS2_V7.1 (SEQ ID NO: 11), CNS2_V11 (SEQ ID NO: 5) and CNS2_V10 (SEQ ID NO: 6) are shown in FIG. Figure 1B As shown. CNS2_V4 and CNS2_V4.1 showed pan-neuronal expression in all brain regions. CNS2_V3 and CNS2_V3.3 showed forebrain and cerebellum-enriched expression. CNS2_V7, CNS2_V7.1, and CNS2_V11 showed forebrain-enriched expression. CNS2_V10 showed cerebellum and hippocampus-enriched expression.

[0251] For quantification of colocalization analysis, only cells expressing a given reporter gene were counted using the corresponding color channel, and only cells expressing the pan-neuronal marker NeuN (i.e., neurons) were independently identified using the corresponding color channel. The ratio of cells co-expressing both markers to the total number of cells expressing the reporter gene alone was then calculated and reported as the mean ± sem (i.e., specificity). The ratio of cells co-expressing both markers to the total number of cells expressing the pan-neuronal marker was also calculated and reported as the mean ± sem (i.e., sensitivity). Quantification was performed using a minimum of two independent biological replicates, and all replicates were highly consistent. Corresponding to Figure 1A The results of the data in Figure 2A , Figure 2B and Figure 2C as shown in .

[0252] Corresponds to Figure 1B The results of the data in Figure 3A-3D . Shown are the results of quantitative analysis of the intensity, specificity and sensitivity of transgene expression after systemic injection of adult mice with PHP.eB capsid and incubation for 3 weeks. Figure 3A Shown is the percent specificity of transgene expression (dTomato) to neurons in the somatosensory cortex (determined by NeuN expression). Figure 3B Shown are the percent sensitivity (percentage of the neuronal population captured) of transgene expression in neurons within the somatosensory cortex following systemic administration. Figure 3C The percent sensitivity of transgene expression across cortical layers within the somatosensory cortex is shown. Figure 3D The percent sensitivity of transgene expression in neurons within the striatum is shown.

[0253] Following systemic delivery of rAAV (ie, through the body by injection in the circulatory system), these sequences did not elicit transgene expression in any organ except the brain.

[0254] Intraventricular viral injection

[0255] For ICV injection of postnatal mice, 5E+10 produced viral particles with AAV capsid AAV9 were injected on PND 1. After 4 weeks of incubation, animals were collected on postnatal day 29 (PND29).

[0256] Analysis of early postnatal ICV injections of regulatory elements in AAV9 driving dTomato or dCas9

[0257] Figure 4A and Figure 4B Representative images of transgene expression following ICV injection at P1 and 4 weeks of incubation using AAV9 capsids are shown in (animals collected at P29). Figure 4AImages showing expression of a fluorescent reporter gene (dTomato) driven by CNS2_V4.1 (SEQ ID NO: 10) in the brain. Figure 4B Shown is expression analysis of dCas9 transgene expression driven by CNS2_V4.1 (SEQ ID NO: 10) detected by qPCR.

[0258] The data show that the regulatory elements retain their identity across different injection methods, injection ages, transgenes, and serotypes. The CNS2_V4.1 regulatory elements showed the same expression profile after ICV injection of AAV9 capsids at PND1 compared to systemic injection of AAV PHP.eB capsids in adults.

Claims

1. A vector comprising (i) a heterologous nucleotide sequence; and (ii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer, wherein the one or more regions have at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

2. The vector of claim 1, wherein the vector further comprises a promoter.

3. The vector according to claim 2, comprising in the order of 5' to 3': (i) the promoter; (ii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and (iii) the heterologous nucleotide sequence.

4. The vector according to claim 2, comprising in the order of 5' to 3': (i)(a) the nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and (ii) said promoter; and (iii) the heterologous nucleotide sequence.

5. The vector according to claim 2, comprising in the order of 5' to 3': (i) the promoter; (ii) the heterologous nucleotide sequence; and (iii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

6. The vector of any one of claims 1-5, wherein the vector is a non-viral vector.

7. The vector of claim 6, wherein the non-viral vector is a plasmid vector.

8. The vector of any one of claims 1-5, wherein the vector is a viral vector.

9. The vector of claim 8, wherein the viral vector is an adeno-associated virus (AAV) vector or an adenoviral vector.

10. The vector of claim 9, wherein the AAV vector comprises a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR flanking the heterologous nucleotide sequence, a promoter and (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or higher sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

11. The vector of claim 10, comprising, in 5' to 3' order: (i) the 5' AAV ITR; (ii) the promoter; (iii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (iv) the heterologous nucleotide sequence; and (v) the 3' AAV ITR.

12. The vector of claim 10, comprising, in 5' to 3' order: (i) the 5' AAV ITR; (ii) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (iii) the promoter; (iv) the heterologous nucleotide sequence; and (v) the 3' AAV ITR.

13. The vector of claim 10, comprising, in 5' to 3' order: (i) the 5' AAV ITR; (ii) the promoter; (iii) the heterologous nucleotide sequence; (iv) (a) the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (b) the reverse nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (d) the reverse complementary nucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and (v) the 3' AAV ITR.

14. The vector of any one of claims 2 to 13, wherein the promoter comprises an RNA-polymerase II binding site, a TATA box, a TF2B recognition element (BRE), a motif 10 element (MTE), or a downstream promoter element (DPE).

15. The vector of any one of claims 2-13, wherein the promoter is a minimal promoter. The vector of claim 15 , wherein the minimal promoter is the human β-globin minimal promoter.

17. The vector of any one of claims 1-16, wherein the vector further comprises an enhancer.

18. The vector of any one of claims 1-17, wherein the vector further comprises or encodes a polyadenylation signal sequence.

19. The vector of any one of claims 1 to 18, wherein the vector further comprises or encodes a Kozak sequence.

20. The vector of any one of claims 1-19, wherein the vector further comprises or encodes a woodchuck hepatitis virus post-transcriptional element (WPRE), a hepatitis B virus post-transcriptional regulatory element, an RNA transport element (RTE), a WPRE3 or a wsl3 regulatory element.

21. The vector of any one of claims 1-20, wherein the vector further comprises an artificial intron.

22. The vector of any one of claims 1-21, wherein the vector further encodes a 2A self-cleaving peptide.

23. The vector of any one of claims 1-22, wherein the vector further comprises or encodes an internal ribosome entry site (IRES).

24. The vector of any one of claims 1-23, wherein the heterologous nucleotide sequence encodes a protein.

25. The vector of any one of claims 1-23, wherein the heterologous nucleotide sequence encodes a gene silencing nucleic acid.

26. The vector of any one of claims 1-23, wherein the heterologous nucleotide sequence encodes a CRISPR-associated protein.

27. The vector of claim 26, wherein the heterologous nucleotide sequence further encodes a guide RNA (gRNA).

28. A viral particle comprising the vector of any one of claims 1-5 and 8-27.

29. The viral particle of claim 28, wherein the viral particle is a recombinant adenoviral particle.

30. The viral particle of claim 28, wherein the viral particle is a recombinant AAV (rAAV) particle.

31. The viral particle of claim 30, wherein the rAAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Particles of serotype Anc80, TM-AAV6, AAV-PHP.A, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV2-TLH, or AAV2-GMN.

32. A population of viral particles comprising a plurality of the viral particles of any one of claims 28-32.

33. A pharmaceutical composition comprising the vector of any one of claims 1-27, the viral particle of any one of claims 28-32 or the population of claim 32, and a pharmaceutically acceptable carrier, excipient or diluent.

34. A nanoparticle comprising the carrier of any one of claims 1-7 and 14-27.

35. A cell comprising the vector of any one of claims 1-27 or the viral particle of any one of claims 28-32.

36. The cell of claim 35, wherein the cell is a mammalian cell or an insect cell.

37. A method for producing rAAV particles, the method comprising: (i) culturing the cell of claim 35 or 36 under conditions that allow packaging of the rAAV particles; and (ii) harvesting the cultured host cells or culture medium for collecting the rAAV particles.

38. A method for selectively expressing a heterologous nucleotide sequence in neurons of the central nervous system (CNS) of a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 1-27, the viral particle of any one of claims 28-32, the population of claim 32, the pharmaceutical composition of claim 33, or the nanoparticle of claim 34.

39. The method of claim 38, wherein expression of the heterologous nucleotide sequence is enriched in a specific region of the CNS.

40. The method of claim 38, wherein the heterologous nucleotide sequence expression is enriched in neurons of one or more of: forebrain, cerebellum, and spinal cord.

41. The method of claim 38, wherein expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain and the cerebellum.

42. The method of claim 38, wherein expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain.

43. The method of claim 38, wherein expression of the heterologous nucleotide sequence is enriched in neurons of the cerebellum.

44. The method of claim 38, wherein expression of the heterologous nucleotide sequence is enriched in neurons of the spinal cord.

45. The method of claim 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10, and wherein expression of the heterologous nucleotide sequence is enriched in neurons of the CNS.

46. ​​The method of claim 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:9, and wherein expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain and the cerebellum.

47. The method of claim 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:11, and wherein expression of the heterologous nucleotide sequence is enriched in neurons of the forebrain.

48. The method of claim 38, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 6, and wherein expression of the heterologous nucleotide sequence is enriched in neurons of the cerebellum and hippocampus.

49. The method of any one of claims 38-48, wherein the vector, the viral particle, the population, the pharmaceutical composition, or the nanoparticle is administered to the subject intravenously.

50. The method of any one of claims 38-48, wherein the vector, the viral particle, the population, the pharmaceutical composition, or the nanoparticle is administered to the subject via injection into the CNS.

Images