Nucleic acid regulatory elements for gene expression in central nervous system and methods of use

Abstract

The present application relates to nucleic acid regulatory elements (NAREs) capable of enhancing expression of genes in the central nervous system. The present application also relates to methods of employing the NARE and uses of the NARE. Expression cassettes and vectors containing the NARE are also disclosed. These are particularly useful for applications using gene therapy.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of the earlier filing dates of U.S. Provisional Patent Application No. 63 / 379,138, filed on October 11, 2022, and U.S. Provisional Patent Application No. 63 / 496,554, filed on April 17, 2023, under 35 U.S.C.§119(e). The U.S. Provisional Patent Applications are hereby incorporated by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The content of the electronic sequence listing (SeqList - 162027.54376.xml; size: 166,758 bytes; and creation date: October 6, 2023) is hereby incorporated by reference in its entirety herein. Technical field

[0005] This application relates to nucleic acid regulatory elements that are capable of enhancing gene expression in a variety of tissues or particularly in tissues including the CNS (central nervous system). This application also relates to methods of using these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. These are particularly useful for applications using gene therapy. Background art

[0006] A promoter is a DNA region that initiates gene transcription. Since the promoter region controls when and where a gene of interest is expressed in an organism, the promoter is a key element for regulating the level and specificity of transgene expression, particularly in the context of gene therapy.

[0007] The use of engineered nucleic acid regulatory elements (NAREs) (which may include various components, including promoters, enhancers, etc.) specifically customized for a given gene therapy provides multiple benefits. First, optimized NAREs allow for the gene expression levels required for a particular therapeutic gene. Second, engineered NAREs with enhanced potency allow for the administration of a smaller amount of gene therapy vector, thus reducing immune responses and associated safety risks. Third, for some gene therapies, it may be desirable to confine gene expression to one or more specific tissues. Thus, the use of nucleic acid regulatory elements with tissue-specific expression can limit unwanted transgene expression and promote persistent transgene expression in the tissue of interest. Such tissue-specific NAREs can be used to eliminate the need for tissue-specific viral capsids for gene delivery (or can be used in combination with tissue-specific viral capsids). Fourth, selection of an appropriate NARE also allows for control of the kinetics of gene expression, and thus influences the persistence of gene therapy. Finally, it may be desirable to engineer the NAREs to be of reduced size (without sacrificing strength or specificity) in order to effectively package larger transgene cargo into viral vectors.

[0008] Although attempts have been made to characterize and optimize NARE sequences through conventional low-throughput assays (rational design) or newer high-throughput methods (such as MPRA), these methods still require costly and time-consuming in vitro optimization. Thus, there is a need for more efficient methods of engineering NAREs, as well as nucleic acid regulatory elements with enhanced potency, reduced size, and / or tissue specificity. SUMMARY OF THE INVENTION

[0009] Provided herein are nucleic acid regulatory elements (NAREs), including NAREs particularly suitable for expression of an operably linked sequence (e.g., a protein or RNA coding sequence) in the central nervous system. Also provided herein are methods of using the NAREs and uses of the NAREs. For example, provided herein are methods for expressing a transgene operably linked to one or more of the nucleic acid regulatory elements disclosed herein. Also provided are expression cassettes and vectors containing the NAREs.

[0010] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53 (see Table 4). In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53. In an embodiment, a polynucleotide sequence is provided that comprises a NARE sequence selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53.

[0011] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of: B29, B30, B32, B35, B36, B39, B40-B44, and B46-B52-B53. In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of: B29, B30, B32, B35, B36, B39, B40-B44, and B46-B52-B53. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: B29, B30, B32, B35, B36, B39, B40-B44, and B46-B52-B53.

[0012] In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a sequence that is at least 80% identical to a NARE selected from the group consisting of B32, B36, and B48 - B50. In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a sequence that is at least 90% identical to a NARE selected from the group consisting of B32, B36, and B48 - B50. In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a NARE selected from the group consisting of B32, B36, and B48 - B50.

[0013] In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a sequence that is at least 80% identical to the NARE sequence provided in Table 4. In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a sequence that is at least 90% identical to the NARE sequence provided in Table 4. In an embodiment, a polynucleotide is provided, the polynucleotide comprising the NARE sequence provided in Table 4.

[0014] In one aspect, a NARE is provided, the NARE comprising:

[0015] a) (i) a sequence that is at least 90% identical to SEQ ID NO:36;

[0016] b) (i) a sequence that is at least 90% identical to SEQ ID NO:91; (ii) a sequence that is at least 90% identical to SEQ ID NO:92; (iii) a sequence that is at least 90% identical to SEQ ID NO:93; and (iv) a sequence that is at least 90% identical to SEQ ID NO:94;

[0017] c) (i) a sequence that is at least 90% identical to SEQ ID NO:98; and (ii) a sequence that is at least 90% identical to SEQ ID NO:99;

[0018] d) (i) a sequence that is at least 90% identical to SEQ ID NO:98; and (ii) a sequence that is at least 90% identical to SEQ ID NO:100; or

[0019] e) (i) a sequence that is at least 90% identical to SEQ ID NO:57.

[0020] In some embodiments, the NARE comprises:

[0021] a) (i) a sequence that is at least 95% identical to SEQ ID NO:36;

[0022] b) (i) A sequence that is at least 95% identical to SEQ ID NO: 91; (ii) A sequence that is at least 95% identical to SEQ ID NO: 92; (iii) A sequence that is at least 95% identical to SEQ ID NO: 93; and (iv) A sequence that is at least 95% identical to SEQ ID NO: 94;

[0023] c) (i) A sequence that is at least 95% identical to SEQ ID NO: 98; and (ii) A sequence that is at least 95% identical to SEQ ID NO: 99;

[0024] d) (i) A sequence that is at least 95% identical to SEQ ID NO: 98; and (ii) A sequence that is at least 95% identical to SEQ ID NO: 100; or

[0025] e) (i) A sequence that is at least 95% identical to SEQ ID NO: 57.

[0026] In some embodiments, the NARE comprises:

[0027] a) (i) SEQ ID NO: 36;

[0028] b) (i) SEQ ID NO: 91; (ii) SEQ ID NO: 92; (iii) SEQ ID NO: 93; and (iv) SEQ ID NO: 94;

[0029] c) (i) SEQ ID NO: 98; and (ii) SEQ ID NO: 99;

[0030] d) (i) SEQ ID NO: 98; and (ii) SEQ ID NO: 100; or

[0031] e) (i) SEQ ID NO: 57.

[0032] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 36; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 91-94; (iii) a sequence that is at least 90% identical to SEQ ID NO: 98 or SEQ ID NO: 99; (iv) a sequence that is at least 90% identical to SEQ ID NO: 98 or SEQ ID NO: 100; and (v) a sequence that is at least 90% identical to SEQ ID NO: 57. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 36; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 91-94; (iii) a sequence that is at least 95% identical to SEQ ID NO: 98 or SEQ ID NO: 99; (iv) a sequence that is at least 95% identical to SEQ ID NO: 98 or SEQ ID NO: 100; and (v) a sequence that is at least 95% identical to SEQ ID NO: 57. In some embodiments, a NARE is provided, which comprises: (i) SEQ ID NO: 36; (ii) any one of SEQ ID NOs: 91-94; (iii) SEQ ID NO: 98 or SEQ ID NO: 99; (iv) SEQ ID NO: 98 or SEQ ID NO: 100; and (v) SEQ ID NO: 57.

[0033] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 90% identical to SEQ ID NO: 99 or SEQ ID NO: 100. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 95% identical to SEQ ID NO: 99 or SEQ ID NO: 100. In one embodiment, the NARE comprises: (i) SEQ ID NO: 98; and (ii) SEQ ID NO: 99 or SEQ ID NO: 100.

[0034] On the one hand, a NARE is provided, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 63; and (ii) a sequence that is at least 90% identical to any one of SEQ ID NO: 62, 68, 70, 72, 74, 95 or 96; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 73. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 63; and (ii) a sequence that is at least 95% identical to any one of SEQ ID NO: 62, 68, 70, 72, 74, 95 or 96; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 73. In one embodiment, the NARE comprises: (i) SEQ ID NO: 63; and (ii) any one of SEQ ID NO: 62, 68, 70, 72, 74, 95 or 96; and (iii) optionally, SEQ ID NO: 73.

[0035] On the one hand, a NARE is provided, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 68; (ii) at least 90% identical to SEQ ID NO: 71; and (iii) optionally, a sequence that is at least 90% identical to any one of SEQ ID NO: 87 - 90. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 68; (ii) at least 95% identical to SEQ ID NO: 71; and (iii) optionally, a sequence that is at least 95% identical to any one of SEQ ID NO: 87 - 90. In one embodiment, the NARE comprises: (i) SEQ ID NO: 68; (ii) SEQ ID NO: 71; and (iii) optionally, any one of SEQ ID NO: 87 - 90.

[0036] On the one hand, a NARE is provided, and the NARE comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 83; (ii) a sequence that is at least 90% identical to SEQ ID NO: 84; (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 66 or SEQ ID NO: 86; and (iv) optionally, a sequence that is at least 90% identical to SEQ ID NO: 85. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 83; (ii) a sequence that is at least 95% identical to SEQ ID NO: 84; (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 66 or SEQ ID NO: 86; and (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 85. In one embodiment, the NARE comprises: (i) SEQ ID NO: 83; (i) SEQ ID NO: 84; (iii) optionally, SEQ ID NO: 66 or SEQ ID NO: 86; and (iv) optionally, SEQ ID NO: 85.

[0037] On the one hand, a NARE is provided, and the NARE comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 91; (ii) a sequence that is at least 90% identical to SEQ ID NO: 92; (iii) a sequence that is at least 90% identical to SEQ ID NO: 93; (iv) a sequence that is at least 90% identical to SEQ ID NO: 94; and (v) optionally, a sequence that is at least 90% identical to SEQ ID NO: 63. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 91; (ii) a sequence that is at least 95% identical to SEQ ID NO: 92; (iii) a sequence that is at least 95% identical to SEQ ID NO: 93; (iv) a sequence that is at least 95% identical to SEQ ID NO: 94; and (v) optionally, a sequence that is at least 95% identical to SEQ ID NO: 63. In one embodiment, the NARE comprises: (i) SEQ ID NO: 91; (ii) SEQ ID NO: 92; (iii) SEQ ID NO: 93; (iv) SEQ ID NO: 94; and (v) optionally, SEQ ID NO: 63.

[0038] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NO:75, 76 or 77; (ii) a sequence that is at least 90% identical to SEQ ID NO:104; and (iii) a sequence that is at least 90% identical to SEQ ID NO:103 or 105. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to any one of SEQ ID NO:75, 76 or 77; (ii) a sequence that is at least 95% identical to SEQ ID NO:104; and (iii) a sequence that is at least 95% identical to SEQ ID NO:103 or 105. In one embodiment, the NARE comprises: (i) any one of SEQ ID NO:75, 76 or 77; (ii) SEQ ID NO:104; and (iii) SEQ ID NO:103 or 105.

[0039] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to SEQ ID NO:64; (ii) a sequence that is at least 90% identical to SEQ ID NO:65; (iii) a sequence that is at least 90% identical to SEQ ID NO 66; and (iv) a sequence that is at least 90% identical to SEQ ID NO:67. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO:64; (ii) a sequence that is at least 95% identical to SEQ ID NO:65; (iii) a sequence that is at least 95% identical to SEQ ID NO 66; and (iv) a sequence that is at least 90% identical to SEQ ID NO:67. In one embodiment, the NARE comprises: (i) SEQ ID NO:64; (ii) SEQ ID NO:65; (iii) SEQ ID NO 66; and (iv) SEQ ID NO:67.

[0040] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 73; (ii) a sequence that is at least 90% identical to any one of SEQ ID NO: 75, 76 or 77; and (iii) a sequence that is at least 90% identical to SEQ ID NO: 78. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 73; (ii) a sequence that is at least 95% identical to any one of SEQ ID NO: 75, 76 or 77; and (iii) a sequence that is at least 95% identical to SEQ ID NO: 78. In one embodiment, the NARE comprises: (i) SEQ ID NO: 73; (ii) any one of SEQ ID NO: 75, 76 or 77; and (iii) SEQ ID NO: 78.

[0041] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 79; and (ii) a sequence that is at least 90% identical to SEQ ID NO: 80. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 79; and (ii) a sequence that is at least 95% identical to SEQ ID NO: 80. In one embodiment, the NARE comprises: (i) SEQ ID NO: 79; and (ii) SEQ ID NO: 80.

[0042] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 81; and (ii) a sequence that is at least 90% identical to SEQ ID NO: 82. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 81; and (ii) a sequence that is at least 95% identical to SEQ ID NO: 82. In one embodiment, the NARE comprises: (i) SEQ ID NO: 81; and (ii) SEQ ID NO: 82.

[0043] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NO:75, 76 or 77; and (ii) a sequence that is at least 90% identical to SEQ ID NO:104. In one embodiment, the NARE comprises: (i) a sequence that is at least 95% identical to any one of SEQ ID NO:75, 76 or 77; and (ii) a sequence that is at least 95% identical to SEQ ID NO:104. In one embodiment, the NARE comprises: (i) any one of SEQ ID NO:75, 76 or 77; and (ii) SEQ ID NO:104.

[0044] On the one hand, a NARE is provided, which comprises a sequence that is at least 90% identical to any one of SEQ ID NO:3 - 5, 7 - 9, 11, 14, 15, 18 and 23 - 61. In one embodiment, the NARE comprises a sequence that is at least 95% identical to any one of SEQ ID NO:3 - 5, 7 - 9, 11, 14, 15, 18 and 23 - 61. In one embodiment, the NARE comprises any one of SEQ ID NO:3 - 5, 7 - 9, 11, 14, 15, 18 and 23 - 61. In one embodiment, the NARE comprises a sequence that is at least 90% identical to SEQ ID NO:36, 40, 55, 56 or 57. In one embodiment, the NARE comprises a sequence that is at least 95% identical to SEQ ID NO:36, 40, 55, 56 or 57. In one embodiment, the NARE comprises any one of SEQ ID NO:36, 40, 55, 56 or 57.

[0045] On the one hand, an expression construct is provided, which comprises the NARE disclosed herein and a transgene operably linked thereto. In one embodiment, the expression construct further comprises a polyadenylation sequence.

[0046] On the one hand, a vector is provided, which comprises the expression construct disclosed herein. In one embodiment, the vector is a non - viral vector. In one embodiment, the vector is a viral vector. In one embodiment, the vector is an adeno - associated virus (AAV) vector. In one embodiment, the vector comprises a nucleic acid sequence that comprises (i) the expression construct disclosed herein, and (ii) one or more inverted terminal repeats (ITRs). In one embodiment, the vector comprises a nucleic acid sequence that comprises a 5' ITR and a 3' ITR. In some embodiments, the 5' ITR and the 3' ITR are derived from AAV serotype AAV2.

[0047] On the one hand, a cell is provided, which cell comprises an expression construct disclosed herein or a vector disclosed herein. In one embodiment, the cell is a neuronal cell.

[0048] On the one hand, a pharmaceutical composition is provided, which pharmaceutical composition comprises (i) an expression construct disclosed herein or a vector disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0049] On the one hand, a method for expressing a transgene in a cell comprising an expression construct disclosed herein or a vector disclosed herein is provided. On the one hand, a method for regulating the expression of a transgene in a cell comprising an expression construct disclosed herein or a vector disclosed herein is provided. In one embodiment, the cell is a neuronal cell.

[0050] On the one hand, provided herein is a method for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject an expression construct disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein. On the one hand, provided herein is a method for treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, the method comprising administering to the subject an expression construct disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein. In one embodiment, the subject has a mutation in the ALS2 gene, VAPB gene, SETX gene, TDP-43 gene, FUS / TLS gene, C9orf72 gene, and / or OPTN gene. In one embodiment, the subject is human. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1A and Figure 1B . Dual reporter design and measurement. Figure 1A Illustrates the design of the dual reporter construct. Candidate NAREs were cloned upstream (5′) of the mClover3 coding sequence. Each construct contains a constant region that includes the tdTomato transgene used as a normalization control. Figure 1B Provides an example of flow cytometry data obtained with the dual reporter system, where mClover3 expression in tdTomato+ cells reflects the level of promoter activity.

[0052] Figure 2A 、 Figure 2B 、 Figure 2C 、 Figure 2D 、 Figure 2E and Figure 2F . Potency of selected NAREs in neuronal cells. Figure 2A 、 Figure 2C andFigure 2D . Potency of selected NAREs measured using a dual reporter assay in transfected N2a cells (mouse neuroblastoma cell line). Figure 2B : Potency of selected NAREs in transfected BE2M17 cells (neuroblast cell line isolated from the brain of a 2-year-old male patient with neuroblastoma). Figure 2E : Potency of selected NAREs in transfected BE2M17 cells. Figure 2F : Potency of selected promoters in transfected N2a cells. Detailed Description

[0053] Nucleic acid regulatory elements (NAREs), including promoters, are essential components of gene therapy, controlling the expression level and persistence of therapeutic genes. NAREs can drive cell-specific expression of transgenes independent of, for example, capsid selection. Incorporating stronger NAREs can increase potency and efficacy at lower viral vector doses, potentially reducing safety risks, immune responses, and vector production costs. Additionally, reducing the NARE size while maintaining strength and specificity allows for efficient packaging of larger transgenes or expression cassettes into AAVs. This application provides a library of NAREs to improve the safety, efficacy, and persistence of therapeutic transgene expression.

[0054] Although multiple NAREs and particularly promoter sequences have been characterized / optimized through conventional low-throughput assays (rational design) or newer high-throughput methods such as MPRA, these methods still require expensive and time-consuming in vitro optimization. Here, using an advanced artificial intelligence model, a convolutional neural network (CNN) was repurposed and optimized to predict promoter potency. A library of NAREs was computationally constructed by cloning all known enhancer elements reported in the ENCODE database upstream of efficient, compact constitutive promoters. Subsequently, the correct spacing between enhancer elements that performed particularly well and promoter sequences was optimized. At the same time, in silico saturation mutagenesis was performed, whereby all possible point mutations within the promoter sequence were introduced and those with the greatest impact on promoter potency were selected. Elements that performed particularly well were generated and tested in vitro. While most NAREs showed better or equivalent performance compared to the original promoter, the best enhancer elements and point mutations were selected and incorporated in a second round of optimization. After synthesizing and testing this new set in vitro, enhanced NAREs were obtained that exhibited increased potency.

[0055] NARE

[0056] This document provides NAREs, which may include promoters and / or enhancers as part of their sequences. Traditionally, a promoter is defined as the DNA region that initiates transcription. A promoter includes specific DNA motifs accessible by transcription factors (TFs) and their complexes. On the other hand, an enhancer is defined as the DNA region that amplifies transcription initiation by directly interacting with the target promoter. Similarly, enhancer sequences far from the target promoter contain DNA motifs that serve as binding sites for TFs and cofactors. Sometimes, the term promoter can be used as a shorthand to refer to a nucleic acid sequence containing multiple regulatory elements. For example, the control promoter CAG contains the CMV enhancer, the β-actin promoter region, and an intron, but can also be referred to as a promoter. Specifically, the CAG promoter consists of: (1) the cytomegalovirus (CMV) early enhancer element, (2) the promoter, the first exon, and the first intron of the chicken β-actin gene, and (3) the splice acceptor of the rabbit β-globin gene. As used herein, the term "nucleic acid regulatory element" can refer to a promoter as defined in the traditional sense, as well as a combination of elements containing a promoter and / or other nucleic acid regulatory elements that regulate the expression of a sequence encoding an RNA or protein operably linked to the element. A nucleic acid regulatory element can be or include, for example, one or more of a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0057] As used herein, the term "NARE" can refer to a promoter as defined in the traditional sense, as well as a combination of nucleic acid regulatory elements containing a promoter and / or other nucleic acid regulatory elements that regulate the expression of a gene operably linked to the element. A nucleic acid regulatory element can be or include, for example, one or more of a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0058] As used herein, a "transgene" refers to, for example, a gene (specifically the coding sequence of the gene) transferred into one or more cells of an organism using the vectors described herein. A transgene can encode a protein or RNA that is normally expressed in the cells of the target organism, or can encode a protein or RNA from a different organism. A transgene can be integrated into the genome of the target cell or can exist as part of an extrachromosomal expression construct.

[0059] As used herein, "operatively linked" means that a first molecule is joined to a second molecule, where the molecules are arranged such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, if a NARE regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, then the NARE is operatively linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are joined such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion, then they are operatively linked to each other. Two transcriptional regulatory elements can be operatively linked to each other by a linker nucleic acid (e.g., intervening non-coding nucleic acid) or can be operatively linked to each other in the absence of intervening nucleotides.

[0060] The present invention provides NAREs that are particularly suitable for driving expression in the CNS. As used herein, NAREs starting with "B" are particularly useful for the expression of genes in the CNS (including in neuronal cells). CNS NAREs can be CNS-specific, meaning that they show a significant increase or preferential expression of an operatively linked transgene in the CNS compared to other tissues. Tables 2 and 4 provide NAREs that are particularly suitable for driving expression in the CNS.

[0061] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a NARE provided in Table 2 or Table 4. In an embodiment, a polynucleotide is provided that comprises one or more NAREs provided in Table 2 or Table 4.

[0062] In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a NARE selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b and B53. In an embodiment, a polynucleotide sequence is provided, the polynucleotide sequence comprising a NARE sequence selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b and B53.

[0063] As used herein, the term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that are aligned for purposes of comparison. For example, when a position in the compared nucleotide sequences is occupied by the same base, the molecules are identical at that position. The degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. Methods and computer programs for determining sequence identity and similarity are publicly available, including but not limited to the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990)) and the ALIGN program (version 2.0). The well-known Smith-Waterman algorithm can also be used to determine similarity. The BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0, ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions and other modifications when comparing sequences.

[0064] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of: B29, B30, B32, B35, B36, B39, B40 - B44, and B46 - B52 - B53. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: B29, B30, B32, B35, B36, B39, B40 - B44, and B46 - B52 - B53.

[0065] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of: B32, B36, and B48 - B50. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: B32, B36, and B48 - B50.

[0066] In an embodiment, the NARE comprises an intron or a portion of an intron. The intron or portion of the intron can be selected from: ACTA1 intron, ACTA1e intron, ACTC1 intron, ACTC1.3 intron, ACTC1e intron, ACTC1e - ACTC1p intron, ALDOA intron, APOC intron, APOC1 intron, ATF5 intron, CAMK2A intron, CBA intron, chimeric intron, CMV - rabbit β - globin intron, CRYAB intron, DES.4 intron, EEF1A1 intron, EEF1B2 intron, EF1a intron, FHL1 intron, FLOT1 intron, FXYD1 intron, GFAP intron, HBB intron, hCPE intron, hEf1a2 - intron, HPD intron, IFI27L2 intron, murine IgG chimeric intron, MVM intron, MVMi - AATp intron, MVMi - SynE - mTTRp intron, rabbit β - globin intron, RBP4 intron, RPL26 intron, RPL27 intron, S100A6 intron, sEEF1A1 intron, SV40 intron, TMSB10 intron, and UCHL1 intron.

[0067] In an embodiment, the NARE comprises an enhancer or a part of an enhancer. The enhancer or the part of the enhancer can be selected from: ACTA1 enhancer, ACTC1 enhancer, CKM enhancer, CMV enhancer, CMV enhancer, MCK enhancer, mDES enhancer, mDES.1 enhancer, minCKM enhancer, minCKM2 enhancer, minDes enhancer, NRGN enhancer 1.1, NRGN enhancer 1.2, NRGN enhancer 2.1, NRGN enhancer 2.2, SV40 enhancer and SV40 enhancer (SV40e).

[0068] In an embodiment, the nucleic acid regulatory element comprises a UTR or a part of a UTR. The UTR or the part of the UTR can be selected from: CTNNB1 UTR, hEf1a2-utr1, hEf1a2-utr2, hNSE utr1, hNSE utr2, hSyn1_utr1, hSyn1_utr2, HTLV 5'UTR, HTLV 5'UTR, L21 UTR, TMSB10_utr1 and TMSB10_utr2.

[0069] In some embodiments, the NARE comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to a sequence selected from the group consisting of: SEQ ID NO:3-5, 7-9, 11, 14, 15, 18 and 23-61.

[0070] In some embodiments, the NARE comprises a sequence selected from the group consisting of: SEQ ID NO:3-5, 7-9, 11, 14, 15, 18 and 23-61.

[0071] In some embodiments, the NARE comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to a sequence selected from the group consisting of: SEQ ID NO:24, 25, 33-39, 42-45, 47, 49-54, 57 and 58.

[0072] In some embodiments, the NARE comprises a sequence selected from the group consisting of: SEQ ID NO:24, 25, 33-39, 42-45, 47, 49-54, 57 and 58.

[0073] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 63; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 62, 68, 70, 72, 74, 95, or 96; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 73.

[0074] In some embodiments, the NARE comprises: (i) SEQ ID NO: 62; (ii) any one of SEQ ID NOs: 62, 68, 70, 72, 74, 95, or 96; and (iii) optionally, SEQ ID NO: 73.

[0075] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 62; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 63.

[0076] In some embodiments, the NARE comprises: (i) SEQ ID NO: 62; and (ii) SEQ ID NO: 63.

[0077] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:64; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:65; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO 66; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67.

[0078] In some embodiments, the NARE comprises: (i) SEQ ID NO:64; (ii) SEQ ID NO:65; (iii) SEQ ID NO:66; and (iv) SEQ ID NO:67.

[0079] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:70; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63.

[0080] In some embodiments, the NARE comprises: (i) SEQ ID NO:70; and (ii) SEQ ID NO:63.

[0081] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:68; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63.

[0082] In some embodiments, the NARE comprises: (i) SEQ ID NO:68; and (ii) SEQ ID NO:63.

[0083] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:68; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:71; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs:87 - 90.

[0084] In some embodiments, the NARE comprises: (i) SEQ ID NO:68; (ii) SEQ ID NO:71; and (iii) optionally, any one of SEQ ID NOs:87 - 90.

[0085] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:68; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:71.

[0086] In some embodiments, the NARE comprises: (i) SEQ ID NO:68; and (ii) SEQ ID NO:71.

[0087] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:63; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:72.

[0088] In some embodiments, the NARE comprises: (i) SEQ ID NO:63; and (ii) SEQ ID NO:72.

[0089] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63.

[0090] In some embodiments, the NARE comprises: (i) SEQ ID NO:73; (ii) SEQ ID NO:74; and (iii) SEQ ID NO:63.

[0091] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:75, 76, or 77; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:78.

[0092] In some embodiments, the NARE comprises: (i) SEQ ID NO:73; (ii) any one of SEQ ID NO:75, 76, or 77; and (iii) SEQ ID NO:78.

[0093] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:75; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:78.

[0094] In some embodiments, the NARE comprises: (i) SEQ ID NO:73; (ii) SEQ ID NO:75; and (iii) SEQ ID NO:78.

[0095] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:80.

[0096] In some embodiments, the NARE comprises: (i) SEQ ID NO:79; and (ii) SEQ ID NO:80.

[0097] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:81; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:82.

[0098] In some embodiments, the NARE comprises: (i) SEQ ID NO:81; and (ii) SEQ ID NO:82.

[0099] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:84; (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:66 or SEQ ID NO:86; and (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:85.

[0100] In some embodiments, the NARE comprises: (i) SEQ ID NO:83; (ii) SEQ ID NO:84; (iii) optionally, SEQ ID NO:66 or SEQ ID NO:86; and (iv) optionally, SEQ ID NO:85.

[0101] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:84.

[0102] In some embodiments, the NARE comprises: (i) SEQ ID NO:83; and (ii) SEQ ID NO:84.

[0103] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:84; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:85; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:86.

[0104] In some embodiments, the NARE comprises: (i) SEQ ID NO:83; (ii) SEQ ID NO:84; (iii) SEQ ID NO:85; and (iv) SEQ ID NO:86.

[0105] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:84; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:66; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:86.

[0106] In some embodiments, the NARE comprises: (i) SEQ ID NO:83; (ii) SEQ ID NO:84; (iii) SEQ ID NO:66; and (iv) SEQ ID NO:86.

[0107] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 68; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 71; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 87.

[0108] In some embodiments, the NARE comprises: (i) SEQ ID NO: 68; (ii) SEQ ID NO: 71; and (iii) SEQ ID NO: 87.

[0109] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 68; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 71; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 88.

[0110] In some embodiments, the NARE comprises: (i) SEQ ID NO: 68; (ii) SEQ ID NO: 71; and (iii) SEQ ID NO: 88.

[0111] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:68; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:71; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:89.

[0112] In some embodiments, the NARE comprises: (i) SEQ ID NO:68; (ii) SEQ ID NO:71; and (iii) SEQ ID NO:89.

[0113] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:68; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:71; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:90.

[0114] In some embodiments, the NARE comprises: (i) SEQ ID NO:68; (ii) SEQ ID NO:71; and (iii) SEQ ID NO:90.

[0115] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 93; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 94; and (v) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 63.

[0116] In some embodiments, the NARE comprises: (i) SEQ ID NO: 91; (ii) SEQ ID NO: 92; (iii) SEQ ID NO: 93; (iv) SEQ ID NO: 94; and (v) optionally, SEQ ID NO: 63.

[0117] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 93; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 94.

[0118] In some embodiments, the NARE comprises: (i) SEQ ID NO:91; (ii) SEQ ID NO:92; (iii) SEQ ID NO:93; and (iv) SEQ ID NO:94.

[0119] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:91; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:92; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:93; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:94; and (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63.

[0120] In some embodiments, the NARE comprises: (i) SEQ ID NO:91; (ii) SEQ ID NO:92; (iii) SEQ ID NO:93; (iv) SEQ ID NO:94; and (v) SEQ ID NO:63.

[0121] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:95.

[0122] In some embodiments, the NARE comprises: (i) SEQ ID NO:63; and (ii) SEQ ID NO:95.

[0123] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:96.

[0124] In some embodiments, the NARE comprises: (i) SEQ ID NO:63; and (ii) SEQ ID NO:96.

[0125] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:98; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:99 or SEQ ID NO:100.

[0126] In some embodiments, the NARE comprises: (i) SEQ ID NO:98; and (ii) SEQ ID NO:99 or SEQ ID NO:100.

[0127] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:98; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:99.

[0128] In some embodiments, the NARE comprises: (i) SEQ ID NO:98; and (ii) SEQ ID NO:99.

[0129] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:98; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:100.

[0130] In some embodiments, the NARE comprises: (i) SEQ ID NO:98; and (ii) SEQ ID NO:100.

[0131] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:75, 76, or 77; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:103 or 105.

[0132] In some embodiments, the NARE comprises: (i) any one of SEQ ID NO:75, 76, or 77; (ii) SEQ ID NO:104; and (iii) SEQ ID NO:103 or 105.

[0133] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:103 or 105.

[0134] In some embodiments, the NARE comprises: (i) SEQ ID NO:76; (ii) SEQ ID NO:104; and (iii) SEQ ID NO:103 or 105.

[0135] In some embodiments, the NARE comprises (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:75, 76, or 77; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:103; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104.

[0136] In some embodiments, the NARE comprises: (i) any one of SEQ ID NO:75, 76, or 77; (ii) SEQ ID NO:103; and (iii) SEQ ID NO:104.

[0137] In some embodiments, the NARE comprises (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:103; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104.

[0138] In some embodiments, the NARE comprises: (i) SEQ ID NO:76; (ii) SEQ ID NO:103; and (iii) SEQ ID NO:104.

[0139] In some embodiments, the NARE comprises (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:75, 76, or 77; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:105.

[0140] In some embodiments, the NARE comprises: (i) any one of SEQ ID NO:75, 76, or 77; (ii) SEQ ID NO:104; and (iii) SEQ ID NO:105.

[0141] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:105.

[0142] In some embodiments, the NARE comprises: (i) SEQ ID NO:76; (ii) SEQ ID NO:104; and (iii) SEQ ID NO:105.

[0143] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:75, 76, or 77; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104.

[0144] In some embodiments, the NARE comprises: (i) any one of SEQ ID NO:75, 76, or 77; and (ii) SEQ ID NO:104.

[0145] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:77; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:104.

[0146] In some embodiments, the NARE comprises: (i) SEQ ID NO:77; and (ii) SEQ ID NO:104.

[0147] Provided herein is a method for expressing a transgene in the CNS, wherein the transgene is operably linked to the NARE disclosed herein.

[0148] Provided herein is a method for expressing a transgene in neuronal cells, wherein the transgene is operably linked to the NARE disclosed herein.

[0149] The present disclosure provides a method for expressing a transgene in the CNS, wherein the transgene is operably linked to a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the NARE provided in Table 2 or Table 4. The present disclosure provides a method for expressing a transgene in the CNS, wherein the transgene is operably linked to the NARE provided in Table 2 or Table 4. The present disclosure provides a method for expressing a transgene in the CNS, wherein the transgene is operably linked to a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid regulatory element selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53. The present disclosure provides a method for expressing a transgene in the CNS, wherein the transgene is operably linked to a NARE selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53. The present disclosure provides a method for expressing a transgene in a neuronal cell, wherein the transgene is operably linked to a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the NARE provided in Table 2 or Table 4. The present disclosure provides a method for expressing a transgene in a neuronal cell, wherein the transgene is operably linked to the NARE provided in Table 2 or Table 4.

[0150] The present disclosure provides a method for expressing a transgene in a neuronal cell, wherein the transgene is operably linked to a NARE having a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53. The present disclosure provides a method for expressing a transgene in a neuronal cell, wherein the transgene is operably linked to a NARE selected from the group consisting of: B2_L21, B2_L21_v2, B3, B4-CPGless-L21, B4_L21, B5, B6, B6_L21, B8_L21, B9-B12, B19-B36, B36_v2, B37-B40, B40_v2, B41, B41_V2, B42-B52, B52b, and B53.

[0151] Add any content related to the main embodiments in the Summary of the Invention here.

[0152] In some embodiments, the transgene is UPF1 or BDNF.

[0153] The present disclosure also provides NAREs for use as references or controls. Some NAREs provide constitutive expression of an operably linked transgene, i.e., the expression of the transgene is maintained at a constant level. Constitutive NAREs can drive the expression of an operably linked transgene in a variety of cell types and tissues. As used herein, a promoter starting with "C" is a constitutive NARE. The constitutive NAREs disclosed herein can be used for gene expression in the liver, muscle, and / or central nervous system. Table 1 provides the sequences of the control NAREs.

[0154] NARE modification

[0155] Those skilled in the art will understand that certain modifications can be made to the NAREs disclosed herein without eliminating the ability of the NAREs to drive gene expression in the desired tissues / cell types. Various in vitro and in vivo methods are known in the art for confirming that modified NAREs are still capable of driving gene expression, including but not limited to the methods used herein. For example, the strength of a NARE can be evaluated by operably linking the NARE to a transgene encoding a protein and measuring transgene expression, such as by detecting the mRNA encoding the protein, or by measuring the presence or activity of the protein, such as by ELISA, Western Blot, fluorescence, enzymatic activity of the protein, etc.

[0156] Provided herein are NAREs comprising one or more components. Provided herein are NAREs comprising one or more of the sequence components (or sequence variants thereof) listed in any of Tables 1-4. Provided herein are NAREs comprising one or more of the sequence components (or sequence variants thereof) listed in Table 4. Provided herein are NAREs comprising one or more components (or sequence variants thereof) of the full-length NAREs provided in Table 4. By way of non-limiting example only, a NARE is provided that comprises one or more components (or sequence variants thereof) of NAREB2_L21_v2: (1) NSEmin; and (2) L21. In an embodiment, a NARE is provided that comprises one or more components (or sequence variants thereof) of the full-length NAREs provided in Table 4, whereby the components (or sequence variants thereof) are arranged 5' to 3' as shown in Table 4. In an embodiment, a NARE is provided that comprises one or more components (or sequence variants thereof) of the full-length NAREs provided in Table 4, whereby the components (or sequence variants thereof) are arranged in a different order 5' to 3' than shown in Table 4.

[0157] In an embodiment, in the NAREs of Table 2 or Table 4, one or more components have been replaced with different sequences, including components (or sequence variants thereof) from different NAREs of Table 2 or Table 4. In addition, NAREs comprising components (or sequence variants thereof) derived from two or more NAREs disclosed in Table 2 or Table 4 are also contemplated.

[0158] In addition, NARE variants were also considered, which include sequences other than the elements of NARE disclosed herein (e.g., added to either end or inserted within the NARE sequence). Similarly, NARE variants were provided, in which certain sequences have been removed from the NARE disclosed herein (e.g., as terminal or internal deletions). In an embodiment, a NARE is provided that includes one or more components without any additional nucleic acid sequences joining the components of the NARE. In an embodiment, a NARE is provided that includes one or more components, whereby the individual components are linked by additional nucleic acid sequences. These additional nucleic acid sequences may or may not be related to the expression of an operably linked transgene. In an embodiment, some components are directly linked, while other components are linked to other components via intervening sequences. Thus, variants of the NAREs disclosed in Table 2 or Table 4 are considered, in which additional sequences are added between different components. Further considered are variants of the NAREs disclosed in Table 2 or Table 4, which disclose the same components of a given NARE in Table 2 or Table 4, but differ in the sequences linking the individual components.

[0159] Nucleic acid constructs and vectors

[0160] In one aspect, nucleic acid constructs and vectors are provided, as well as their use for introducing a transgene or expression construct into a cell. The nucleic acid construct includes an expression construct, including a plasmid. The term "expression construct" refers to a recombinant polynucleotide construct that includes a nucleic acid encoding an RNA capable of being transcribed in a cell. Methods for constructing expression constructs and plasmids by standard recombinant techniques are known in the art.

[0161] In some embodiments, the vector contains a recombinant DNA construct that includes additional DNA elements, including DNA segments that provide for DNA replication in a host cell and expression of a target gene at an appropriate level in a target cell. As used herein, "vector" means an agent that contains a polynucleotide to be delivered to a host cell in vitro, ex vivo, or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA microcircles, or viruses (including virus-derived sequences). A vector can also refer to a virus particle that contains a nucleic acid to be delivered to a host cell in vitro, ex vivo, or in vivo. In some embodiments, a vector refers to a virus particle that contains a recombinant viral genome, wherein the viral genome contains one or more ITRs and a transgene.

[0162] In one embodiment, the vector is a viral vector or a combination of multiple viral vectors. In one aspect, a vector is provided that contains any nucleic acid construct disclosed herein.

[0163] The present disclosure provides nucleic acid constructs and vectors that comprise a NARE disclosed herein operably linked to a transgene. In embodiments, a nucleic acid construct or vector disclosed herein comprises additional regulatory elements including, but not limited to, promoters, enhancers, translation initiation signals, introns, and / or splicing enhancers. In embodiments, a nucleic acid construct or vector disclosed herein comprises a polyadenylation sequence. In embodiments, a nucleic acid construct or vector disclosed herein comprises an internal ribosome entry site (IRES). IRES sequences can be used to produce more than one polypeptide from a single gene transcript. The IRES (or other suitable sequence) is used to produce a protein containing more than one polypeptide chain, or to express two different proteins in or from the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE, and ganglion cells. In one embodiment, the IRES is located 3' of the transgene.

[0164] Viral vector

[0165] Viral vectors for expressing a target gene in a target cell, tissue, or organism are known in the art and include, for example, AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric viruses, mosaic viruses, or pseudotyped viruses and / or viruses containing exogenous proteins, synthetic polymers, nanoparticles, or small molecules).

[0166] AAV vector

[0167] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires a helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames that encode the Rep and Cap proteins, respectively. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. The functions of these proteins are mainly to regulate AAV replication and to rescue AAV and integrate it into the host cell chromosome. The Cap reading frame encodes three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the viral particle capsid. More than 80% of the total protein in the AAV viral particle comprises VP3. Flanking the rep and cap open reading frames at the 5' and 3' termini are inverted terminal repeats (ITRs) that are approximately 145 bp in length. The two ITRs are the only cis-elements required for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter gene transgene.

[0168] The recombinant adeno-associated virus "rAAV" vector includes any vector derived from any adeno-associated virus serotype. The rAAV vector may have one or more of the AAV wild-type genes that are entirely or partially deleted, preferably the Rep and / or Cap genes, but retains the functional flanking ITR sequences.

[0169] In some embodiments, the viral vector is an rAAV virion that contains an rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome contains the nucleic acid construct disclosed herein.

[0170] In some embodiments, the viral vector disclosed herein contains a nucleic acid that includes AAV 5' ITR and 3' ITR located 5' and 3' of the sequence encoding the transgene, respectively. In an embodiment, the transgene is UPF1, BDNF, or ATP7B. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5' ITR and 3' ITR sequences arranged in tandem, such as 5' to 3' or head-to-tail or in another alternative configuration. In other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITR, or to have 5' ITR (or conversely, 3' ITR) located both 5' and 3' of the transgene. The ITR sequences may be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences may be present. The ITR need not be a wild-type nucleotide sequence and may be altered (e.g., by nucleotide insertion, deletion, or substitution) as long as the sequence provides functional rescue, replication, and packaging. The ITR may be selected from AAV2, or from other AAV serotypes as described herein.

[0171] In some embodiments, a vector containing a nucleic acid sequence is provided, the nucleic acid sequence including (i) the nucleic acid construct disclosed herein and (ii) one or more inverted terminal repeats (ITRs). In one embodiment, the nucleic acid sequence includes a 5' ITR and a 3' ITR. In one embodiment, the 5' ITR and the 3' ITR are derived from the adeno-associated virus (AAV) serotype AAV2.

[0172] In some embodiments, the viral vector is an AAV vector, such as AAV1 (i.e., AAV containing AAV1 ITR and AAV1 capsid protein), AAV2 (i.e., AAV containing AAV2 ITR and AAV2 capsid protein), AAV3 (i.e., AAV containing AAV3 ITR and AAV3 capsid protein), AAV4 (i.e., AAV containing AAV4 ITR and AAV4 capsid protein), AAV5 (i.e., AAV containing AAV5 ITR and AAV5 capsid protein), AAV6 (i.e., AAV containing AAV6 ITR and AAV6 capsid protein), AAV7 (i.e., AAV containing AAV7 ITR and AAV7 capsid protein), AAV8 (i.e., AAV containing AAV8 ITR and AAV8 capsid protein), AAV9 (i.e., AAV containing AAV9 ITR and AAV9 capsid protein), AAVrh74 (i.e., AAV containing AAVrh74 ITR and AAVrh74 capsid protein), AAVrh.8 (i.e., AAV containing AAVrh.8 ITR and AAVrh.8 capsid protein), or AAVrh.10 (i.e., AAV containing AAVrh.10 ITR and AAVrh.10 capsid protein).

[0173] In some embodiments, the viral vector is a pseudotyped AAV vector that contains ITRs from one AAV serotype and a capsid protein from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., AAV containing AAV2 ITR and AAV9 capsid protein). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., AAV containing AAV2 ITR and AAV10 capsid protein).

[0174] In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., AAV containing AAV2 ITR and AAV7m8 capsid protein).

[0175] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein of a chimeric body containing one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In an embodiment, the capsid is a variant AAV capsid, such as AAV2 variant rAAV2-retro (SEQ ID NO:44, from WO 2017 / 218842, incorporated herein by reference).

[0176] In some embodiments, the AAV vector contains two or more capsid proteins selected from different serotypes. In some embodiments, the AAV vector contains rAAV2-retro and AAVrh.10 capsid proteins. In some embodiments, the AAV vector contains rAAV2-retro and AAVrh.10 capsid proteins at ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively. In some embodiments, the AAV vector contains AAVrh.10 and rAAV2-retro capsid proteins at ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively.

[0177] In some embodiments, a mixture of (1) an AAV vector containing rAAV2-retro and (2) an AAV vector containing AAVrh.10 is used. In some embodiments, the ratios of (1) the AAV vector containing rAAV2-retro and (2) the AAV vector containing AAVrh.10 are 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively. In some embodiments, the ratios of (1) the AAV vector containing AAVrh.10 and (2) the AAV vector containing rAAV2-retro are 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively.

[0178] Other viral vectors

[0179] Other viral vectors include adenovirus (AV) vectors, such as those based on human adenovirus type 2 and human adenovirus type 5, which have been rendered replication-deficient by deletions in the E1 and E3 regions. A transcription cassette can be inserted into the E1 region, thereby generating a recombinant E1 / E3-deleted AV vector. Adenovirus vectors also include helper-dependent high-capacity adenovirus vectors (also known as high-capacity "gutless" or "gutted" vectors), which do not contain viral coding sequences. These vectors contain the cis-acting elements required for viral DNA replication and packaging, mainly the inverted terminal repeats (ITRs) and the packaging signal (CY). These helper-dependent AV vector genomes have the potential to carry exogenous DNA ranging from a few hundred base pairs to approximately 36 kb.

[0180] Alternatively, other systems such as lentiviral vectors can be used. Lentivirus-based systems can transduce both non-dividing and dividing cells, making them useful for applications targeting non-dividing cells such as those in the CNS. Lentiviral vectors are derived from the human immunodeficiency virus and, like the virus, integrate into the host genome, thereby providing the potential for very long-term gene expression.

[0181] Cationic lipids, polymers, or both can also be used as vectors to introduce polynucleotides (including plasmids, YACs, minichromosomes, and microcircles) carrying a target gene containing an expression cassette into cells or organisms by non-viral vector systems. Conjugated poly-L-lysine (PLL) polymers and polyethyleneimine (PEI) polymer systems can also be used to deliver the vectors to cells. Other methods for delivering vectors to cells include hydrodynamic injection, electroporation, and the use of ultrasound, both for cell culture and for organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012; 1:27), which is incorporated herein by reference.

[0182] rAAV virion production

[0183] The rAAV viral particles disclosed herein can be constructed and produced using the materials and methods described herein as well as those known to those of skill in the art. Such engineering methods for constructing any embodiment of the present disclosure are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989); and International Patent Publication No. WO 95 / 13598. In addition, methods suitable for generating rAAV cassettes in an adenovirus capsid have been described in U.S. Patent Nos. 5,856,152 and 5,871,982.

[0184] Briefly, to package the rAAV genome into rAAV viral particles, a host cell containing sequences necessary for expressing AAV rep and AAV cap or functional fragments thereof, as well as helper genes necessary for AAV production, is used. The AAV rep and cap sequences are obtained from an AAV source as identified herein. The AAV rep and cap sequences can be introduced into the host cell in any manner known to those of skill in the art, including but not limited to transfection, electroporation, liposome delivery, membrane fusion techniques, high-velocity DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into the host cell by one or more nucleic acid molecules and stably exist as episomes in the cell. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has rep and cap sequences that are transiently expressed in the host cell. For example, a nucleic acid molecule that can be used for such transfection contains, from 5' to 3', a NARE promoter, an optional spacer inserted between the start site of the promoter and the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.

[0185] The rep and cap sequences, along with their expression control sequences, can be provided on a single vector, or each sequence can be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences can be provided on a vector containing other DNA sequences to be introduced into the host cell. Preferably, the promoter used for such constructs can be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins can be in any form that transfers these components into the host cell. Ideally, such a molecule is in plasmid form, which can contain other non-viral sequences, such as those of marker genes. Such a molecule does not contain AAV ITRs and generally does not contain AAV packaging sequences. To avoid the occurrence of homologous recombination, other viral sequences, especially those of adenovirus, are avoided in such plasmids. The plasmid is ideally constructed such that it can be stably transfected into cells.

[0186] Although the molecule providing the rep and cap can be transiently transfected into the host cell, it is preferred to stably transform the host cell with the sequences necessary for expressing functional rep / cap proteins in the host cell, such as as an episome or by integration into the host cell's chromosome. Depending on the promoter controlling the expression of such stable transfection in the host cell, the rep / cap proteins can be transiently expressed (e.g., by using an inducible promoter).

[0187] The methods for constructing the embodiments of the present disclosure are conventional genetic engineering or recombinant engineering techniques, such as those described in the above references. For example, rAAV can be produced using the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.) with a triple transfection method according to the manufacturer's instructions. See also Herzog et al., 1999, Nature Medic., 5(1):56 - 63, for the method used in the following examples, using a plasmid with a transgene, a helper plasmid containing AAV rep and cap, and a plasmid providing adenovirus helper functions for E2A, E4Orf6, and VA. Although this specification provides illustrative examples of specific constructs, using the information provided herein, those skilled in the art can select and design other suitable constructs using the selection of spacers, promoters, and other elements (including at least one translation start and stop signal) and optionally adding polyadenylation sites.

[0188] rAAV viral particles can be produced by culturing host cells containing an rAAV virus as described herein, the host cells containing an rAAV genome to be packaged into the rAAV viral particles, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression. Suitable viral helper genes (such as adenovirus E2A, E4 Orf6 and VA, and other possible helper genes) can be provided to the culture in a variety of ways known in the art, preferably on a separate plasmid. Thereafter, recombinant AAV viral particles directing the expression of the transgene are isolated from the cells or cell culture in the absence of contaminating helper virus or wild-type AAV.

[0189] Expression of the transgene can be measured in a manner known in the art. For example, target cells can be infected in vitro and the copy number of the transgene in the cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression levels can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and protein expression levels can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or by specific methods detailed in the examples below.

[0190] Cell

[0191] In embodiments, the nucleic acid constructs and vectors disclosed herein are used to deliver a NARE operably linked to a transgene to a cell. Cells containing a NARE, nucleic acid construct or vector disclosed herein are provided. In embodiments, the cell is a neuronal cell. The cell can be a mammalian cell. The cell can be a human cell. The cell may be isolated.

[0192] Pharmaceutical composition

[0193] Pharmaceutical compositions are provided herein, the pharmaceutical compositions comprising a nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.

[0194] Preferably, the nucleic acid constructs or vectors disclosed herein are assayed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0195] The formulation of the nucleic acid constructs or vectors disclosed herein involves the use of pharmaceutically and / or physiologically acceptable agents or carriers, particularly agents or carriers suitable for injection, such as buffered saline or other buffers (e.g., HEPES), to maintain the pH at an appropriate physiological level. The nucleic acid constructs or vectors disclosed herein can be formulated into pharmaceutical compositions. In addition to the carrier, these compositions can also contain pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials can be determined by a skilled person according to the route of administration. The pharmaceutical compositions are generally in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, which is incorporated herein by reference. Saline solutions, magnesium chloride, dextrose or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, can be included. In some cases, surfactants, such as Pluronic acid (PF68) at 0.001%, can be used. In some cases, Ringer's injection solution, lactated Ringer's injection solution, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.

[0196] A pharmaceutical composition comprising the nucleic acid constructs or vectors disclosed herein can be formulated with one or more pharmaceutically acceptable excipients, which can be pharmaceutically acceptable materials, compositions, or agents, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate, or zinc stearate or stearic acid), solvents, or encapsulating materials, which are involved in carrying or transporting a therapeutic compound for administration to a subject, swelling agents, salts, surfactants, and / or preservatives. Some examples of materials that can be used as pharmaceutically acceptable excipients include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations.

[0197] An expander is a compound that increases the mass of a pharmaceutical preparation and contributes to the physical structure of the preparation in freeze-dried form. Suitable expanders according to the present invention include mannitol, glycine, polyethylene glycol, and sorbitol.

[0198] The use of a surfactant can reduce the aggregation of the reconstituted protein and / or reduce the formation of microparticles in the reconstituted preparation. The amount of surfactant added should be such that it reduces the aggregation of the reconstituted protein and minimizes the formation of microparticles after reconstitution. Suitable surfactants according to the present invention include polysorbates (such as polysorbate 20 or 80); poloxamers (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-betaine (such as lauramidopropyl); myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl or sodium methyl oleoyl taurate; and copolymers of polyethylene glycol, polypropylene glycol, and ethylene glycol and propylene glycol (such as Pluronics, PF68, etc.).

[0199] Preservatives can be used in the preparations of the present invention. Suitable preservatives for the preparations of the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl-dimethyl ammonium chlorides, where the alkyl is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols (such as phenol, butanol, and benzyl alcohol), alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, such as "Remington’s Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Edition, Mack Publishing Company, Easton, Pa., (1995).

[0200] For delayed release, the nucleic acid constructs or vectors disclosed herein can be included in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from a biocompatible polymer or in a liposomal carrier system according to methods known in the art.

[0201] If the carrier is intended for long-term storage, it can be frozen in the presence of glycerol.

[0202] Method

[0203] The present disclosure provides methods for inducing the expression of a transgene in a cell and / or tissue. The present disclosure provides a method for inducing the expression of a transgene, the method comprising providing a cell comprising a nucleic acid construct comprising a NARE disclosed herein operably linked to a transgene, and culturing the cell under conditions that permit the expression of the transgene. In an embodiment, the cell is a neuronal cell. The present disclosure provides a method for inducing the expression of a transgene in vivo. The present disclosure provides a method for inducing the expression of a transgene in vitro. The present disclosure provides a method for inducing the expression of a transgene ex vivo.

[0204] The present disclosure provides methods for treating a disease or disorder in a subject in need thereof using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein. In an embodiment, the disease or disorder is a neurological disease or disorder.

[0205] In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals include, but are not limited to, human or non-human mammals such as cattle, equines, canines, sheep, or felines, among others. An individual and a patient are also subjects herein.

[0206] As used herein, the term "treat / treated / treating / treatment" refers to therapeutic treatment, where the goal is to slow down (alleviate) an undesired physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of the present disclosure, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms; reduction in the degree of a condition, disorder, or disease; stabilization (i.e., not worsening) of the state of a condition, disorder, or disease; delay in the onset or slowing of the progression of a condition, disorder, or disease; improvement in one or more symptoms of a condition, disorder, or disease state; and remission (whether partial or total) or enhancement or improvement of a condition, disorder, or disease. Treatment includes eliciting a clinically significant response without undue side effects. Treatment also includes prolonging survival as compared to the expected survival period in the absence of treatment. The terms "prevent / prevention," etc. refer to acting before the onset of an apparent disease or disorder to prevent the development of the disease or disorder or to minimize the degree of the disease or disorder, or to slow its progression.

[0207] In some embodiments, treatment refers to increasing survival (e.g., survival time). For example, treatment can result in an increase in the patient's life expectancy. In some embodiments, compared to the average life expectancy of one or more untreated control individuals with a neurological disorder (including but not limited to amyotrophic lateral sclerosis (ALS)), treatment results in an increase in the patient's life expectancy of more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more. In some embodiments, compared to the average life expectancy of one or more untreated control individuals with a neurological disorder such as ALS, treatment results in an increase in the patient's life expectancy of more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or longer. In some embodiments, treatment results in long-term survival of the patient. As used herein, the term "long-term survival" refers to a survival time or life expectancy of more than about 40 years, 45 years, 50 years, 55 years, 60 years or longer.

[0208] In embodiments, the subject is at risk of developing ALS. In some cases, the subject to be treated is genetically predisposed to develop ALS. For example, the subject to be treated has a mutation in the SOD1 gene, ALS2 gene, VAPB gene, SETX gene, TDP-43 gene, FUS / TLS gene, C9orf72 gene, and / or OPTN gene. In embodiments, the subject does not have a mutation in the SOD1 gene.

[0209] Methods of administration include but are not limited to intracisternal, intraventricular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intrathecal, intravaginal, transdermal, rectal, by inhalation, or topically (especially to the ear, nose, eye, or skin). The mode of administration is at the discretion of the practitioner.

[0210] In some cases, the nucleic acid constructs or vectors described herein are administered locally. By way of example, this can be achieved by local infusion during surgery, topical application (e.g., in a cream or lotion), by injection, via a catheter, via a suppository or enema, or via an implant, which is a porous, non-porous, or gel-like material, including membranes, such as silicone rubber membranes or fibers. In some cases, the nucleic acid constructs or vectors described herein are introduced into the central nervous system, the circulatory system, or the gastrointestinal tract by any suitable route, including intraventricular injection, intrathecal injection, paravertebral injection, epidural injection, enema, and injection adjacent to a peripheral nerve.

[0211] The compositions described herein can be administered in a single dose or in multiple doses. Such compositions can be administered at regular time intervals depending on the nature, severity, and extent of the disorder of the subject. In some embodiments, a therapeutically effective amount of the nucleic acid construct or vector is administered intrathecally at regular time intervals (e.g., once a year, once every six months, once every five months, once every three months, once every two months, monthly, once every two weeks, or once a week).

[0212] The amount of the nucleic acid construct or vector described herein effective to treat a disease can be determined using standard clinical techniques known to those of skill in the art. In addition, in vitro or in vivo assays can optionally be used to assist in identifying the optimal dosage range. The exact dosage to be employed will also depend on the route of administration, the disorder, the severity of the disorder being treated, and various physical factors associated with the individual being treated, and can be determined at the discretion of the healthcare practitioner.

[0213] An effective amount of rAAV carrying a nucleic acid sequence encoding a transgene (including but not limited to UPF1, ATP7B, and BDNF) under the control of a NARE can be, for example, in the range of about 1×10 9 to about 1×10 14 rAAV genome particles (vg) / kg body weight. "Genome particles" are defined herein as AAV capsids containing single-stranded DNA molecules, which can be quantified by sequence-specific methods such as qPCR or ddPCR. In some embodiments, rAAV is administered at about 1×10 12 to about 1×10 13 rAAV vg / kg body weight. In some embodiments, rAAV is administered at about 5×10 11 to about 5×10 12 vg / mL cerebrospinal fluid (CSF) volume. In some embodiments, rAAV is administered at a total of about 7.5×10 13 to 7.5×1014 Administration of vg.

[0214] In some embodiments, rAAV is administered to an animal at about 1×10 11 to about 1x10 14 rAAV genome particles (vg) / kg body weight.

[0215] In some embodiments, the rAAV genome particles are provided in a volume between about 20 μL and about 50 mL. In some embodiments, the rAAV genome particles are provided in a volume between about 30 μL and about 30 mL. In some embodiments, the rAAV genome particles are provided in a volume of about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 μL. In some embodiments, the rAAV genome particles are provided in a volume of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 mL.

[0216] The attending physician may also select other doses within these ranges. It should be understood that for any particular subject, a specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the nucleic acid construct or vector, and the dosage ranges described herein are only exemplary and are not intended to limit the scope or practice of the claimed invention.

[0217] The nucleic acid constructs or vectors disclosed herein can also be advantageously provided ex vivo to cells, which are then administered to a subject. Methods of treating diseases by implanting cells that have been modified to express a recombinant protein are also well known. See, for example, U.S. Patent No. 5,399,346, which discloses methods for introducing nucleic acids into primary human cells for introduction into the human body. Although human cells are preferably used for ex vivo therapy in some embodiments, other cells (such as bacterial cells) can also be implanted into the vascular system of a subject to provide a sustained release of a therapeutic agent. See, for example, U.S. Patent Nos. 4,309,776 and 5,704,910.

[0218] This application is related to U.S. Provisional Application No. 63 / 379,109, entitled "UPF1 EXPRESSION CONSTRUCTS", U.S. Provisional Application No. 63 / 379,113, entitled "ATP7B GENE THERAPY", and U.S. Provisional Application No. 63 / 379,114, entitled "BDNF GENE THERAPY", all of which were filed by the same applicant on October 11, 2022, and each of which is hereby incorporated by reference in its entirety.

[0219] It should be understood that the present invention is not limited to the specific molecules, compositions, methods, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein may be used to practice or test embodiments of the present invention. It should also be understood that the disclosure of the present invention in this specification includes all possible combinations of such specific features. For example, where a specific feature is disclosed in the context of a particular aspect or embodiment of the present invention or a particular claim, said feature may also be used in combination with and / or in the context of other particular aspects and embodiments of the present invention to the extent possible, and is generally used in the present invention.

[0220] In cases where a method involving two or more defined steps is mentioned herein, the defined steps may be performed in any order or simultaneously (unless the context excludes such possibilities), and the method may include one or more other steps performed before any defined step, between two defined steps, or after all defined steps (unless the context excludes those possibilities).

[0221] All other cited patents and applications are hereby incorporated by reference in their entirety. Further, when the definition or use of a term in a reference incorporated by reference herein is inconsistent with or contradicts the definition of said term provided herein, the definition of said term provided herein shall prevail and the definition of said term in the reference shall not apply.

[0222] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be construed as limiting or defining the entire scope of the present invention.

[0223] Examples

[0224] Example 1: NARE Design

[0225] Potential NARE candidates were also identified from genes highly expressed in human target tissue (brain). Subsequently, promoter regions were defined or cis-regulatory elements were identified based on chromatin marks, accessibility, conservation, and other genome-wide datasets.

[0226] Incorporate ten - base element (MTE) / downstream promoter element (DPE) modifications to increase transcription, and incorporate various cellular, viral, or synthetic 5′UTR motifs to increase transcription / translation. The choice of introns has changed, for example, including short cellular introns that naturally have unique independent transcription start sites, indicating the presence of a promoter within this particular intron. Additionally, the introns are inserted at different positions within the sequence, and / or the introns are truncated while maintaining splicing elements. Furthermore, single or multiple point mutations are introduced to increase expression or prevent inactivation. Additionally, different transcriptional enhancers are introduced upstream of the core promoter. In some cases, transcriptional motif insertions, replacements, and / or shufflings are employed, and CpG sequences are removed to reduce immunogenicity while maintaining promoter activity.

[0227] Gene regulatory elements were also optimized by: (i) identifying, shuffling, and cloning highly expressed transcription factor binding sites (TFBS) upstream of the core promoter; (ii) using artificial intelligence (AI) algorithms to iteratively mutate and score the promoter.

[0228] Example 2: Dual - reporter protein expression assay

[0229] To test the ability of NARE candidates to drive gene expression, a dual - reporter gene assay using flow cytometry was utilized. Figure 1A Diagrams of the dual - reporter gene expression constructs and assays are provided. Different NARE candidate sequences were cloned upstream of a transgene encoding mClover3 (a green / yellow fluorescent protein), which in turn is upstream of the woodchuck hepatitis virus post - transcriptional regulatory element (WPRE250) and a synthetic polyadenylation (polyA) signal. mClover3 fluorescence serves as a measure of NARE strength. The plasmid also contains a separate expression cassette (from 5’ to 3’: SV40 promoter - tdTomato (a red fluorescent protein) - SV40 polyA), which is the same in all NARE test constructs ( Figure 1A ). tdTomato fluorescence serves as an internal normalization control, which can account for variations in transfection efficiency.

[0230] Use Transfection reagent was used to transiently transfect various cell lines with a dual-reporter gene plasmid. After 48 hours, the cells were trypsinized and resuspended for flow cytometry. The raw data was processed, and only live single cells were selected for analysis. A sample containing untransfected cells was used to set the gating for the tdTomato signal. Then, this gating was applied to all live single cells to identify tdTomato-positive (and thus transfected) cells. Subsequently, mClover3 fluorescence was determined for the tdTomato-positive population. Since transfection varied slightly between different experiments, the relative NARE potency was calculated by the ratio of mClover3 fluorescence to tdTomato fluorescence. NARE candidates were tested together with known constitutive and tissue-specific reference promoters such as CAG, CMV, or AAT.

[0231] Example 3: Intensities of Different NAREs in Neuronal Cells

[0232] Materials and Methods

[0233] Cell culture of neuronal cell lines

[0234] The Neuro2A (N2A), BE2-M17, and SH-SY5Y immortalized cell lines were maintained at 37 °C in 5% CO2. N2A is a murine neuroblastoma cell line. BE2-M17 is a human neuroblastoma cell line. SH-SY5Y is a tertiary subcloned cell line derived from the SK-N-SH neuroblastoma cell line. The TurboFect TM reagent was used to transiently transfect the cells with the plasmid. Two days later, the culture medium and cell lysates were collected for protein analysis. After 30 passages, a new cell aliquot was thawed and passaged twice, and then used for subsequent experiments.

[0235] Primary neuron culture

[0236] Timed pregnant female mice at embryonic day 16 (E16) were anesthetized with CO2 and sacrificed by cervical dislocation. In a dissection hood, 24 - 26 embryos were collected per experiment through an incision in the mother's abdomen, removed from the amniotic sac, and decapitated in ice - cold Hank's balanced salt solution (HBSS). Using fine scissors and forceps, the brain was rapidly dissected, the meninges were cleared from the cortex, and it was separated under a dissecting microscope. The cortex was collected in ice - cold HBSS and kept on ice until all embryos were dissected. In a tissue culture hood, the HBSS was removed, and the cortical tissue was digested with 0.25% trypsin - EDTA at 37°C for 12 min, followed by DNase1 treatment at 37°C for 10 min. Serial wet trituration was performed with a 25 - ml serological pipette, followed by wet trituration with 10 - and 5 - ml serological pipettes to dissociate the tissue. The cell suspension was washed once with DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin and passed through a 40 - μM cell strainer, then counted on a hemocytometer. Single cells were seeded at a density of 400,000 cells per well in wells coated with poly - d - lysine (0.1 mg ml -1 ). The cells were grown in neurobasal medium supplemented with B27 supplement, N2 supplement, and 0.5 mM l - glutamine and maintained at 37°C in 5% CO2. Half of the medium was changed every 3 - 4 days to feed the culture.

[0237] Primary neuron transduction

[0238] On day 1 in vitro, AAV2 / 1 particles were added to the neuronal medium at multiplicities of infection (MOIs) of 10,000 and 50,000. After 72 h, the medium was changed according to the standard neuronal protocol.

[0239] Results

[0240] Creating stronger neuronal NAREs enables higher expression of therapeutic transgenes at lower viral doses. Lower dosing can prevent severe adverse events such as dorsal root ganglion toxicity. Therefore, a library of nucleic acid regulatory elements suitable for gene expression in the central nervous system was developed. Compared with commonly used reference promoters (NSE, Syn, CAMKIIα), these NAREs showed strong expression in mouse N2A cells. Specifically, these identified NAREs were equal to or greater than CMV in promoter strength, and some were even stronger than CAG ( Figure 2A , Figure 2B , Figure 2C and Figure 2D)。Neuron-specific enolase (NSE) is a neuron-specific promoter that contains a TATA-like sequence, lacks a CAAT box, and has sequences for an AP-1 binding motif, an AP-2 binding element, an SP-1 binding sequence, and a cAMP response element. Syn (or the human synapsin 1 promoter) and α-calcium / calmodulin-dependent kinase II (CAMKIIα) are also neuron-specific promoters.

[0241] Figure 2E and Figure 2F shows the potency of additional NAREs in human and murine neuronal cell lines.

[0242] Table 1. Control NARE. SN = SEQ ID NO.

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] Table 2. NARE.

[0249]

[0250]

[0251] Table 3. NARE components

[0252]

[0253]

[0254] Table 4. NARE and NARE components. SN = SEQ ID NO.

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

Claims

1. A nucleic acid regulatory element (NARE), the NARE comprising: a) (i) a sequence that is at least 90% identical to SEQ ID NO: 36; b) (i) a sequence that is at least 90% identical to SEQ ID NO: 91; (ii) a sequence that is at least 90% identical to SEQ ID NO: 92; (iii) a sequence that is at least 90% identical to SEQ ID NO: 93; and (iv) a sequence that is at least 90% identical to SEQ ID NO: 94; c) (i) a sequence that is at least 90% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 90% identical to SEQ ID NO: 99; d) (i) a sequence that is at least 90% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 90% identical to SEQ ID NO: 100; or e) (i) a sequence that is at least 90% identical to SEQ ID NO:

57.

2. The NARE according to claim 1, the NARE comprising: a) (i) a sequence that is at least 95% identical to SEQ ID NO: 36; b) (i) a sequence that is at least 95% identical to SEQ ID NO: 91; (ii) a sequence that is at least 95% identical to SEQ ID NO: 92; (iii) a sequence that is at least 95% identical to SEQ ID NO: 93; and (iv) a sequence that is at least 95% identical to SEQ ID NO: 94; c) (i) a sequence that is at least 95% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 95% identical to SEQ ID NO: 99; d) (i) a sequence that is at least 95% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 95% identical to SEQ ID NO: 100; or e) (i) a sequence that is at least 95% identical to SEQ ID NO:

57.

3. The NARE according to claim 1, the NARE comprising: a) (i) SEQ ID NO: 36; b) (i) SEQ ID NO: 91; (ii) SEQ ID NO: 92; (iii) SEQ ID NO: 93; and (iv) SEQ ID NO: 94; c) (i) SEQ ID NO: 98; and (ii) SEQ ID NO: 99; d) (i) SEQ ID NO: 98; and (ii) SEQ ID NO: 100; or e) (i) SEQ ID NO:

57.

4. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 90% identical to SEQ ID NO: 99 or SEQ ID NO:

100.

5. The NARE according to claim 4, wherein the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 98; and (ii) a sequence that is at least 95% identical to SEQ ID NO: 99 or SEQ ID NO:

100.

6. The NARE according to claim 4, wherein the NARE comprises: (i) SEQ ID NO: 98; and (ii) SEQ ID NO: 99 or SEQ ID NO:

100.

7. A NARE, wherein the NARE comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 63; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 62, 68, 70, 72, 74, 95 or 96; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO:

73.

8. The NARE according to claim 7, wherein the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 63; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 62, 68, 70, 72, 74, 95 or 96; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO:

73.

9. The NARE according to claim 7, wherein the NARE comprises: (i) SEQ ID NO: 63; (ii) any one of SEQ ID NOs: 62, 68, 70, 72, 74, 95 or 96; and (iii) optionally, SEQ ID NO:

73.

10. A NARE, wherein the NARE comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 68; (ii) at least 90% identical to SEQ ID NO: 71; and (iii) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 87 - 90.

11. The NARE according to claim 10, wherein the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 68; (ii) at least 95% identical to SEQ ID NO: 71; and (iii) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 87 - 90.

12. The NARE according to claim 10, wherein the NARE comprises: (i) SEQ ID NO: 68; (ii) SEQ ID NO: 71; and (iii) optionally, any one of SEQ ID NOs: 87 - 90.

13. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 83; (ii) a sequence that is at least 90% identical to SEQ ID NO: 84; (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 66 or SEQ ID NO: 86; and (iv) optionally, a sequence that is at least 90% identical to SEQ ID NO:

85.

14. The NARE according to claim 13, the NARE comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 83; (ii) a sequence that is at least 95% identical to SEQ ID NO: 84; (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 66 or SEQ ID NO: 86; and (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO:

85.

15. The NARE according to claim 13, the NARE comprising: (i) SEQ ID NO: 83; (i) SEQ ID NO: 84; (iii) optionally, SEQ ID NO: 66 or SEQ ID NO: 86; and (iv) optionally, SEQ ID NO:

85.

16. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 91; (ii) a sequence that is at least 90% identical to SEQ ID NO: 92; (iii) a sequence that is at least 90% identical to SEQ ID NO: 93; (iv) a sequence that is at least 90% identical to SEQ ID NO: 94; and (v) optionally, a sequence that is at least 90% identical to SEQ ID NO:

63.

17. The NARE according to claim 16, the NARE comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 91; (ii) a sequence that is at least 95% identical to SEQ ID NO: 92; (iii) a sequence that is at least 95% identical to SEQ ID NO: 93; (iv) a sequence that is at least 95% identical to SEQ ID NO: 94; and (v) optionally, a sequence that is at least 95% identical to SEQ ID NO:

63.

18. The NARE according to claim 16, the NARE comprising: (i) SEQ ID NO: 91; (ii) SEQ ID NO: 92; (iii) SEQ ID NO: 93; (iv) SEQ ID NO: 94; and (v) optionally, SEQ ID NO:

63.

19. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to any one of SEQ ID NO:75, 76 or 77; (ii) a sequence that is at least 90% identical to SEQ ID NO:104; and (iii) a sequence that is at least 90% identical to SEQ ID NO:103 or 105.

20. The NARE according to claim 19, the NARE comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NO:75, 76 or 77; (ii) a sequence that is at least 95% identical to SEQ ID NO:104; and (iii) a sequence that is at least 95% identical to SEQ ID NO:103 or 105.

21. The NARE according to claim 19, the NARE comprising: (i) any one of SEQ ID NO:75, 76 or 77; (ii) SEQ ID NO:104; and (iii) SEQ ID NO:103 or 105.

22. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO:64; (ii) a sequence that is at least 90% identical to SEQ ID NO:65; (iii) a sequence that is at least 90% identical to SEQ ID NO 66; and (iv) a sequence that is at least 90% identical to SEQ ID NO:

67.

23. The NARE according to claim 22, the NARE comprising: (i) a sequence that is at least 95% identical to SEQ ID NO:64; (ii) a sequence that is at least 95% identical to SEQ ID NO:65; (iii) a sequence that is at least 95% identical to SEQ ID NO 66; and (iv) a sequence that is at least 90% identical to SEQ ID NO:

67.

24. The NARE according to claim 22, the NARE comprising: (i) SEQ ID NO:64; (ii) SEQ ID NO:65; (iii) SEQ ID NO 66; and (iv) SEQ ID NO:

67.

25. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO:73; (ii) a sequence that is at least 90% identical to any one of SEQ ID NO:75, 76 or 77; and (iii) a sequence that is at least 90% identical to SEQ ID NO:

78.

26. The NARE according to claim 25, the NARE comprising: (i) a sequence that is at least 95% identical to SEQ ID NO:73; (ii) a sequence that is at least 95% identical to any one of SEQ ID NO:75, 76 or 77; and (iii) a sequence that is at least 95% identical to SEQ ID NO:

78.

27. The NARE according to claim 25, wherein the NARE comprises: (i) SEQ ID NO: 73; (ii) any one of SEQ ID NO: 75, 76 or 77; and (iii) SEQ ID NO:

78.

28. A NARE, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 79; and (ii) a sequence that is at least 90% identical to SEQ ID NO:

80.

29. The NARE according to claim 28, wherein the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 79; and (ii) a sequence that is at least 95% identical to SEQ ID NO:

80.

30. The NARE according to claim 28, wherein the NARE comprises: (i) SEQ ID NO: 79; and (ii) SEQ ID NO:

80.

31. A NARE, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 81; and (ii) a sequence that is at least 90% identical to SEQ ID NO:

82.

32. The NARE according to claim 31, wherein the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 81; and (ii) a sequence that is at least 95% identical to SEQ ID NO:

82.

33. The NARE according to claim 31, wherein the NARE comprises: (i) SEQ ID NO: 81; and (ii) SEQ ID NO:

82.

34. A NARE, comprising: (i) a sequence that is at least 90% identical to any one of SEQ ID NO: 75, 76 or 77; and (ii) a sequence that is at least 90% identical to SEQ ID NO:

104.

35. The NARE according to claim 34, wherein the NARE comprises: (i) a sequence that is at least 95% identical to any one of SEQ ID NO: 75, 76 or 77; and (ii) a sequence that is at least 95% identical to SEQ ID NO:

104.

36. The NARE according to claim 34, wherein the NARE comprises: (i) any one of SEQ ID NO: 75, 76 or 77; and (ii) SEQ ID NO:

104.

37. A NARE, comprising a sequence that is at least 90% identical to any one of SEQ ID NO: 3 - 5, 7 - 9, 11, 14, 15, 18 and 23 - 61.

38. The NARE according to claim 37, wherein the NARE comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 3 - 5, 7 - 9, 11, 14, 15, 18 and 23 - 61.

39. The NARE according to claim 37, wherein the NARE comprises any one of SEQ ID NO: 3 - 5, 7 - 9, 11, 14, 15, 18 and 23 - 61.

40. The NARE as claimed in claim 37, wherein the NARE comprises a sequence that is at least 90% identical to SEQ ID NO: 36, 40, 55, 56 or 57.

41. The NARE as claimed in claim 40, wherein the NARE comprises a sequence that is at least 95% identical to SEQ ID NO: 36, 40, 55, 56 or 57.

42. The NARE as claimed in claim 40, wherein the NARE comprises any one of SEQ ID NO: 36, 40, 55, 56 or 57.

43. An expression construct, the expression construct comprising the NARE as claimed in any one of the preceding claims and an operably linked transgene.

44. The expression construct as claimed in claim 43, wherein the expression construct further comprises a polyadenylation sequence.

45. A vector, the vector comprising the expression construct as claimed in claim 43.

46. The vector as claimed in claim 45, wherein the vector is a non-viral vector.

47. The vector as claimed in claim 45, wherein the vector is a viral vector.

48. The vector as claimed in claim 47, wherein the vector is an adeno-associated (AAV) vector.

49. A vector comprising a nucleic acid sequence, the nucleic acid sequence comprising (i) the expression construct as claimed in claim 43, and (ii) one or more inverted terminal repeats (ITRs).

50. The vector as claimed in claim 49, wherein the nucleic acid sequence comprises a 5' ITR and a 3' ITR.

51. The vector as claimed in claim 50, wherein the 5' ITR and the 3' ITR are derived from AAV serotype AAV2.

52. A cell, the cell comprising the expression construct as claimed in claim 43 or the vector as claimed in any one of claims 45 - 51.

53. The cell as claimed in claim 52, wherein the cell is a neuronal cell.

54. A pharmaceutical composition, the pharmaceutical composition comprising (i) the expression construct as claimed in claim 43 or the vector as claimed in any one of claims 45 - 51, and (ii) a pharmaceutically acceptable excipient.

55. A method for expressing a transgene in a cell, the cell comprising the expression construct as claimed in claim 43 or the vector as claimed in any one of claims 45 - 51.

56. A method for regulating transgene expression in a cell, the cell comprising the expression construct as claimed in claim 43 or the vector as claimed in any one of claims 45 - 51.

57. The method as claimed in claim 55 or 56, wherein the cell is a neuronal cell.

58. A method for treating a subject in need thereof for a neurological disease or disorder, the method comprising administering to the subject the expression construct as claimed in claim 43, the vector as claimed in any one of claims 45 - 51 or the pharmaceutical composition as claimed in claim 54.

59. A method for treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, the method comprising administering to the subject an expression construct as claimed in claim 43, a vector as claimed in any one of claims 45 - 51, or a pharmaceutical composition as claimed in claim 54.

60. The method as claimed in claim 59, wherein the subject has a mutation in the ALS2 gene, the VAPB gene, the SETX gene, the TDP - 43 gene, the FUS / TLS gene, the C9orf72 gene, and / or the OPTN gene.

61. The method as claimed in claim 60, wherein the subject is human.

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