Nucleic acid regulatory elements for gene expression in muscle and methods of use

Abstract

This application relates to nucleic acid regulatory elements that can constitutively enhance gene expression in various tissues, or in particular in tissues including muscle. This application further relates to methods that utilize 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.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing dates of U.S. Provisional Patent Application No. 63 / 379,138, filed October 11, 2022, and U.S. Provisional Patent Application No. 63 / 496,554, filed April 17, 2023, which are hereby incorporated by reference in their entireties.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (SeqList-162027.54176.xml, size: 290,976 bytes, and creation date: October 10, 2023) are incorporated herein by reference in their entirety.

[0003] This application relates to nucleic acid regulatory elements that can enhance gene expression in various tissues, particularly tissues including muscle. This application further relates to methods that utilize 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 technology]

[0004] A promoter is a DNA region at which transcription of a gene is initiated. Because the promoter region controls when and where a gene of interest is expressed within an organism, promoters are important elements for regulating the level and specificity of transgene expression, especially in the context of gene therapy.

[0005] The use of engineered nucleic acid regulatory elements (NAREs) (which may contain various components, including promoters, enhancers, etc.) specifically tailored to a given gene therapy offers various advantages. First, optimized NAREs enable the desired gene expression level for a particular therapeutic gene. Second, engineered NAREs with increased potency allow for the administration of smaller amounts of gene therapy vectors, thus reducing immune responses and associated safety risks. Third, for some gene therapies, it is desirable for gene expression to be restricted to specific tissue(s). Therefore, the use of nucleic acid regulatory elements with tissue-specific expression can not only limit undesired transgene expression but also promote sustained transgene expression in the tissue(s) or tissue of interest. Such tissue-specific NAREs can be used to eliminate the need for tissue-specific viral capsids used for gene delivery (or can be used in combination with tissue-specific viral capsids). Fourth, selecting an appropriate NARE also allows for control of the kinetics of gene expression, which in turn affects the durability of the gene therapy. Finally, it may be desirable to engineer NAREs of reduced size (without sacrificing strength or specificity) to allow efficient packaging of larger transgene cargoes into viral vectors.

[0006] While efforts have been made to characterize and optimize NARE sequences through traditional low-throughput analysis (rational design) or newer high-throughput methodologies (e.g., MPRA), these approaches still require expensive and time-consuming in vitro optimization. Therefore, more efficient methods for engineering NAREs, as well as nucleic acid regulatory elements with enhanced potency, reduced size, and / or tissue specificity, are needed. Summary of the Invention

[0007] Provided herein are nucleic acid regulatory elements (NAREs) that include NAREs that are particularly suitable for expression of operably linked sequences (e.g., protein or RNA coding sequences) in muscle. Also provided herein are methods for expressing a transgene operably linked to one or more of the nucleic acid regulatory elements disclosed herein.

[0008] In embodiments, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE provided in Table 4 or 6. In embodiments, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE provided in Table 4 or 6. In embodiments, a polynucleotide is provided that comprises a NARE provided in Table 4 or 6.

[0009] In embodiments, there is provided a polynucleotide sequence comprising a sequence at least 80% identical to a NARE selected from the group consisting of M1 to M18, M20, M21, M23, M24, M32 to M49, M51 to M70, and M74 to M120 (listed in Table 6). In embodiments, there is provided a polynucleotide sequence comprising a sequence at least 90% identical to a NARE selected from the group consisting of M1 to M18, M20, M21, M23, M24, M32 to M49, M51 to M70, and M74 to M120. In embodiments, there is provided a polynucleotide sequence comprising a NARE selected from the group consisting of M1 to M18, M20, M21, M23, M24, M32 to M49, M51 to M70, and M74 to M120.

[0010] In embodiments, polynucleotide sequences are provided that comprise a sequence at least 80% identical to a NARE selected from the group consisting of M21, M23 to M28, M32, M34 to M36, M51, M53, M55, M57, M59, M61, and M63 (listed in Table 6). In embodiments, polynucleotide sequences are provided that comprise a sequence at least 90% identical to a NARE selected from the group consisting of M21, M23 to M28, M32, M34 to M36, M51, M53, M55, M57, M59, M61, and M63. In embodiments, polynucleotide sequences are provided that comprise a NARE selected from the group consisting of M21, M23 to M28, M32, M34 to M36, M51, M53, M55, M57, M59, M61, and M63.

[0011] In embodiments, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of M21 and M24. In embodiments, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of M21 and M24. In embodiments, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of M21 and M24.

[0012] In embodiments, there is provided a polynucleotide sequence comprising a sequence at least 80% identical to a NARE selected from the group consisting of M21, M24 to M27, M30, and M32 to M36 (listed in Table 6). In embodiments, there is provided a polynucleotide sequence comprising a sequence at least 90% identical to a NARE selected from the group consisting of M21, M24 to M27, M30, and M32 to M36. In embodiments, there is provided a polynucleotide sequence comprising a NARE selected from the group consisting of M21, M24 to M27, M30, and M32 to M36.

[0013] In embodiments, there is provided a polynucleotide sequence comprising a sequence that is at least 80% identical to a NARE selected from the group consisting of M49, M51, M61, M98, M99, and M111-M115 (listed in Table 6). In embodiments, there is provided a polynucleotide sequence comprising a sequence that is at least 90% identical to a NARE selected from the group consisting of M49, M51, M61, M98, M99, and M111-M115. In embodiments, there is provided a polynucleotide sequence comprising a NARE selected from the group consisting of M49, M51, M61, M98, M99, and M111-M115.

[0014] In embodiments, one or more NAREs disclosed herein are operably linked to a transgene.

[0015] In one aspect, a NARE is provided, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 132, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 156-163.

[0016] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 132, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 156-163. In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 132, and (ii) any one of SEQ ID NOs: 156-163.

[0017] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 164, (ii) a sequence that is at least 90% identical to SEQ ID NO: 134, (iii) a sequence that is at least 90% identical to SEQ ID NO: 135, and (vi) a sequence that is at least 90% identical to SEQ ID NO: 148 or 149. In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 164, (ii) a sequence that is at least 95% identical to SEQ ID NO: 134, (iii) a sequence that is at least 95% identical to SEQ ID NO: 135, and (vi) a sequence that is at least 95% identical to SEQ ID NO: 148 or 149. In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 164, (ii) SEQ ID NO: 134, (iii) SEQ ID NO: 135, and (vi) SEQ ID NO: 148 or 149.

[0018] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 148 or 149. In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 148 or 149. In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 148 or 149.

[0019] In one aspect, a NARE is provided that comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 154 or 189; (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 220; and (iv) a sequence that is at least 90% identical to SEQ ID NO: 150.

[0020] In some embodiments, a NARE is provided that comprises: (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 154 or 189; (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 220; and (iv) a sequence that is at least 95% identical to SEQ ID NO: 150.

[0021] In some embodiments, a NARE is provided that comprises: (i) any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, SEQ ID NO: 154 or 189; (iii) optionally, SEQ ID NO: 220; and (iv) SEQ ID NO: 150.

[0022] In one aspect, a NARE is provided, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 131; (ii) a sequence that is at least 90% identical to SEQ ID NO: 126 or 213; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 200.

[0023] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 131, (ii) a sequence that is at least 95% identical to SEQ ID NO: 126 or 213, and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 200.

[0024] In some embodiments, a NARE is provided that comprises: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126 or 213; and (iii) optionally, SEQ ID NO: 200.

[0025] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 131, (ii) a sequence that is at least 90% identical to SEQ ID NO: 126, and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 200.

[0026] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 131, (ii) a sequence that is at least 95% identical to SEQ ID NO: 126, and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 200.

[0027] In some embodiments, a NARE is provided that comprises: (i) SEQ ID NO: 131, (ii) SEQ ID NO: 126, and (iii) optionally, SEQ ID NO: 200.

[0028] In one aspect, a NARE is provided, comprising (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 190-199, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 155.

[0029] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 190-199, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 155.

[0030] In some embodiments, a NARE is provided that comprises (i) any one of SEQ ID NOs: 190-199, and (ii) SEQ ID NO: 155.

[0031] In one aspect, a NARE is provided, comprising (i) an array that is at least 90% identical to any one of SEQ ID NOs: 190-199, and (ii) an array that is at least 90% identical to any one of SEQ ID NOs: 128-130.

[0032] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 190-199, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 128-130.

[0033] In some embodiments, a NARE is provided that comprises (i) any one of SEQ ID NOs: 190-199, and (ii) any one of SEQ ID NOs: 128-130.

[0034] In one aspect, a NARE is provided, comprising (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 128-130, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

[0035] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 128-130, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

[0036] In some embodiments, a NARE is provided that comprises (i) any one of SEQ ID NOs: 128-130, and (ii) any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

[0037] In one aspect, a NARE is provided, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 121; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 122 or 154.

[0038] In some embodiments, a NARE is provided that comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 121; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 122 or 154.

[0039] In some embodiments, a NARE is provided that comprises: (i) SEQ ID NO: 121; (ii) any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, SEQ ID NO: 122 or 154.

[0040] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 121, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 166-169, 171, or 172.

[0041] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 121, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 166-169, 171, or 172.

[0042] In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 166-169, 171, or 172.

[0043] In some embodiments, a NARE is provided that comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 122 or 154; (ii) a sequence that is at least 90% identical to SEQ ID NO: 121; and (iii) a sequence that is at least 90% identical to SEQ ID NO: 167.

[0044] In some embodiments, a NARE is provided that comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 122 or 154; (ii) a sequence that is at least 95% identical to SEQ ID NO: 121; and (iii) a sequence that is at least 90% identical to SEQ ID NO: 167.

[0045] In some embodiments, a NARE is provided that comprises: (i) SEQ ID NO: 122 or 154, (ii) SEQ ID NO: 121, and (iii) SEQ ID NO: 167.

[0046] In one aspect, a NARE is provided, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 122; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 173-179, or 188; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 207.

[0047] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 122, (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 173-179, or 188, and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 207.

[0048] In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 122, (ii) any one of SEQ ID NOs: 173-179, or 188, and (iii) optionally, SEQ ID NO: 207.

[0049] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 122, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 173-179.

[0050] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 122, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 173-179.

[0051] In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 122, and (ii) any one of SEQ ID NOs: 173-179.

[0052] In one aspect, a NARE is provided, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 185-187.

[0053] In some embodiments, a NARE is provided that comprises (ii) a sequence that is at least 95% identical to SEQ ID NO: 184, and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 185-187.

[0054] In some embodiments, a NARE is provided that comprises SEQ ID NO: 184, and (ii) any one of SEQ ID NOs: 185-187.

[0055] In one aspect, a NARE is provided, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 180, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 181 or 182.

[0056] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 180, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 181 or 182.

[0057] In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 180, and (ii) any one of SEQ ID NOs: 181 or 182.

[0058] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 183, (ii) a sequence that is at least 90% identical to SEQ ID NO: 182, and (iii) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

[0059] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 183, (ii) a sequence that is at least 95% identical to SEQ ID NO: 182, and (iii) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

[0060] In some embodiments, a NARE is provided that comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) optionally, any one of SEQ ID NOs: 200, 201, 205, or 208.

[0061] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 183, (ii) a sequence that is at least 90% identical to SEQ ID NO: 182, and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

[0062] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 183, (ii) a sequence that is at least 95% identical to SEQ ID NO: 182, and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

[0063] In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 183, (ii) SEQ ID NO: 182, and (iii) any one of SEQ ID NOs: 200, 201, 205, or 208.

[0064] In one aspect, a NARE is provided, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 154 or 164, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 128-130.

[0065] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 154 or 164, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 128-130.

[0066] In some embodiments, a NARE is provided that comprises (i) SEQ ID NO: 154 or 164, and (ii) any one of SEQ ID NOs: 128-130.

[0067] In one aspect, a NARE is provided, comprising: (i) a sequence at least 90% identical to SEQ ID NO: 170; (ii) optionally, a sequence at least 90% identical to SEQ ID NO: 188; and (iii) a sequence at least 90% identical to SEQ ID NO: 203 or 204.

[0068] In some embodiments, a NARE is provided that comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 170, (ii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 188, and (iii) a sequence that is at least 95% identical to SEQ ID NO: 203 or 204.

[0069] In some embodiments, a NARE is provided that comprises: (i) SEQ ID NO: 170; (ii) optionally, SEQ ID NO: 188; and (iii) SEQ ID NO: 203 or 204.

[0070] In one aspect, (a) (i) a sequence that is at least 90% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 149; (b) (i) a sequence at least 90% identical to SEQ ID NO: 164, (ii) a sequence at least 90% identical to SEQ ID NO: 134, (iii) a sequence at least 90% identical to SEQ ID NO: 135, and (vi) SEQ ID NO: 149; (c) (i) a sequence that is at least 90% identical to SEQ ID NO: 165, (ii) a sequence that is at least 90% identical to SEQ ID NO: 154, (iii) a sequence that is at least 90% identical to SEQ ID NO: 220, and (iv) a sequence that is at least 90% identical to SEQ ID NO: 150; (d) (i) a sequence that is at least 90% identical to SEQ ID NO: 142, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 150; (e) (i) a sequence that is at least 90% identical to SEQ ID NO: 217, (ii) a sequence that is at least 90% identical to SEQ ID NO: 189, and (iii) a sequence that is at least 90% identical to SEQ ID NO: 150; (f) (i) a sequence that is at least 90% identical to SEQ ID NO: 196, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 155; (g) (i) a sequence that is at least 90% identical to SEQ ID NO: 194, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 155, or (h) A NARE is provided, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 187.

[0071] In some embodiments, (a) (i) a sequence that is at least 95% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 149; (b) (i) a sequence at least 95% identical to SEQ ID NO: 164, (ii) a sequence at least 95% identical to SEQ ID NO: 134, (iii) a sequence at least 95% identical to SEQ ID NO: 135, and (vi) SEQ ID NO: 149; (c) (i) a sequence that is at least 95% identical to SEQ ID NO: 165, (ii) a sequence that is at least 95% identical to SEQ ID NO: 154, (iii) a sequence that is at least 95% identical to SEQ ID NO: 220, and (iv) a sequence that is at least 95% identical to SEQ ID NO: 150; (d) (i) a sequence that is at least 95% identical to SEQ ID NO: 142, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 150; (e) (i) a sequence that is at least 95% identical to SEQ ID NO: 217, (ii) a sequence that is at least 95% identical to SEQ ID NO: 189, and (iii) a sequence that is at least 95% identical to SEQ ID NO: 150; (f) (i) a sequence that is at least 95% identical to SEQ ID NO: 196, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 155; (g) (i) a sequence that is at least 95% identical to SEQ ID NO: 194, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 155, or (h) A NARE is provided, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 187.

[0072] In some embodiments, (a) (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 149; (b) SEQ ID NO: 164, (ii) SEQ ID NO: 134, (iii) SEQ ID NO: 135, and (vi) SEQ ID NO: 149, (c) (i) SEQ ID NO: 165, (ii) SEQ ID NO: 154, (iii) SEQ ID NO: 220, and (iv) SEQ ID NO: 150; (d) (i) SEQ ID NO: 142, and (ii) SEQ ID NO: 150; (e) (i) SEQ ID NO: 217, (ii) SEQ ID NO: 189, and (iii) SEQ ID NO: 150; (f) (i) SEQ ID NO: 196, and (ii) SEQ ID NO: 155; (g) (i) SEQ ID NO: 194, and (ii) SEQ ID NO: 155, or (h) A NARE is provided, comprising: (i) SEQ ID NO: 184; and (ii) SEQ ID NO: 187.

[0073] In one aspect, a NARE is provided that comprises a sequence at least 90% identical to any one of SEQ ID NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, or 74-120. In some embodiments, a NARE is provided that comprises a sequence at least 95% identical to any one of SEQ ID NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, or 74-120. In some embodiments, a NARE is provided that comprises a sequence at least 90% identical to any one of SEQ ID NOs: 1-18 or 32. In some embodiments, a NARE is provided that comprises a sequence at least 95% identical to any one of SEQ ID NOs: 1-18 or 32. In some embodiments, a NARE comprising any one of SEQ ID NOs: 1-18 or 32 is provided.

[0074] In one aspect, an expression construct is provided comprising a NARE disclosed herein and an operably linked transgene. In one embodiment, the expression construct further comprises a polyadenylation sequence.

[0075] In one aspect, a vector is provided comprising an 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 comprising (i) an expression construct disclosed herein and (ii) one or more inverted terminal repeats (ITRs). In one embodiment, the vector comprises a 5'ITR and a 3'ITR. In one embodiment, the 5'ITR and the 3'ITR are derived from AAV serotype AAV2.

[0076] In one aspect, a cell is provided comprising an expression construct or vector disclosed herein. In some embodiments, the cell is a muscle cell.

[0077] In one aspect, a pharmaceutical composition is provided that includes (i) an expression construct disclosed herein or a vector disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0078] In one aspect, a method is provided for expressing a transgene in a cell comprising an expression construct disclosed herein or a vector disclosed herein. In one aspect, a method is provided for regulating transgene expression in a cell comprising an expression construct disclosed herein or a vector disclosed herein. In some embodiments, the cell is a muscle cell. [Brief explanation of the drawings]

[0079] [Figure 1] Dual reporter design and measurements. (a) shows the design of the dual reporter constructs. A candidate NARE was cloned upstream (5') of the mClover3 coding sequence. Each construct contains a constant region containing the tdTomato transgene, which is used as a normalization control. (b) provides example flow cytometry data obtained with the dual reporter system, in which mClover3 expression in tdTomato+ cells reflects the level of NARE activity. [Figure 2A] Generation of a NARE well suited for gene expression in muscle. Example of a nested NARE design (M24). The promoter tMCK was modified by adding MTE / DPE modifications. A synthetic HBG intron variant containing an intron-containing enhancer (SIE) and a modified human desmin promoter was inserted downstream. [Figure 2B] Generation of a NARE well suited for gene expression in muscle. Evolution of a potent NARE, M24, with up to 7-fold increased NARE strength compared to NARE M22 in in vitro transfected C2C12 cells. [Figure 2C]Generation of a NARE well suited for gene expression in muscle. NARE M24 was more potent than the reference promoter tMCK in cultured C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes and AAV8 transduced at an MOI of 10k. Fluorescence of the iRFP713 reporter was measured on the indicated days. [Figure 2D] We generated a NARE well suited for gene expression in muscle. NARE M24 was more potent than the reference tMCK promoter and showed durable expression in primary human myotubes. Primary human myoblasts were differentiated into myotubes and transduced with AAV8 at an MOI of 150k. [Figure 3A] In vivo expression of NAREs M21 and M24. AAV8-iRFP713 containing the tMCK promoter or synthetic M24 NARE, respectively, was injected intramuscularly into 8-week-old male C57B16 mice and imaged using a LICOR Pearl. [Figure 3B] In vivo expression of NAREs M21 and M24. Representative image data showing uninjected and injected hind limbs for AAV8-tMCK and AAV8-M24, respectively, after 7, 10, or 14 days. [Figure 3C] In vivo expression of NARE M21 and M24. Quantification of fluorescence showing that NARE M24 (upper line) induced 8-fold higher expression compared to the reference promoter tMCK (lower line), expression maintained up to 300 days after injection. [Figure 3D] In vivo expression of NAREs M21 and M24. AAV8-eGFP was injected into the gastrocnemius muscle of 8-week-old male C57B16 mice. After 4 weeks, muscles were harvested and sectioned for measurement of native eGFP fluorescence. [Figure 3E] In vivo expression of NAREs M21 and M24. Quantification (FIG. 3E) and representative images (FIG. 3F), scale bar 100 microns. NAREs M21 and M24 were more potent than the reference promoters CAG or CK8e in mouse gastrocnemius muscle. [Figure 3F]In vivo expression of NAREs M21 and M24. Quantification (FIG. 3E) and representative images (FIG. 3F), scale bar 100 microns. NAREs M21 and M24 were more potent than the reference promoters CAG or CK8e in mouse gastrocnemius muscle. [Figure 4] Efficacy of selected NAREs in muscle cells. A library of NAREs was generated containing NAREs derived from genes highly expressed in human skeletal muscle, whereby NAREs were modified with various elements to increase NARE potency. NARE sizes ranged from 450 bp to 1.6 kb. Dual reporter plasmids carrying different NARE candidates were transiently transfected into C2C12 cells. GFP expression levels in transfected (RFP+) cells were quantified by flow cytometry. [Figure 5A] Efficacy of selected NAREs in muscle cells. Figures 9A, 9B, and 9C: Creation of synthetic enhancers via shuffling transcription factor (TF) motifs to increase expression in muscle. Figure 5A: Diagram for creating enhancers placed upstream of a minimal promoter. Transcription factor binding sites (e.g., muscle-enriched sites: MPEX, MEF-1, MEF-2, MEF-3, MAPF, and strong ubiquitous sites: Trex, TEF, SRE, SP1, CREBH, GATA-2, CAATbox, AP-1, AP-2) were randomly ligated and inserted upstream of a minimal promoter (C6) and the mClover3 transgene to test enhancer function. [Figure 5B] Efficacy of selected NAREs in muscle cells. Figures 9A, 9B, and 9C: Creation of synthetic enhancers via shuffling transcription factor (TF) motifs to increase expression in muscle. Figure 5B: Representative sequencing showed that the majority of synthetic sequences were under 200 bp. [Figure 5C]Efficacy of selected NAREs in muscle cells. Figures 9A, 9B, and 9C: Creation of synthetic enhancers via shuffling transcription factor (TF) motifs to increase expression in muscle. Figure 5C: Relative expression from synthetic enhancers bound to promoter C6 normalized to the parent NARE in co-transfected C2C12 cells. The synthetic fragment increased expression up to 8-fold from the minimal NARE in mouse C2C12 muscle cells. [Figure 5D] Potency of selected NAREs in muscle cells. Representative images of particularly well-performing NAREs identified by the screen (right) compared to the C6 promoter (left) in transfected C2C12 cells. [Figure 5E] Efficacy of Selected NAREs in Muscle Cells. The synthetic fragments identified above can be used in combination with other NAREs to generate potent, novel NAREs suitable for expression in muscle. Expression of these NAREs was measured using a dual reporter assay in transfected C2C12 cells. [Figure 5F] Efficacy of selected NAREs in muscle cells. Efficacy of NARE M51 compared to selected reference promoters in transfected C2C12 cells. [Figure 5G] Potency of selected NAREs in muscle cells. Potency of the CK8e-derived NARE M49 in transfected C2C12 cells. [Figure 5H] Potency of selected NAREs in muscle cells. Potency of minMCK-derived NARE M51 in transfected C2C12 cells. [Figure 5I] Efficacy of selected NAREs in muscle cells. Primary human myotubes were transduced with AAV8-miRFP713-WPRE-SV40 polyA containing the indicated NAREs. [Figure 5J] Efficacy of selected NAREs in muscle cells. Primary human myotubes were transduced with AAV8-miRFP713-WPRE-SV40 polyA containing the indicated NAREs. [Figure 5K]Potency of selected NAREs in muscle cells. Potency of hDes-derived NAREs compared to the indicated reference promoters using dual reporter assays in C2C12 cells. [Figure 5L] Potency of selected NAREs in muscle cells. Potency of tMCK-derived NAREs compared to the indicated reference promoters using a dual reporter assay in C2C12 myoblasts. DETAILED DESCRIPTION OF THE INVENTION

[0080] Nucleic acid regulatory elements (NAREs), including promoters, are essential components of gene therapy that control the expression level and durability of therapeutic genes. NAREs can, for example, drive cell-specific expression of transgenes independently of capsid selection. Incorporation of stronger NAREs can increase potency and efficacy at lower viral vector doses, thereby potentially reducing safety risks, immune responses, and the cost of vector production. Furthermore, reducing 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 nucleic acid regulatory elements for improving the safety, efficacy, and durability of therapeutic transgene expression.

[0081] Many NAREs, particularly promoter sequences, have been characterized and optimized through traditional low-throughput analysis (rational design) or newer high-throughput methodologies (e.g., MPRA), but these approaches still require expensive and time-consuming in vitro optimization. Here, we used an advanced artificial intelligence model to repurpose convolutional neural networks (CNNs) and optimize them to predict NARE potency. A library of nucleic acid regulatory elements was computationally constructed by cloning all known enhancer elements reported in the ENCODE database upstream of strong, small, constitutive promoters. The precise spacing between the enhancer elements and promoter sequences that performed particularly well was then optimized. In parallel, in silico saturation mutagenesis was performed, whereby all possible point mutations within the NARE sequences were introduced and those with the best effect on NARE potency were selected. Specifically, well-functioning NAREs were produced and tested in vitro. While most nucleic acid regulatory elements performed better or equivalently to the original promoter, the best enhancer elements and point mutations were selected and combined in a second optimization round. After synthesis and testing of this new set in vitro, enhanced nucleic acid regulatory elements were obtained that exhibited increased potency.

[0082] NARE Provided herein are NAREs that may contain promoters and / or enhancers as part of their sequences. Conventionally, a promoter is defined as a DNA region where transcription is initiated. A promoter contains specific DNA motifs that can be accessed by transcription factors (TFs) and their complexes. On the other hand, an enhancer is defined as a DNA region that amplifies transcription initiation by directly interacting with a target promoter. Similarly, enhancer sequences distal to a target promoter contain DNA motifs that act as binding sites for TFs and cofactors. The term promoter can sometimes be used as a shortened form to refer to a nucleic acid sequence containing multiple regulatory elements. For example, CAG, which contains a CMV enhancer, a β-actin promoter region, and an intron, can be referred to as a promoter. Specifically, the CAG promoter consists of (1) a cytomegalovirus (CMV) early enhancer element, (2) the promoter, the first exon and first intron of the chicken β-actin gene, and (3) the splice acceptor of the rabbit β-globin gene.

[0083] As used herein, the term "NARE" can refer to a promoter, as defined in the conventional sense, as well as a combination of nucleic acid regulatory elements, including promoters and / or other nucleic acid regulatory elements, that regulate expression of a gene operably linked to the element(s). A nucleic acid regulatory element can be, or can include, for example, a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0084] As used herein, "transgene" refers to a gene (particularly the coding sequence of a gene) that is transferred into one or more cells of an organism, for example, using a vector described herein. A transgene can encode a protein or RNA that is normally expressed in the cells of the target organism, or it can encode a protein or RNA from a different organism. A transgene can be integrated into the genome of the target cell or can be present as part of an extrachromosomal expression construct.

[0085] As used herein, "operably linked" refers to a first molecule linked to a second molecule, the molecules being positioned so that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single, continuous 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, the NARE is operably linked to the transcribable polynucleotide molecule. In addition, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without any intervening nucleotides.

[0086] Provided herein are engineered regulatory elements that can drive transgene expression in a variety of different tissues, including muscle. The NARE engineering platform described herein has resulted in a large number of NAREs that can be used to drive the expression of multiple therapeutic transgenes.

[0087] Provided herein is a NARE that is particularly suitable for driving expression in muscle.As used herein, the NARE that begins with "M" is particularly useful for expressing genes in muscle.Muscle NAREs can be muscle-specific, meaning that they show significantly increased or preferential expression of operably linked transgenes in muscle compared to other tissues.

[0088] Tables 4 and 6 provide NAREs that are particularly suitable for driving expression in muscle.

[0089] In embodiments, polynucleotide sequences are provided that comprise 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 4 and Table 6. In embodiments, polynucleotides are provided that comprise one or more NAREs provided in Table 4 and Table 6.

[0090] As used herein, the term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides.Identity can be determined by comparing the position of each sequence that can be aligned for comparison purposes.For example, if the position of the nucleotide sequence being compared is occupied by the same base, the molecules are identical at that position.The degree of identity between nucleic acid sequences 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 both 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 may also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894, BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions, and other modifications when comparing sequences.

[0091] In embodiments, there is provided a 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 M1 to M18, M20, M21, M23, M24, M32 to M49, M51 to M70, and M74 to M120, as listed in Table 6. In embodiments, there is provided a polynucleotide sequence comprising a NARE selected from the group consisting of M1 to M18, M20, M21, M23, M24, M32 to M49, M51 to M70, and M74 to M120.

[0092] In embodiments, provided are polynucleotide sequences 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 M21, M23 to M28, M32, M34 to M36, M51, M53, M55, M57, M59, M61, and M63, as listed in Table 6. In embodiments, provided are polynucleotide sequences comprising a NARE selected from the group consisting of M21, M23 to M28, M32, M34 to M36, M51, M53, M55, M57, M59, M61, and M63.

[0093] In embodiments, there is provided a 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 M21 and M24. In embodiments, there is provided a polynucleotide sequence comprising a NARE selected from the group consisting of M21 and M24.

[0094] In embodiments, provided are polynucleotide sequences 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 M21, M24 to M27, M30, and M32 to M36, as listed in Table 6. In embodiments, provided are polynucleotide sequences comprising a NARE selected from the group consisting of M21, M24 to M27, M30, and M32 to M36.

[0095] In embodiments, 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 M49, M51, M61, M98, M99, and M111-M115 (listed in Table 6).

[0096] In embodiments, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of M49, M51, M61, M98, M99, and M111-M115.

[0097] Provided herein are methods for expressing a transgene in a muscle cell, wherein the transgene is operably linked to a NARE disclosed herein, which is particularly suitable for driving expression in muscle.

[0098] Provided herein is a method for expressing a transgene in a muscle 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 a NARE provided herein. Provided herein is a method for expressing a transgene in a muscle cell, wherein the transgene is operably linked to a NARE provided herein.

[0099]

[0010] Provided herein are methods for expressing a transgene in a muscle 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 a NARE selected from the group consisting of M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120.

[0011] Provided herein are methods for expressing a transgene in a muscle cell, wherein the transgene is operably linked to a NARE selected from the group consisting of M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120.

[0100] 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 NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, and 74-120.

[0101] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, and 74-120.

[0102] 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 NOs: 1-18 and 32.

[0103] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NOs: 1-18 and 32.

[0104] In some embodiments, the NARE is (i) 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 SEQ ID NO: 164; (ii) 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 SEQ ID NO: 134. (iii) sequences that are 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 SEQ ID NO: 135; and (vi) sequences that are 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 SEQ ID NO: 148 or 149.

[0105] In some embodiments, the NARE comprises (i) SEQ ID NO: 164, (ii) SEQ ID NO: 134, (iii) SEQ ID NO: 135, and (vi) SEQ ID NO: 148 or 149.

[0106] In some embodiments, the NARE comprises: (i) 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 SEQ ID NO: 164; (ii) 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 SEQ ID NO: 134; (iii) 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 SEQ ID NO: 135; and (vi) 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 SEQ ID NO: 148.

[0107] In some embodiments, the NARE comprises (i) SEQ ID NO: 164, (ii) SEQ ID NO: 134, (iii) SEQ ID NO: 135, and (vi) SEQ ID NO: 148.

[0108] In some embodiments, the NARE comprises: (i) 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 SEQ ID NO: 164; (ii) 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 SEQ ID NO: 134; (iii) 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 SEQ ID NO: 135; and (vi) 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 SEQ ID NO: 149.

[0109] In some embodiments, the NARE comprises (i) SEQ ID NO: 164, (ii) SEQ ID NO: 134, (iii) SEQ ID NO: 135, and (vi) SEQ ID NO: 149.

[0110] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 218, and (ii) 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 SEQ ID NO: 148 or 149.

[0111] In some embodiments, the NARE comprises (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 148 or 149.

[0112] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 218, and (ii) 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 SEQ ID NO: 148.

[0113] In some embodiments, the NARE comprises (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 148.

[0114] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 218, and (ii) 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 SEQ ID NO: 149.

[0115] In some embodiments, the NARE comprises (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 149.

[0116] In some embodiments, the NARE is (i) 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 any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, 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, or at least 98% identical to SEQ ID NOs: 154 or 189. (iii) optionally, 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 SEQ ID NO: 220; and (iv) 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 SEQ ID NO: 150.

[0117] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, SEQ ID NO: 154 or 189; (iii) optionally, SEQ ID NO: 220; and (iv) SEQ ID NO: 150.

[0118] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 133, and (ii) 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 SEQ ID NO: 150.

[0119] In some embodiments, the NARE comprises (i) SEQ ID NO: 133, and (ii) SEQ ID NO: 150.

[0120] In some embodiments, the NARE comprises: (i) 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 SEQ ID NO: 165; (ii) 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 SEQ ID NO: 154; (iii) 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 SEQ ID NO: 220; and (iv) 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 SEQ ID NO: 150.

[0121] In some embodiments, the NARE comprises (i) SEQ ID NO: 165, (ii) SEQ ID NO: 154, (iii) SEQ ID NO: 220, and (iv) SEQ ID NO: 150.

[0122] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 142, and (ii) 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 SEQ ID NO: 150.

[0123] In some embodiments, the NARE comprises (i) SEQ ID NO: 142, and (ii) SEQ ID NO: 150.

[0124] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 217, (ii) 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 SEQ ID NO: 189, and (iii) 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 SEQ ID NO: 150.

[0125] In some embodiments, the NARE comprises (i) SEQ ID NO: 217, (ii) SEQ ID NO: 189, and (iii) SEQ ID NO: 150.

[0126] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 any one of SEQ ID NOs: 156-163.

[0127] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) any one of SEQ ID NOs: 156-163.

[0128] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 156.

[0129] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 156.

[0130] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 157.

[0131] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 158.

[0132] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 158.

[0133] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 159.

[0134] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 159.

[0135] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 160.

[0136] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 160.

[0137] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 161.

[0138] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 161.

[0139] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 162.

[0140] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 162.

[0141] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 132, and (ii) 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 SEQ ID NO: 163.

[0142] In some embodiments, the NARE comprises (i) SEQ ID NO: 132, and (ii) SEQ ID NO: 163.

[0143] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 131; (ii) 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 SEQ ID NO: 126 or 213; and (iii) optionally, 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 SEQ ID NO: 200.

[0144] In some embodiments, the NARE comprises (i) SEQ ID NO: 131, (ii) SEQ ID NO: 126 or 213, and (iii) optionally, SEQ ID NO: 200.

[0145] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 131, and (ii) 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 SEQ ID NO: 126, and (iii) optionally, 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 SEQ ID NO: 200.

[0146] In some embodiments, the NARE comprises (i) SEQ ID NO: 131, (ii) SEQ ID NO: 126, and (iii) optionally, SEQ ID NO: 200.

[0147] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 131, and (ii) 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 SEQ ID NO: 126.

[0148] In some embodiments, the NARE comprises (i) SEQ ID NO: 131, and (ii) SEQ ID NO: 126.

[0149] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 131; (ii) 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 SEQ ID NO: 126; and (iii) 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 SEQ ID NO: 200.

[0150] In some embodiments, the NARE comprises (i) SEQ ID NO: 131, (ii) SEQ ID NO: 126, and (iii) SEQ ID NO: 200.

[0151] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 131; (ii) 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 SEQ ID NO: 213; and (iii) 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 SEQ ID NO: 200.

[0152] In some embodiments, the NARE comprises (i) SEQ ID NO: 131, (ii) SEQ ID NO: 213, and (iii) SEQ ID NO: 200.

[0153] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 190-199, and (ii) 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 SEQ ID NO: 155.

[0154] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 190-199, and (ii) SEQ ID NO: 155.

[0155] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 196, and (ii) 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 SEQ ID NO: 155.

[0156] In some embodiments, the NARE comprises (i) SEQ ID NO: 196, and (ii) SEQ ID NO: 155.

[0157] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 190, and (ii) 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 SEQ ID NO: 155.

[0158] In some embodiments, the NARE comprises (i) SEQ ID NO: 190, and (ii) SEQ ID NO: 155.

[0159] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 191, and (ii) 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 SEQ ID NO: 155.

[0160] In some embodiments, the NARE comprises (i) SEQ ID NO: 191, and (ii) SEQ ID NO: 155.

[0161] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 192, and (ii) 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 SEQ ID NO: 155.

[0162] In some embodiments, the NARE comprises (i) SEQ ID NO: 192, and (ii) SEQ ID NO: 155.

[0163] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 193, and (ii) 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 SEQ ID NO: 155.

[0164] In some embodiments, the NARE comprises (i) SEQ ID NO: 193, and (ii) SEQ ID NO: 155.

[0165] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 194, and (ii) 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 SEQ ID NO: 155.

[0166] In some embodiments, the NARE comprises (i) SEQ ID NO: 194, and (ii) SEQ ID NO: 155.

[0167] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 195, and (ii) 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 SEQ ID NO: 155.

[0168] In some embodiments, the NARE comprises (i) SEQ ID NO: 195, and (ii) SEQ ID NO: 155.

[0169] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 197, and (ii) 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 SEQ ID NO: 155.

[0170] In some embodiments, the NARE comprises (i) SEQ ID NO: 197, and (ii) SEQ ID NO: 155.

[0171] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 198, and (ii) 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 SEQ ID NO: 155.

[0172] In some embodiments, the NARE comprises (i) SEQ ID NO: 198, and (ii) SEQ ID NO: 155.

[0173] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 199, and (ii) 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 SEQ ID NO: 155.

[0174] In some embodiments, the NARE comprises (i) SEQ ID NO: 199, and (ii) SEQ ID NO: 155.

[0175] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 190-199, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0176] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 190-199, and (ii) any one of SEQ ID NOs: 128-130.

[0177] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 196, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0178] In some embodiments, the NARE comprises (i) SEQ ID NO: 196, and (ii) any one of SEQ ID NOs: 128-130.

[0179] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 190, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0180] In some embodiments, the NARE comprises (i) SEQ ID NO: 190, and (ii) any one of SEQ ID NOs: 128-130.

[0181] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 191, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0182] In some embodiments, the NARE comprises (i) SEQ ID NO: 191, and (ii) any one of SEQ ID NOs: 128-130.

[0183] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 192, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0184] In some embodiments, the NARE comprises (i) SEQ ID NO: 192, and (ii) any one of SEQ ID NOs: 128-130.

[0185] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 193, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0186] In some embodiments, the NARE comprises (i) SEQ ID NO: 193, and (ii) any one of SEQ ID NOs: 128-130.

[0187] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 194, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0188] In some embodiments, the NARE comprises (i) SEQ ID NO: 194, and (ii) any one of SEQ ID NOs: 128-130.

[0189] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 195, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0190] In some embodiments, the NARE comprises (i) SEQ ID NO: 195, and (ii) any one of SEQ ID NOs: 128-130.

[0191] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 197, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0192] In some embodiments, the NARE comprises (i) SEQ ID NO: 197, and (ii) any one of SEQ ID NOs: 128-130.

[0193] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 198, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0194] In some embodiments, the NARE comprises (i) SEQ ID NO: 198, and (ii) any one of SEQ ID NOs: 128-130.

[0195] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 199, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0196] In some embodiments, the NARE comprises (i) SEQ ID NO: 199, and (ii) any one of SEQ ID NOs: 128-130.

[0197] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 190-199, and (ii) 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 SEQ ID NO: 130.

[0198] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 190-199, and (ii) SEQ ID NO: 130.

[0199] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 196, and (ii) 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 SEQ ID NO: 130.

[0200] In some embodiments, the NARE comprises (i) SEQ ID NO: 196, and (ii) SEQ ID NO: 130.

[0201] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 190, and (ii) 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 SEQ ID NO: 130.

[0202] In some embodiments, the NARE comprises (i) SEQ ID NO: 190, and (ii) SEQ ID NO: 130.

[0203] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 191, and (ii) 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 SEQ ID NO: 130.

[0204] In some embodiments, the NARE comprises (i) SEQ ID NO: 191, and (ii) SEQ ID NO: 130.

[0205] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 192, and (ii) 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 SEQ ID NO: 130.

[0206] In some embodiments, the NARE comprises (i) SEQ ID NO: 192, and (ii) SEQ ID NO: 130.

[0207] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 193, and (ii) 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 SEQ ID NO: 130.

[0208] In some embodiments, the NARE comprises (i) SEQ ID NO: 193, and (ii) SEQ ID NO: 130.

[0209] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 194, and (ii) 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 SEQ ID NO: 130.

[0210] In some embodiments, the NARE comprises (i) SEQ ID NO: 194, and (ii) SEQ ID NO: 130.

[0211] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 195, and (ii) 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 SEQ ID NO: 130.

[0212] In some embodiments, the NARE comprises (i) SEQ ID NO: 195, and (ii) SEQ ID NO: 130.

[0213] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 197, and (ii) 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 SEQ ID NO: 130.

[0214] In some embodiments, the NARE comprises (i) SEQ ID NO: 197, and (ii) SEQ ID NO: 130.

[0215] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 198, and (ii) 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 SEQ ID NO: 130.

[0216] In some embodiments, the NARE comprises (i) SEQ ID NO: 198, and (ii) SEQ ID NO: 130.

[0217] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 199, and (ii) 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 SEQ ID NO: 130.

[0218] In some embodiments, the NARE comprises (i) SEQ ID NO: 199, and (ii) SEQ ID NO: 130.

[0219] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, and 221.

[0220] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

[0221] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 137.

[0222] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 137.

[0223] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 138.

[0224] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 138.

[0225] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 219.

[0226] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 219.

[0227] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 212.

[0228] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 212.

[0229] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 211.

[0230] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 211.

[0231] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 143.

[0232] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 143.

[0233] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 221.

[0234] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 221.

[0235] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 123.

[0236] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 123.

[0237] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 127.

[0238] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 127.

[0239] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 139.

[0240] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 139.

[0241] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 136.

[0242] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 136.

[0243] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 141.

[0244] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 141.

[0245] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 209.

[0246] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 209.

[0247] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 124.

[0248] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 124.

[0249] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 125.

[0250] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 125.

[0251] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 152.

[0252] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 152.

[0253] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 140.

[0254] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 140.

[0255] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 145.

[0256] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 145.

[0257] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 146.

[0258] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 146.

[0259] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 147.

[0260] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 147.

[0261] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 153.

[0262] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 153.

[0263] In some embodiments, the NARE comprises (i) 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 any one of SEQ ID NOs: 128-130, and (ii) 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 SEQ ID NO: 210.

[0264] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 128-130, and (ii) SEQ ID NO: 210.

[0265] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

[0266] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

[0267] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 137.

[0268] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 137.

[0269] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 138.

[0270] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 138.

[0271] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 219.

[0272] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 219.

[0273] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 212.

[0274] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 212.

[0275] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 211.

[0276] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 211.

[0277] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 143.

[0278] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 143.

[0279] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 221.

[0280] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 221.

[0281] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 123.

[0282] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 123.

[0283] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 127.

[0284] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 127.

[0285] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 139.

[0286] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 139.

[0287] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 136.

[0288] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 136.

[0289] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 141.

[0290] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 141.

[0291] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 209.

[0292] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 209.

[0293] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 124.

[0294] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 124.

[0295] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 125.

[0296] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 125.

[0297] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 152.

[0298] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 152.

[0299] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 140.

[0300] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 140.

[0301] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 145.

[0302] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 145.

[0303] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 146.

[0304] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 146.

[0305] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 147.

[0306] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 147.

[0307] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 153.

[0308] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 153.

[0309] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 128, and (ii) 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 SEQ ID NO: 210.

[0310] In some embodiments, the NARE comprises (i) SEQ ID NO: 128, and (ii) SEQ ID NO: 210.

[0311] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121; (ii) 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 any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, 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 SEQ ID NO: 122 or 154.

[0312] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121; (ii) 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 any one of SEQ ID NOs: 166-168, 171, 172; and (iii) optionally, 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 SEQ ID NO: 122 or 154.

[0313] In some embodiments, the NARE comprises (ii) SEQ ID NO: 121, (iii) any one of SEQ ID NOs: 166-169, 171, 172, and (iii) optionally, SEQ ID NO: 122 or 154.

[0314] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, (ii) any one of SEQ ID NOs: 166-168, 171, 172, and (iii) optionally, SEQ ID NO: 122 or 154.

[0315] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 any one of SEQ ID NOs: 166-169, 171, or 172.

[0316] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 any one of SEQ ID NOs: 166-168, 171, or 172.

[0317] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) any one of SEQ ID NOs: 166-169, 171, or 172.

[0318] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) any one of SEQ ID NOs: 166-168, 171, or 172.

[0319] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122 or 154; (ii) 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 any one of SEQ ID NO: 121; and (iii) 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 SEQ ID NO: 167.

[0320] In some embodiments, the NARE comprises (i) SEQ ID NO: 122 or 154, (ii) SEQ ID NO: 121, and (iii) SEQ ID NO: 167.

[0321] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 SEQ ID NO: 166.

[0322] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 166.

[0323] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 SEQ ID NO: 167.

[0324] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 167.

[0325] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 SEQ ID NO: 168 or 169.

[0326] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 168 or 169.

[0327] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 SEQ ID NO: 168.

[0328] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 168.

[0329] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 SEQ ID NO: 171.

[0330] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 171.

[0331] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 154, (ii) 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 SEQ ID NO: 121, and (iii) 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 SEQ ID NO: 167.

[0332] In some embodiments, the NARE comprises (i) SEQ ID NO: 154, (ii) SEQ ID NO: 121, and (iii) SEQ ID NO: 167.

[0333] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, (ii) 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 SEQ ID NO: 121, and (iii) 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 SEQ ID NO: 167.

[0334] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, (ii) SEQ ID NO: 121, and (iii) SEQ ID NO: 167.

[0335] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 121, and (ii) 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 SEQ ID NO: 172.

[0336] In some embodiments, the NARE comprises (i) SEQ ID NO: 121, and (ii) SEQ ID NO: 172.

[0337] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122; (ii) 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 any one of SEQ ID NOs: 173-179, or 188; and (iii) optionally, 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 SEQ ID NO: 207.

[0338] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122; (ii) 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 any one of SEQ ID NOs: 173, 174, 176, 178, 179, or 188; and (iii) optionally, 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 SEQ ID NO: 207.

[0339] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, (ii) any one of SEQ ID NOs: 173-179, or 188, and (iii) optionally, SEQ ID NO: 207.

[0340] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, (ii) any one of SEQ ID NOs: 173, 174, 176, 178, 179, or 188, and (iii) optionally, SEQ ID NO: 207.

[0341] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 any one of SEQ ID NOs: 173-179.

[0342] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 any one of SEQ ID NOs: 173, 174, 176, 178, or 179.

[0343] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) any one of SEQ ID NOs: 173-179.

[0344] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) any one of SEQ ID NOs: 173, 174, 176, 178, or 179.

[0345] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 173.

[0346] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 173.

[0347] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 174 or 175.

[0348] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 174 or 175.

[0349] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 174.

[0350] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 174.

[0351] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 176 or 177.

[0352] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 176 or 177.

[0353] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 176.

[0354] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 176.

[0355] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 178.

[0356] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 178.

[0357] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, and (ii) 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 SEQ ID NO: 179.

[0358] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, and (ii) SEQ ID NO: 179.

[0359] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, (ii) 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 SEQ ID NO: 188, and (iii) 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 SEQ ID NO: 207.

[0360] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, (ii) SEQ ID NO: 188, and (iii) SEQ ID NO: 207.

[0361] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 184, and (ii) 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 any one of SEQ ID NOs: 185-187.

[0362] In some embodiments, the NARE comprises (i) SEQ ID NO: 184, and (ii) any one of SEQ ID NOs: 185-187.

[0363] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 184, and (ii) 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 SEQ ID NO: 185.

[0364] In some embodiments, the NARE comprises (i) SEQ ID NO: 184, and (ii) SEQ ID NO: 185.

[0365] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 184, and (ii) 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 SEQ ID NO: 186.

[0366] In some embodiments, the NARE comprises (i) SEQ ID NO: 184, and (ii) SEQ ID NO: 186.

[0367] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 184, and (ii) 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 SEQ ID NO: 187.

[0368] In some embodiments, the NARE comprises (i) SEQ ID NO: 184, and (ii) SEQ ID NO: 187.

[0369] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 180, and (ii) 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 SEQ ID NO: 181 or 182.

[0370] In some embodiments, the NARE comprises (i) SEQ ID NO: 180, and (ii) SEQ ID NO: 181 or 182.

[0371] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 180, and (ii) 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 SEQ ID NO: 181.

[0372] In some embodiments, the NARE comprises (i) SEQ ID NO: 180, and (ii) SEQ ID NO: 181.

[0373] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 180, and (ii) 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 SEQ ID NO: 182.

[0374] In some embodiments, the NARE comprises (i) SEQ ID NO: 180, and (ii) SEQ ID NO: 182.

[0375] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183; (ii) 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 SEQ ID NO: 182; and (iii) optionally, 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 any one of SEQ ID NOs: 200, 201, 205, or 208.

[0376] In some embodiments, the NARE comprises (i) SEQ ID NO: 183, (ii) SEQ ID NO: 182, and (iii) optionally, any one of SEQ ID NOs: 200, 201, 205, or 208.

[0377] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183; (ii) 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 SEQ ID NO: 182; and (iii) 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 any one of SEQ ID NOs: 200, 201, 205, or 208.

[0378] In some embodiments, the NARE comprises any one of (i) SEQ ID NO: 183, (ii) SEQ ID NO: 182, and (iii) SEQ ID NO: 200, 201, 205, or 208.

[0379] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183, and (ii) 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 SEQ ID NO: 182.

[0380] In some embodiments, the NARE comprises (i) SEQ ID NO: 183, and (ii) SEQ ID NO: 182.

[0381] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183, (ii) 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 SEQ ID NO: 182, and (iii) 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 SEQ ID NO: 200.

[0382] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183, (ii) 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 SEQ ID NO: 182, and (iii) 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 SEQ ID NO: 205.

[0383] In some embodiments, the NARE comprises (i) SEQ ID NO: 183, (ii) SEQ ID NO: 182, and (iii)) SEQ ID NO: 205.

[0384] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183, (ii) 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 SEQ ID NO: 182, and (iii) 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 SEQ ID NO: 208.

[0385] In some embodiments, the NARE comprises (i) SEQ ID NO: 183, (ii) SEQ ID NO: 182, and (iii)) SEQ ID NO: 208.

[0386] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 183, (ii) 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 SEQ ID NO: 182, and (iii) 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 SEQ ID NO: 201.

[0387] In some embodiments, the NARE comprises (i) SEQ ID NO: 183, (ii) SEQ ID NO: 182, and (iii)) SEQ ID NO: 201.

[0388] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 170; (ii) optionally, 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 SEQ ID NO: 188; and (iii) 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 SEQ ID NO: 203 or 204.

[0389] In some embodiments, the NARE comprises (i) SEQ ID NO: 170, (ii) optionally SEQ ID NO: 188, and (iii) SEQ ID NO: 203 or 204.

[0390] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 170, and (ii) 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 SEQ ID NO: 203.

[0391] In some embodiments, the NARE comprises (i) SEQ ID NO: 170, and (ii) SEQ ID NO: 203.

[0392] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 122, (ii) 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 SEQ ID NO: 188, and (iii) 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 SEQ ID NO: 204.

[0393] In some embodiments, the NARE comprises (i) SEQ ID NO: 122, (ii) SEQ ID NO: 188, and (iii) SEQ ID NO: 204.

[0394] In some embodiments, the NARE comprises (i) at least one copy of 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 SEQ ID NO: 154 or 164, and (ii) 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 any one of SEQ ID NOs: 128-130. In some embodiments, the NARE comprises (i) SEQ ID NO: 154 or 164, and (ii) any one of SEQ ID NOs: 128-130.

[0395] In some embodiments, the NARE comprises (i) at least one copy of 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 SEQ ID NO: 154, and (ii) 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 any one of SEQ ID NOs: 128-130. In some embodiments, the NARE comprises (i) a first 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 SEQ ID NO: 154, (ii) a second 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 SEQ ID NO: 154, and (iii) 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 any one of SEQ ID NOs: 128-130.

[0396] In some embodiments, the NARE comprises (i) at least one copy of SEQ ID NO: 154, and (ii) any one of SEQ ID NOs: 128-130. In some embodiments, the NARE comprises (i) a first sequence comprising SEQ ID NO: 154, (ii) a second sequence comprising SEQ ID NO: 154, and (iii) any one of SEQ ID NOs: 128-130.

[0397] In some embodiments, the NARE comprises (i) at least one copy of 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 SEQ ID NO: 154, and (ii) 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 SEQ ID NO: 128. In some embodiments, the NARE comprises (i) a first 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 SEQ ID NO: 154, (ii) a second 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 SEQ ID NO: 154, and (iii) 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 SEQ ID NO: 128.

[0398] In some embodiments, the NARE comprises (i) at least one copy of SEQ ID NO: 154, and (ii) SEQ ID NO: 128. In some embodiments, the NARE comprises (i) a first sequence comprising SEQ ID NO: 154, (ii) a second sequence comprising SEQ ID NO: 154, and (iii) SEQ ID NO: 128.

[0399] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 164, and (ii) 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 any one of SEQ ID NOs: 128-130.

[0400] In some embodiments, the NARE comprises (i) SEQ ID NO: 164, and (ii) any one of SEQ ID NOs: 128-130.

[0401] In some embodiments, the NARE comprises (i) 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 SEQ ID NO: 164, and (ii) 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 SEQ ID NO: 129.

[0402] In some embodiments, the NARE comprises (i) SEQ ID NO: 164, and (ii) SEQ ID NO: 129.

[0403] In embodiments, the NARE comprises an intron or a portion of an intron, such as an 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 beta globulin intron, CRYAB intron, DES.4 intron, EEF1A1 intron, EEF1B2 intron, EF1a intron, FHL1 intron, FLOT 1 intron, FXYD1 intron, GFAP intron, HBB intron, hCPE intron, hEf1a2-intron, HPD intron, IFI27L2 intron, mouse IgG chimeric intron, MVM intron, MVMi-AATp intron, MVMi-SynE-mTTRp intron, Rabit beta globin intron, RBP4 intron, RPL26 intron, RPL27 intron, S100A6 intron, sEEF1A1 intron, SV40 intron, TMSB10 intron, and UCHL1 intron.

[0404] In embodiments, the NARE comprises an enhancer or a portion of an enhancer, which may be selected from an ACTA1 enhancer, an ACTC1 enhancer, a CKM enhancer, a CMV enhancer, a CMV enhancer, an MCK enhancer, an mDES enhancer, an mDES.1 enhancer, a minCKM enhancer, a minCKM2 enhancer, a minDes enhancer, an NRGN enhancer 1.1, an NRGN enhancer 1.2, an NRGN enhancer 2.1, an NRGN enhancer 2.2, an SV40 enhancer, and an SV40 enhancer (SV40e).

[0405] In embodiments, the NARE comprises a UTR or a portion of a UTR. The UTR or portion of a UTR may 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.

[0406] NAREs used as references or controls are also provided herein. Some NAREs are provided for constitutive expression of an operably linked transgene, i.e., for maintaining the expression of the transgene at a constant level. Constitutive NAREs can drive the expression of an operably linked transgene in various cell types and tissues. As used herein, NAREs beginning with "C" are constitutive NAREs. The constitutive NAREs disclosed herein may be useful for expressing genes in muscle.

[0407] NARE Qualification Those skilled in the art will understand that certain modifications can be made to the NAREs disclosed herein without eliminating the ability of the NARE to drive gene expression in the desired tissue / cell type. Various in vitro and in vivo methods for confirming that a modified NARE is still able to drive gene expression are known in the art, including, but not limited to, the methods used herein. For example, the strength of a NARE can be assessed by operably linking the NARE to a transgene encoding a protein and measuring transgene expression, e.g., by detecting mRNA encoding the protein, or by measuring the presence or activity of the protein, e.g., by ELISA, Western blot, fluorescence, enzymatic activity of the protein, etc.

[0408] Provided herein are NAREs comprising one or more components. Provided herein are NAREs comprising one or more sequence components (or sequence variants thereof) listed in any one of Tables 4-6. Provided herein are NAREs comprising one or more sequence components (or sequence variants thereof) listed in Table 4. Provided herein are NAREs comprising one or more components (or sequence variants thereof) of a full-length NARE provided in Table 6. Simply by way of illustration and non-limiting example, provided are NAREs comprising one or more components (or sequence variants thereof) of NARE M37 (CK8<em>d2 ): (1) CK8e.1, and (2) md2. In embodiments, provided are NAREs comprising one or more components (or sequence variants thereof) of a full-length NARE provided in Table 4 or Table 6, whereby the components (or sequence variants thereof) are arranged 5' to 3' as shown in Table 6. In embodiments, NAREs are provided that comprise one or more components of a full-length NARE provided in Table 6 (or sequence variants thereof), whereby the components (or sequence variants thereof) are not arranged 5' to 3' as shown in Table 6, but are arranged in a different order.

[0409] In embodiments, in a NARE of Table 4 or Table 6, one or more of the components are replaced with a different sequence, including components (or sequence variants thereof) from a different NARE of Table 4 or Table 6. Additionally, NAREs that include components (or sequence variants thereof) from two or more NAREs disclosed in Tables 4-6 are also contemplated.

[0410] Furthermore, NARE variants are contemplated that contain sequences in addition to the elements of the NAREs disclosed herein (e.g., added to either end of the NARE sequence or inserted within the NARE sequence). Similarly, NARE variants are provided in which specific sequences have been removed from the NAREs disclosed herein (e.g., as terminal or internal deletions). In embodiments, NAREs comprising one or more components are provided without any additional nucleic acid sequence(s) linking the components of the NARE. In embodiments, NAREs comprising one or more components are provided, whereby individual components are connected 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 embodiments, some of the components are directly linked, while other components are linked to other components via intervening sequences. Thus, variants of the NAREs disclosed in Tables 4 or 6 are contemplated, in which additional sequences have been added between different components. Variants of the NAREs disclosed in Tables 4 or 6 are further contemplated, which disclose the same components of a given NARE in Tables 4 or 6, but differ in the sequence(s) connecting the individual components.

[0411] Nucleic Acid Constructs and Vectors In one aspect, nucleic acid constructs and vectors for introducing transgenes or expression constructs into cells, and their uses are provided. Nucleic acid constructs include expression constructs, including plasmids. The term "expression construct" refers to a recombinant polynucleotide construct that includes a nucleic acid encoding an RNA that can be transcribed in a cell. Methods for constructing expression constructs and plasmids via standard recombinant techniques are known in the art.

[0412] In some embodiments, a vector comprises a recombinant DNA construct containing additional DNA elements, including DNA segments that provide appropriate levels of DNA replication in host cells and target gene expression in target cells. As used herein, "vector" refers to a vehicle containing a polynucleotide delivered to a host cell, either in vitro, ex vivo, or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA minicircles, or viruses (containing viral-derived sequences). A vector may also refer to a virion containing a nucleic acid delivered to a host cell, either in vitro, ex vivo, or in vivo. In some embodiments, a vector refers to a virion containing a recombinant viral genome, where the viral genome includes one or more ITRs and a transgene.

[0413] In one embodiment, the vector is a viral vector or a combination of viral vectors. In one aspect, a vector is provided comprising any of the nucleic acid constructs disclosed herein.

[0414] Provided herein are nucleic acid constructs and vectors comprising a NARE disclosed herein operably linked to a transgene. In embodiments, the nucleic acid constructs or vectors disclosed herein contain additional regulatory elements, including, but not limited to, a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer. In embodiments, the nucleic acid constructs or vectors disclosed herein contain a polyadenylation sequence. In embodiments, the nucleic acid constructs or vectors disclosed herein contain an internal ribosome entry site (IRES). IRES sequences can be used to produce multiple polypeptides from a single gene transcript. IRES (or other suitable sequences) can be used to produce proteins containing multiple polypeptide chains or to express two different proteins from or within 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.

[0415] viral vectors Viral vectors for expressing target genes in target cells, tissues, or organisms are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric, mosaic, or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles, or small molecules).

[0416] AAV vectors Adeno-associated viruses (AAVs) are small, single-stranded DNA viruses that require a helper virus to promote efficient replication. The 4.7-kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames encoding 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. These proteins primarily function in AAV replication and rescue, as well as in regulating AAV integration into host cell chromosomes. 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 virion capsid. VP3 accounts for more than 80% of the total protein in AAV virions. Adjacent to the 5' and 3' ends of the Rep and Cap open reading frames are inverted terminal repeats (ITRs) approximately 145 bp long. The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with therapeutic or reporter transgenes.

[0417] Recombinant adeno-associated virus "rAAV" vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have one or more of the AAV wild-type genes, preferably the Rep and / or Cap genes, deleted in whole or in part, but retain functional flanking ITR sequences.

[0418] In some embodiments, the viral vector is a rAAV virion comprising a rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome comprises a nucleic acid construct disclosed herein.

[0419] In some embodiments, the viral vectors disclosed herein comprise a nucleic acid comprising AAV 5' and 3' ITRs located 5' and 3', respectively, of a sequence encoding a transgene. In embodiments, the transgene is UPF1, BDNF, or ATP7B. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5' and 3' ITR sequences arranged in tandem, e.g., 5'-3', or head-to-tail, or in another alternative arrangement. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs or to have the 5' ITR (or conversely, the 3' ITR) located both 5' and 3' to the transgene. The ITR sequences may be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences may be present. The ITRs need not be wild-type nucleotide sequences and can be modified (e.g., by nucleotide insertion, deletion, or substitution) so long as the sequences provide functional rescue, replication, and packaging. The ITRs can be selected from AAV2 or from other AAV serotypes, as described herein.

[0420] In some embodiments, a vector is provided comprising a nucleic acid sequence comprising (i) a nucleic acid construct disclosed herein and (ii) one or more inverted terminal repeats (ITRs). In one embodiment, the nucleic acid sequence comprises a 5'ITR and a 3'ITR. In one embodiment, the 5'ITR and the 3'ITR are derived from adeno-associated virus (AAV) serotype AAV2.

[0421] In some embodiments, the viral vector is an AAV vector, e.g., AAV1 (i.e., AAV containing AAV1 ITRs and AAV1 capsid protein), AAV2 (i.e., AAV containing AAV2 ITRs and AAV2 capsid protein), AAV3 (i.e., AAV containing AAV3 ITRs and AAV3 capsid protein), AAV4 (i.e., AAV containing AAV4 ITRs and AAV4 capsid protein), AAV5 (i.e., AAV containing AAV5 ITRs and AAV5 capsid protein), AAV6 (i.e., AAV containing AAV6 ITRs and AAV6 capsid protein), AAV7 (i.e., AAV containing AAV7 ITRs and AAV7 capsid protein), AAV8 (i.e., AAV containing AAV8 ITRs and AAV8 capsid protein), AAV9 (i.e., AAV9 ITRs and AAV9 capsid protein), AAVrh.74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein).

[0422] In some embodiments, the viral vector is a pseudotyped AAV vector containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins).

[0423] In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV containing AAV2 ITRs and AAV 7m8 capsid proteins).

[0424] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, incorporated herein by reference).

[0425] 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 a ratio 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-retrocapsid proteins at a ratio 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.

[0426] In some embodiments, a mixture of (1) an AAV vector comprising rAAV2-retro and (2) an AAV vector comprising AAVrh.10 is used. In some embodiments, a ratio 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, of (1) an AAV vector comprising rAAV2-retro and (2) an AAV vector comprising AAVrh.10 is used. In some embodiments, a ratio 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, of (1) an AAV vector comprising AAVrh.10 and (2) an AAV vector comprising rAAV2-retro is used.

[0427] Other viral vectors 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 deletion of the E1 and E3 regions. A transcription cassette can be inserted into the E1 region to obtain an E1 / E3-deleted recombinant AV vector. Adenovirus vectors also include helper-dependent, large-capacity adenovirus vectors (also known as large-capacity, "gutless" or "gutted" vectors) that do not contain viral coding sequences. These vectors contain cis-acting elements necessary for viral DNA replication and packaging, primarily inverted terminal repeats (ITRs) and packaging signals (CYs). These helper-dependent AV vector genomes have the potential to carry foreign DNA from several hundred base pairs to approximately 36 kb.

[0428] Alternatively, other systems, such as lentiviral vectors, can be used. Lentiviral-based systems can transduce not only non-dividing cells but also dividing cells, making them useful for targeting non-dividing cells in the CNS, for example. Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome, providing the potential for very long-term gene expression.

[0429] Polynucleotides, including plasmids, YACs, minichromosomes, and minicircles, carrying target genes containing expression cassettes can also be introduced into cells or organisms by non-viral vector systems, for example, using cationic lipids, polymers, or both as carriers. Conjugated poly-L-lysine (PLL) polymer and polyethyleneimine (PEI) polymer systems can also be used to deliver vectors into cells. Other methods for delivering vectors into cells include hydrodynamic injection and electroporation, as well as the use of ultrasound, for both cell cultures and 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.

[0430] rAAV virion production The rAAV virions disclosed herein can be constructed and produced using materials and methods described herein and known to those of skill in the art. Such engineering methods used to construct 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. Additionally, suitable methods for producing rAAV cassettes within adenovirus capsids are described in U.S. Patent Nos. 5,856,152 and 5,871,982.

[0431] Briefly, to package the rAAV genome into rAAV virions, host cells containing sequences necessary for expressing AAV rep and AAV cap or functional fragments thereof, as well as helper genes essential for AAV production, are used. The AAV rep and cap sequences are obtained from AAV sources found herein. The AAV rep and cap sequences can be introduced into host cells by any method known to those skilled in the art, including, but not limited to, transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into host cells via one or more nucleic acid molecules and stably present in the cells as episomes. In another embodiment, the rep and cap sequences are stably integrated into the cell's genome. In another embodiment, the rep and cap sequences are transiently expressed in the host cell. For example, a nucleic acid molecule useful for such transfection comprises, from 5' to 3', a promoter, an optional spacer inserted between the promoter and the start of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.

[0432] The rep and cap sequences, along with their expression control sequences, may be provided on a single vector, or each sequence may be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences may be provided on a vector containing other DNA sequences that can be introduced into host cells. Preferably, the promoter used in this construct may be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins may be in any form that transfers these components into host cells. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as sequences of marker genes. This molecule does not contain AAV ITRs and generally does not contain AAV packaging sequences. To avoid the occurrence of homologous recombination, other viral sequences, particularly adenoviral sequences, are avoided in this plasmid. Desirably, this plasmid is constructed so that it can be stably transfected into cells.

[0433] Although the molecules providing rep and cap can be transiently transfected into host cells, it is preferred that the host cells be stably transformed with the sequences necessary to express functional rep / cap proteins in the host cell, e.g., as an episome or by integration into the host cell chromosome. Depending on the promoter controlling expression in such stably transfected host cells, the rep / cap proteins can be transiently expressed (e.g., through the use of an inducible promoter).

[0434] The methods used to construct embodiments of the present disclosure are conventional genetic or recombinant engineering techniques, as described in the references above. For example, rAAV can be produced using a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.), according to the manufacturer's instructions. See Herzog et al., 1999, Nature Medic., 5(1):56-63, for the method used in the following examples, which employs a plasmid carrying the transgene, a helper plasmid containing AAV rep and cap, and a plasmid supplying the adenoviral helper functions of E2A, E4Orf6, and VA. While the present specification provides illustrative examples of specific constructs using the information provided herein, those skilled in the art will be able to select and design other suitable constructs using their selection of spacers, promoters, and other elements, including at least one translational start and stop signal, and the optional addition of a polyadenylation site.

[0435] rAAV virions can be produced by culturing host cells containing the rAAV viruses described herein, which contain the rAAV genome packaged into rAAV virions, AAV rep sequences, and AAV cap sequences, under the control of regulatory sequences that direct their expression. Suitable viral helper genes, such as adenovirus E2A, E4Orf6, and VA, among other possible helper genes, can be provided to the culture by various methods known in the art, preferably on separate plasmids. Recombinant AAV virions directing the expression of the transgene are then isolated from the cells or cell culture in the absence of contaminating helper virus or wild-type AAV.

[0436] The expression of the transgene can be measured by methods 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). The RNA expression level can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the protein expression level can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by specific methods detailed in the following examples.

[0437] cell In embodiments, the nucleic acid constructs and vectors disclosed herein are used to deliver a NARE operably linked to a transgene to a cell. Provided herein are cells comprising a NARE, nucleic acid construct, or vector disclosed herein. In embodiments, the cell is a muscle cell. The cell may be a mammalian cell. The cell may be a human cell. The cell may be isolated.

[0438] Pharmaceutical Composition Provided herein is a pharmaceutical composition comprising a nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.

[0439] The nucleic acid constructs or vectors disclosed herein are preferably evaluated for contamination by conventional methods and then formulated into pharmaceutical compositions suitable for storage and / or administration to a patient.

[0440] Formulation of the nucleic acid constructs or vectors disclosed herein involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly buffered saline or other buffers, such as HEPES, suitable for subretinal injection, to maintain pH at an appropriate physiological level. The nucleic acid constructs or vectors disclosed herein can be formulated into pharmaceutical compositions. These compositions may contain, in addition to the vector, pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives known to those skilled in the art. Such materials must be non-toxic and not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials can be determined by those skilled in the art depending on the route of administration. Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, incorporated herein by reference. Physiological saline, magnesium chloride, dextrose, or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. In some cases, surfactants, such as pluronic acid (PF68) 0.001%, may be used. In some cases, Ringer's solution, lactated Ringer's solution, or Hartmann's solution may be used. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0441] Pharmaceutical compositions comprising the nucleic acid constructs or vectors disclosed herein may be formulated with one or more pharmaceutically acceptable excipients, which may be pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, magnesium talc, calcium or zinc stearate, or steric acid), solvents or encapsulating materials, bulking agents, salts, surfactants, and / or preservatives, that are involved in carrying or transporting the therapeutic compound for administration to a subject. Some examples of substances that can act as pharmaceutically acceptable excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, 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, soybean oil, and the like; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffers; and other non-toxic compatible substances used in pharmaceutical formulations.

[0442] Bulking agents are compounds that add mass to a pharmaceutical formulation and contribute to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol, and sorbitol.

[0443] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce particle formation in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes particle formation after reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, or the like. Myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic, PF68, etc.).

[0444] Preservatives may be used in the formulations of the present invention. Suitable preservatives for use in the formulations of the present invention include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl-dimethylammonium chlorides, where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical textbooks, such as "Remington's Pharmaceutical Sciences," The Science and Practice of Pharmacy, 19th Ed., Mack Publishing Company, Easton, Pa., (1995).

[0445] For delayed release, the nucleic acid constructs or vectors disclosed herein may be included in pharmaceutical compositions formulated for delayed release, such as microcapsules formed from biocompatible polymers or liposome carrier systems, according to methods known in the art.

[0446] For long-term storage of the vector, the vector may be frozen in the presence of glycerol.

[0447] method Provided herein are methods for inducing expression of a transgene in cells and / or tissues. Provided herein are methods of inducing expression of a transgene, comprising providing a cell comprising a nucleic acid construct comprising a NARE disclosed herein operably linked to the transgene, and culturing the cell under conditions that allow expression of the transgene. In embodiments, the cell is a muscle cell. Provided herein are methods for inducing expression of a transgene in vivo. Provided herein are methods for inducing expression of a transgene in vitro. Provided herein are methods for inducing expression of a transgene ex vivo.

[0448] Provided herein are methods of treating a disease in a subject in need thereof using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein.

[0449] 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, humans or non-human mammals, such as cows, horses, dogs, sheep, or cats. An "individual" or "patient" is also a subject herein.

[0450] As used herein, the terms "treat," "treated," "treating," or "treatment" refer to therapeutic treatment, the purpose of which is to slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the severity of the condition, disorder, or disease; stabilization (i.e., not worsening) of the pathological condition, disorder, or disease state; delay in the onset of the condition, disorder, or disease, or delay in the progression of the condition, disorder, or disease; improvement in one or more symptoms of the condition, disorder, or disease state; and remission (partial or complete), or improvement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment. The terms "prevent", "preventing", and the like refer to acting before the onset of an overt disease or disorder to prevent the onset of the disease or disorder, minimize the extent of the disease or disorder, or delay the course of its development.

[0451] In some embodiments, treatment refers to increased survival (e.g., survival time). For example, treatment can result in an increased life expectancy of a patient. In some embodiments, treatment can result in an increased life expectancy of 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 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 22%, about 24%, about 26%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 45 ... 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, treatment results in an increase in patient 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 more compared to the life expectancy of one or more control individuals with the disease who are not receiving treatment. 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 greater than about 40, 45, 50, 55, 60 years or more.

[0452] 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 topical, particularly to the ear, nose, eye, or skin. The mode of administration is left to the discretion of the physician.

[0453] In some cases, the nucleic acid constructs or vectors described herein are administered locally. This can be achieved, for example, by local infusion during surgery, by topical application (e.g., cream or lotion), by injection, by catheter, by suppository or enema, or by implant, which is a porous, non-porous, or gel-like material, including membranes such as silastic membranes or fibers. In some situations, the nucleic acid constructs or vectors described herein are introduced into the central nervous system, circulatory system, or gastrointestinal tract by any suitable route, including intraventricular injection, intrathecal injection, paraspinal injection, epidural injection, enema, and injection adjacent to a peripheral nerve.

[0454] The compositions described herein can be administered as a single dose or multiple doses. Such compositions can be administered at regular intervals depending on the nature, severity, and extent of the subject's condition. In some embodiments, a therapeutically effective amount of a nucleic acid construct or vector is administered intrathecally at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, every two months, every other month, every month, every other week, or every week).

[0455] The amount of the nucleic acid construct or vector described herein that is effective in treating a disease can be determined using standard clinical techniques known to those skilled in the art. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose used will also depend on the route of administration, the condition, the severity of the condition being treated, and various physical factors related to the individual being treated, and can be determined according to the judgment of a medical professional.

[0456] 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 is, for example, about 1×10 9 ~Approx. 1×10 14The rAAV may be present in a range of about 1 x 10 rAAV genome particles (vg) / kg body weight. A "genome particle" is defined herein as an AAV capsid containing a single-stranded DNA molecule that can be quantified by a sequence-specific method (such as qPCR or ddPCR). In some embodiments, the rAAV is present in a range of about 1 x 10 12 ~Approx. 1×10 13 In some embodiments, the rAAV is administered at about 5 x 10 11 ~Approx. 5×10 12 In some embodiments, the rAAV is administered in a total of about 7.5 x 10 vg / mL of cerebrospinal fluid (CSF) volume per patient. 13 ~7.5×10 14 It is administered in vg.

[0457] In some embodiments, the rAAV is about 1 x 10 11 ~Approx. 1×10 14 rAAV genome particles (vg) / kg body weight are administered to animals.

[0458] In some embodiments, the rAAV genome particles are provided in a volume of about 20 μL to about 50 mL. In some embodiments, the rAAV genome particles are provided in a volume of about 30 μL to 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, or 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.

[0459] Still other doses within these ranges can be selected by the attending physician. It is understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the nucleic acid constructs or vectors, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

[0460] The nucleic acid constructs or vectors disclosed herein can also be advantageously provided to cells ex vivo, followed by administering the live cells to a subject. Methods for treating disease by transplanting cells modified to express recombinant proteins are also well known. See, for example, U.S. Patent No. 5,399,346, which discloses a method for introducing nucleic acids into primary human cells for introduction into humans. In some embodiments, the use of human cells for ex vivo therapy is preferred, although other cells, such as bacterial cells, may be transplanted into the subject's vasculature to continuously release therapeutic agents. See, for example, U.S. Patent Nos. 4,309,776 and 5,704,910.

[0461] 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 incorporated by reference in its entirety herein.

[0462] It is understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention. Furthermore, it should be understood that the disclosure of the invention herein includes all possible combinations of such particular features. For example, if a particular feature is disclosed in connection with a particular aspect or embodiment of the invention, or with a particular claim, that feature can also be used in combination with and / or in connection with other specific aspects and embodiments of the invention, and in the invention generally, to the extent possible.

[0463] When reference is made herein to a method that includes two or more defined steps, the defined steps can be performed in any order or simultaneously (unless the context excludes this possibility), and the method can include one or more other steps that occur before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes these possibilities).

[0464] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, to the extent that the definition or use of a term in a reference incorporated herein by reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0465] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given, which should not be construed as limiting or defining the entire scope of the invention. [Example]

[0466] Example 1: Design of NARE Materials and Methods Potential NARE candidates were identified from genes highly expressed in humans within the target tissue (muscle). Promoter regions were then defined based on chromatin marks, accessibility, conservation, and other genome-wide datasets, or cis-regulatory elements were identified.

[0467] Motif 10 element (MTE) / downstream promoter element (DPE) modifications were incorporated to increase transcription, as were various cellular, viral, or synthetic 5'UTR motifs to increase transcription / translation. Intron selection was varied to include, for example, short cellular introns that naturally have their own standalone transcription start sites, indicating the presence of a promoter within this particular intron. Additionally, introns were inserted at different positions in the sequence and / or introns were truncated while maintaining splicing elements. Additionally, individual or multiple point mutations were introduced to increase expression or prevent inactivation. Different transcriptional enhancers were also introduced upstream of the core promoter. Insertion, substitution, and / or shuffling of transcriptional motifs were utilized in certain cases, as was removal of CpG sequences to reduce immunogenicity while maintaining promoter activity.

[0468] Gene regulatory elements were also optimized by the following methods: (i) highly expressed transcription factor binding sites (TFBS) upstream of the core promoter were identified, shuffled, and cloned; (ii) iterative mutation and scoring of promoters using artificial intelligence (AI) algorithms.

[0469] Furthermore, nested NAREs were created (Figure 2A). One or more promoters were cloned within the intron of one of the existing promoters, ensuring that the placement did not create unintended translation start sites. In this way, dual, tri, and quad promoters were created, allowing for increased NARE strength while minimizing overall length. For example, in Figure 2A, tMCK was modified by adding MTE / DPE modifications. tMCK is the muscle creatine kinase (MCK) promoter, which was shortened by ligating a triple tandem (206 bp) of MCK enhancers to its 87 bp basal promoter, generating the tMCK (720 bp) NARE. A synthetic HBG intron variant containing an enhancer (SIE) and a modified human desmin promoter was inserted downstream. This nested promoter strategy allows for increased transcriptional output by having multiple promoters, avoiding or minimizing loss of expression due to promoter inactivation. The intron location allows for the generation and processing of multiple transcripts, preventing the potentially deleterious effects of a second promoter sequence on translation rate as it is spliced ​​out in a specific transcript. Additionally, this strategy allows for the targeting of multiple cell types with a single complex promoter, conferring tissue specificity to normally constitutive promoters.

[0470] Generation and screening of synthetic cis-regulatory elements (CREs) by transcription factor shuffling Binding site sequences for tissue-specific and highly expressed transcription factors for a given tissue were printed as a pool of single-stranded oligonucleotides (Figure 5A). The oligonucleotides were annealed to double-stranded oligonucleotides and randomly ligated together overnight. The fragments were then ligated into an AAV plasmid upstream of a small synthetic JeT promoter driving expression of mClover3-WPRE3-syn polyA. This pooled library was transformed into cells, and individual colonies were miniprepped to obtain sufficient DNA for transient transfection experiments.

[0471] Cells of interest were then transiently co-transfected with the TFBS candidate plasmid and mCherry plasmid as a normalization control. After 48 hours, cells were imaged for mClover3 and mCherry fluorescence as described in Example 2. Images were automatically analyzed using CellProfiler to identify red fluorescent protein-positive cells and measure the green fluorescent protein intensity from these cells. The GFP intensity of the candidate TFBS NARE compared to the JeT promoter alone was used to identify positive hits in this screen. Positive hits were then subcloned into a dual reporter assay system to validate the screening results with either JeT or other basal NAREs.

[0472] Example 2: Dual reporter protein expression assay To test the ability of NARE candidates to drive gene expression, we utilized a dual-reporter assay using flow cytometry. A diagram of the dual-reporter expression construct and assay is provided in Figure 1. Different synthetic candidate NARE sequences were cloned upstream of a transgene encoding mClover3 (green / yellow fluorescent protein), which was then positioned upstream of a posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE250) and a synthetic polyadenylation (polyA) signal. mClover3 fluorescence was used as a measure of NARE intensity. The plasmid also contained a separate expression cassette (5' to 3': SV40 promoter-tdTomato (red fluorescent protein)-SV40 polyA), which was the same in all NARE test constructs (Figure 1). tdTomato fluorescence was used as an internal normalization control, allowing us to account for variations in transfection efficiency.

[0473] Various cell lines were transiently transfected with the dual reporter plasmids using Turbofect transfection reagent. After 48 hours, cells were trypsinized and resuspended for flow cytometry. Raw data were processed to select only live single cells for analysis. A gate for tdTomato signal was set using a sample containing untransfected cells. This gate was then applied to all live single cells to identify tdTomato-positive (and therefore transfected) cells. mClover3 fluorescence was then determined for the tdTomato-positive population. Because transfections varied slightly between different experiments, relative NARE potency was calculated by the ratio of mClover3 fluorescence to tdTomato fluorescence. Synthetic candidate NAREs were tested alongside known constitutive and tissue-specific reference promoters, such as CAG, CMV, or AAT.

[0474] Example 3: Intensity of different NAREs in muscle cells (in vitro) Materials and Methods Myotube model The strength of selected muscle-specific NAREs was tested using three cellular models of myotubes. One model is the immortalized C2C12 cell line. C2C12 cells are mouse skeletal myoblasts derived from satellite cells in the thigh muscles of 2-month-old female C3H mice. C2C12 cells were plated on type I collagen-coated vessels and grown in growth medium: DMEM (high glucose, no sodium pyruvate) + 10% fetal bovine serum + 2 mM L-glutamine + 100 units / mL penicillin and 100 μg / mL streptomycin. After reaching confluence, C2C12 cells underwent a medium change to differentiation medium: DMEM (high glucose, no sodium pyruvate) + 2% horse serum + 2 mM L-glutamine + 1 μM insulin + 100 units / mL penicillin and 100 μg / mL streptomycin. C2C12 cells were cultured in differentiation medium for approximately 7 days to form long linear myotubes, which are the result of myoblast fusion during myogenesis.

[0475] The second model used primary human skeletal myoblasts (Thermo Fisher Scientific) derived from a quadriceps muscle biopsy from a human donor lot. Primary human skeletal myoblasts were plated onto type I collagen-coated vessels and subjected to a media change in differentiation medium: DMEM (high glucose, no sodium pyruvate) + 2% horse serum + 2 mM L-glutamine + 1 μM insulin + 100 units / mL penicillin and 100 μg / mL streptomycin. Primary human skeletal myoblasts were cultured in differentiation medium for 2–3 days to rapidly differentiate.

[0476] Alternatively, a third cell model, human induced pluripotent stem cell-derived myoblasts, can be used to test NARE strength in myotubes. hiPSC-derived myoblasts were plated on collagen I-coated vessels and grown in skeletal myoblast medium (AMSBIO). After reaching confluence, hiPSC-derived myoblasts underwent a medium exchange with myotube medium or myotube fusion medium (AMSBIO).

[0477] After successful differentiation of myoblasts into myotubes, cells were transduced with synthetic candidate NAREs driving the expression of fluorescent proteins, such as mClover3 and mIRFP713, via AAV. Candidate NAREs were tested alongside known constitutive and tissue-specific reference promoters, such as CAG, CMV, tMCK, and the creatine kinase 8 promoter (CK8e). Using consistent multiplicities of infection in triplicate samples, AAV candidates were diluted in differentiation medium to replace the medium containing plated adherent myotubes. After 4 hours of incubation at 37°C with 5% CO2, differentiation medium supplemented with 200 nM doxorubicin was added at the same volume as the AAV-containing medium to achieve a final concentration of 100 nM doxorubicin, a drug to improve transduction efficiency. One to two days after infection, the differentiation medium containing AAV and doxorubicin was replaced with regular differentiation medium to remove AAV and doxorubicin. The expression of fluorescent proteins in the myotubes was detected using an Agilent / BioTek Cytation 5 Cell Imaging Multimode Reader. The fluorescence intensity of the myotubes was measured as an indicator of NARE intensity, and the synthetic candidate NAREs were compared among known reference promoters.

[0478] result We developed a library of NAREs suitable for gene expression in muscle.

[0479] Several NAREs were derived from genes highly expressed in human skeletal muscle and modified with various elements to increase NARE strength. The sizes of these NAREs ranged from 450 bp to 1.6 kb. Dual reporter plasmids carrying different NARE candidates were transiently transfected into C2C12 cells. GFP expression levels in transfected (RFP+) cells were quantified by flow cytometry. Relative expression compared to the CAG promoter is shown in Figure 4.

[0480] Using nested promoter designs, we also identified additional NAREs (see Figure 2A for an example of nested promoter design and Figure 2B for the evolution of NARE M24), which is a stronger NARE than tMCK in cultured myotubes generated using C2C12 cells (Figure 2C) and primary human myotubes (Figure 2D).

[0481] NAREs were also generated by creating synthetic enhancers through transcription factor motif shuffling (Figure 5A). Most of the generated synthetic sequences were 200 bp or less (Figure 5B). Expression of a transgene encoding GFP under the control of the synthetic enhancer combined with promoter C6 in C2C12 was normalized to expression of a transgene encoding GFP under the control of a NARE alone (Figures 5C and 5D). Selected hits (MTF1291, MTF1173, MTF1186, MTF1197, MTF1209, MTF1211, MTF1242, MTF1295, MTF1304, and MTF1308) were cloned upstream of either the minCK promoter (M51, M53, M55, M57, M59, M61, M63, M65, M67, and M69) or the Ck8e promoter. (M52, M54, M56, M58, M60, M62, M64, M66, M68, M70).

[0482] The efficacy of selected NAREs in C2C12 cells is shown in Figures 5E and 5F.

[0483] The efficacy of the CK8e-derived NARE M49, which is only 616 bp compared to the parent promoter CK8e (M31), in C2C12 cells is shown in Figure 5G.

[0484] The efficacy of the minMCK-derived NARE M51 (a 366-bp NARE containing an additional enhancer compared to the parent promoter minMCK (M50)) in C2C12 cells is shown in Figure 5H. M51 is 1.5-fold more potent than the 443-bp CK8e promoter.

[0485] The potency of selected NAREs in C2C12 cells is shown in Table 1. The potency of selected NAREs in primary human myotubes is shown in Figure 5I. [Table 1]

[0486] The potency of selected human skeletal α-actin gene (ACTA1)-derived NAREs in C2C12 cells is shown in Figure 5J. The human desmin (hDes) promoter (M19), tMCK promoter (containing a triple tandem copy of the mouse MCK enhancer and mouse MCK basal promoter) (M22), CK8e (M31), hCK promoter (M71), and MHCK7 promoter (M73) served as controls. M98 and M99 are stronger than the hDes, tMCK, hCK, M24, and MHCK7 promoters. The potency of selected NAREs is summarized in Table 2. [Table 2]

[0487] The potency of selected hDes-derived NAREs in C2C12 cells is shown in Figure 5K. M111 (234 bp) is more potent than all tested muscle-specific NAREs.

[0488] The potency of selected tMCK-derived NAREs in C2C12 cells is shown in Figure 5L.

[0489] Example 4: Intensity of different NAREs in muscle cells (in vivo) Materials and Methods Intramuscular injection Male C57BL6 mice, 6-8 weeks old, were kept in standard cages with free access to water and switched from standard chow to a purified diet without alfalfa to minimize background autofluorescence. Mice were anesthetized with 2-3% isoflurane containing 1.5 LPM of oxygen. One leg of each mouse was shaved to remove all fur, cleaned with an alcohol prep pad, and a 27G needle was used to target the gastrocnemius muscle, taking care to avoid any blood vessels. Mice were allowed to recover in a clean, warm cage. Once the mouse was awake and mobile, it was returned to its home cage.

[0490] Intravenous injection Male C57BL6 mice, 6-8 weeks old, were kept in standard cages with free access to water and switched from standard chow to a purified diet without alfalfa to minimize background autofluorescence. Mice were restrained and placed under a warm light to increase vasodilation, and a 27G needle was used to target one of the tail veins. Mice were allowed to recover in a clean, warm cage and, after ensuring there was no bleeding, returned to their home cage.

[0491] Bioimaging Protocol Mice were anesthetized using 2-3% isoflurane with 1.5 LPM oxygen and imaged using a LICOR Pearl System on the white and 700 nm channels. Animals were imaged weekly for 5 weeks, then every 2 months for up to 12 months. Mice were allowed to recover in a clean, warm cage and, once awake and mobile, were returned to their home cage. Data analysis was performed using LICOR Image Studio Software. Regions of interest (ROIs) were selected for background normalization of areas of the animal that did not have fluorescence. ROIs were drawn that included the torso or both paws and normalized to the background ROI. The same region was used across animals for each time point to minimize artifacts in data collection. Absolute fluorescence was recorded for each imaging session.

[0492] eGFP / mClover3 imaging of IM injections Four weeks after injection, animals were euthanized, and gastrocnemius muscles were dissected and instilled overnight in 4% paraformaldehyde in PBS at 4°C. The following morning, the muscles were transferred to 15% sucrose in PBS and incubated overnight at 4°C. The muscles were then transferred to 30% sucrose in PBS and incubated overnight at 4°C. The muscles were then covered with OCT and allowed to equilibrate at room temperature for 1 hour in a cryo-mold. The samples were frozen on dry ice and then stored at -80°C until serially sliced ​​for histological examination using a cryostat. Serial sections, 25 μm thick, were collected at 600 μm intervals to obtain a complete representation of expression throughout the muscle. After sectioning (n=3 per group), slides were air-dried for 10 minutes to complete the mounting process. Slides were then washed three times with PBS, mounted with Prolong antifade solution containing NucBlue, and imaged on a BioTek imager to generate tile scans of all slices.

[0493] eGFP / mClover3 imaging of IV injection Four weeks after injection, animals were euthanized and perfused with PBS followed by 4% paraformaldehyde in PBS. Liver lobes were dissected and further fixed in 4% paraformaldehyde in PBS for 1 hour at room temperature. The liver lobes were then transferred to 15% sucrose in PBS and incubated overnight at 4°C. The following morning, the muscle was transferred to 30% sucrose in PBS and incubated overnight at 4°C. The liver lobes were then covered with OCT and equilibrated in a cryo-mold at room temperature for 1 hour. Samples were frozen on dry ice and then stored at -80°C until serially sliced ​​for histological examination using a cryostat. Serial sections 25µm thick were collected at 1000µm intervals to obtain a complete representation of expression throughout the liver. After sectioning n=3 per group, slides were air-dried for 10 minutes to complete the mounting process. Slides were then washed three times with PBS, mounted with Prolong antifade medium containing NucBlue, and imaged on a BioTek imager to generate tile scans of all slices.

[0494] Western blotting tissue preparation Four weeks after injection, mice were euthanized, and relevant tissues were dissected and flash-frozen in liquid nitrogen. The frozen tissues were then sectioned, and 10 mg sections were dissolved in RIPA buffer and sonicated. The lysates were spun down to remove insoluble material, and protein concentrations were determined using a BCA concentration assay. Proteins were then diluted and normalized to 1 μg / µL using 4x LDS, 10x DTT, and lysis buffer. Samples were denatured at 70-80°C for 10 minutes and stored at -20°C. Samples were then analyzed via Western blotting using a LICOR Odyssey Imager.

[0495] result AAV8-iRFP713 under the control of tMCK or M24 NARE, respectively, was intramuscularly injected into 8-week-old male C57B16 mice and imaged using LICOR Pearl (Figures 3A and 3B). iRFP713 is a near-infrared fluorescent protein. NARE M24 has 8-fold higher expression than tMCK, which was maintained up to 300 days after injection (Figure 3C).

[0496] In another experiment, AAV8-eGFP expressing the NAREs CMV, CAG, tMCK, CK8e, M21, and M24 was injected into the gastrocnemius muscle of 8-week-old male C57B16 mice (Figure 3D). Four weeks later, muscles were harvested and sectioned for measurement of native eGFP fluorescence. NAREs M21 and M24 show increased NARE intensity compared to CAG or CK8e in mouse gastrocnemius muscle (Figures 3E and 3F, Table 3). [Table 3]

[0497] A summary of NARE sequences can be found in Tables 4-6. [Table 4-1] [Table 4-2]

Table 5-1

Table 5-2

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

Table 6-38

Table 6-39

Table 6-40

Table 6-41

Table 6-42

Table 6-43

Table 6-44

Table 6-45

Table 6-46

Table 6-47

Table 6-48

Table 6-49

Table 6-50

Table 6-51

Table 6-52

Table 6-53

Table 6-54

Table 6-55

Table 6-56

Table 6-57

Table 6-58

Table 6-59

Table 6-60

Table 6-61

Table 6-62

Table 6-63

Table 6-64

Table 6-65

Table 6-66

Table 6-67

Table 6-68

Table 6-69

Table 6-70

Table 6-71

Table 6-72

Table 6-73

Table 6-74

Table 6-75

Table 6-76

Table 6-77

Table 6-78

Table 6-79

Table 6-80

Table 6-81

Table 6-82

Table 6-83

Table 6-84

Table 6-85

Table 6-86

Table 6-87

Table 6-88

Table 6-89

Table 6-90

Table 6-91

Table 6-92

Table 6-93

Table 6-94

Table 6-95

Table 6-96

Table 6-97

Table 6-98

Table 6-99

Table 6-100

Table 6-101

Table 6-102

Table 6-103

Table 6-104

Table 6-105

Table 6-106

Table 6-107

Table 6-108

Table 6-109

Table 6-110

Claims

1. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 132, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 156-163.

2. 2. The NARE of claim 1, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 156-163.

3. The NARE of claim 1, comprising: (i) SEQ ID NO: 132; and (ii) any one of SEQ ID NOs: 156-163.

4. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 164; (ii) a sequence that is at least 90% identical to SEQ ID NO: 134; (iii) a sequence that is at least 90% identical to SEQ ID NO: 135; and (vi) a sequence that is at least 90% identical to SEQ ID NO: 148 or 149.

5. 5. The NARE of claim 4, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 164; (ii) a sequence that is at least 95% identical to SEQ ID NO: 134; (iii) a sequence that is at least 95% identical to SEQ ID NO: 135; and (vi) a sequence that is at least 95% identical to SEQ ID NO: 148 or 149.

6. 5. The NARE of claim 4, comprising: (i) SEQ ID NO: 164; (ii) SEQ ID NO: 134; (iii) SEQ ID NO: 135; and (vi) SEQ ID NO: 148 or 149.

7. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 148 or 149.

8. 8. The NARE of claim 7, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 148 or 149.

9. 8. The NARE of claim 7, comprising: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 148 or 149.

10. A NARE comprising: (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 154 or 189; (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 220; and (iv) a sequence that is at least 90% identical to SEQ ID NO:

150.

11. 11. The NARE of claim 10, comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 154 or 189; (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 220; and (iv) a sequence that is at least 95% identical to SEQ ID NO:

150.

12. 11. The NARE of claim 10, comprising: (i) any one of SEQ ID NOs: 133, 142, 165, or 217; (ii) optionally, SEQ ID NO: 154 or 189; (iii) optionally, SEQ ID NO: 220; and (iv) SEQ ID NO:

150.

13. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 131; (ii) a sequence that is at least 90% identical to SEQ ID NO: 126 or 213; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO:

200.

14. 14. The NARE of claim 13, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 131; (ii) a sequence that is at least 95% identical to SEQ ID NO: 126 or 213; and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO:

200.

15. 14. The NARE of claim 13, comprising: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126 or 213; and (iii) optionally, SEQ ID NO:

200.

16. 14. The NARE of claim 13, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 131; (ii) a sequence that is at least 90% identical to SEQ ID NO: 126; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO:

200.

17. 14. The NARE of claim 13, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 131; (ii) a sequence that is at least 95% identical to SEQ ID NO: 126; and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO:

200.

18. 14. The NARE of claim 13, comprising: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126; and (iii) optionally, SEQ ID NO:

200.

19. A NARE comprising (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 190-199, and (ii) a sequence that is at least 90% identical to SEQ ID NO:

155.

20. 20. The NARE of claim 19, comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 190-199; and (ii) a sequence that is at least 95% identical to SEQ ID NO:

155.

21. 20. The NARE of claim 19, comprising: (i) any one of SEQ ID NOs: 190-199; and (ii) SEQ ID NO:

155.

22. A NARE comprising (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 190-199, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 128-130.

23. 23. The NARE of claim 22, comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 190-199; and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 128-130.

24. 23. The NARE of claim 22, comprising: (i) any one of SEQ ID NOs: 190-199; and (ii) any one of SEQ ID NOs: 128-130.

25. A NARE comprising (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 128-130, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

26. 26. The NARE of claim 25, comprising (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 128-130, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

27. 26. The NARE of claim 25, comprising: (i) any one of SEQ ID NOs: 128-130; and (ii) any one of SEQ ID NOs: 123-125, 127, 136-141, 143, 145-147, 152, 153, 209-212, 219, or 221.

28. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 121; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 122 or 154.

29. 29. The NARE of claim 28, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 121; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 122 or 154.

30. 29. The NARE of claim 28, comprising: (i) SEQ ID NO: 121; (ii) any one of SEQ ID NOs: 166-169, 171, or 172; and (iii) optionally, SEQ ID NO: 122 or 154.

31. 29. The NARE of claim 28, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 121, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 166-169, 171, or 172.

32. 29. The NARE of claim 28, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 121, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 166-169, 171, or 172.

33. 29. The NARE of claim 28, comprising: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 166-169, 171, or 172.

34. 29. The NARE of claim 28, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 122 or 154; (ii) a sequence that is at least 90% identical to SEQ ID NO: 121; and (iii) a sequence that is at least 90% identical to SEQ ID NO:

167.

35. 29. The NARE of claim 28, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 122 or 154; (ii) a sequence that is at least 95% identical to SEQ ID NO: 121; and (iii) a sequence that is at least 90% identical to SEQ ID NO:

167.

36. 29. The NARE of claim 28, comprising: (i) SEQ ID NO: 122 or 154, (ii) SEQ ID NO: 121, and (iii) SEQ ID NO:

167.

37. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 122; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 173-179, or 188; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO:

207.

38. 38. The NARE of claim 37, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 122; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 173-179, or 188; and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO:

207.

39. 38. The NARE of claim 37, comprising: (i) SEQ ID NO: 122; (ii) any one of SEQ ID NOs: 173-179, or 188; and (iii) optionally, SEQ ID NO:

207.

40. 38. The NARE of claim 37, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 122, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 173-179.

41. 38. The NARE of claim 37, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 122, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 173-179.

42. 38. The NARE of claim 37, comprising: (i) SEQ ID NO: 122; and (ii) any one of SEQ ID NOs: 173-179.

43. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 185-187.

44. 44. The NARE of claim 43, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 185-187.

45. 44. The NARE of claim 43, comprising (i) SEQ ID NO: 184, and (ii) any one of SEQ ID NOs: 185-187.

46. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 180, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 181 or 182.

47. 47. The NARE of claim 46, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 180, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 181 or 182.

48. 47. The NARE of claim 46, comprising: (i) SEQ ID NO: 180; and (ii) any one of SEQ ID NOs: 181 or 182.

49. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 183; (ii) a sequence that is at least 90% identical to SEQ ID NO: 182; and (iii) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

50. 50. The NARE of claim 49, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 183; (ii) a sequence that is at least 95% identical to SEQ ID NO: 182; and (iii) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

51. 50. The NARE of claim 49, comprising: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) optionally, any one of SEQ ID NOs: 200, 201, 205, or 208.

52. 50. The NARE of claim 49, comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 183; (ii) a sequence that is at least 90% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

53. 50. The NARE of claim 49, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 183; (ii) a sequence that is at least 95% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 200, 201, 205, or 208.

54. 50. The NARE of claim 49, comprising: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) any one of SEQ ID NOs: 200, 201, 205, or 208.

55. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 154 or 164, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 128-130.

56. 56. The NARE of claim 55, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 154 or 164, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 128-130.

57. 56. The NARE of claim 55, comprising: (i) SEQ ID NO: 154 or 164; and (ii) any one of SEQ ID NOs: 128-130.

58. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 170; (ii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 188; and (iii) a sequence that is at least 90% identical to SEQ ID NO: 203 or 204.

59. 59. The NARE of claim 58, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 170; (ii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 188; and (iii) a sequence that is at least 95% identical to SEQ ID NO: 203 or 204.

60. 59. The NARE of claim 58, comprising: (i) SEQ ID NO: 170; (ii) optionally SEQ ID NO: 188; and (iii) SEQ ID NO: 203 or 204.

61. (a) (i) a sequence that is at least 90% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 149; (b) (i) a sequence at least 90% identical to SEQ ID NO: 164, (ii) a sequence at least 90% identical to SEQ ID NO: 134, (iii) a sequence at least 90% identical to SEQ ID NO: 135, and (vi) SEQ ID NO: 149; (c) (i) a sequence that is at least 90% identical to SEQ ID NO: 165, (ii) a sequence that is at least 90% identical to SEQ ID NO: 154, (iii) a sequence that is at least 90% identical to SEQ ID NO: 220, and (iv) a sequence that is at least 90% identical to SEQ ID NO: 150; (d) (i) a sequence that is at least 90% identical to SEQ ID NO: 142, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 150; (e) (i) a sequence that is at least 90% identical to SEQ ID NO: 217, (ii) a sequence that is at least 90% identical to SEQ ID NO: 189, and (iii) a sequence that is at least 90% identical to SEQ ID NO: 150; (f) (i) a sequence that is at least 90% identical to SEQ ID NO: 196, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 155; (g) (i) a sequence at least 90% identical to SEQ ID NO: 194, and (ii) a sequence at least 90% identical to SEQ ID NO: 155, or (h) A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 90% identical to SEQ ID NO:

187.

62. (a) (i) a sequence that is at least 95% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 149; (b) (i) a sequence at least 95% identical to SEQ ID NO: 164, (ii) a sequence at least 95% identical to SEQ ID NO: 134, (iii) a sequence at least 95% identical to SEQ ID NO: 135, and (vi) SEQ ID NO: 149; (c) (i) a sequence that is at least 95% identical to SEQ ID NO: 165, (ii) a sequence that is at least 95% identical to SEQ ID NO: 154, (iii) a sequence that is at least 95% identical to SEQ ID NO: 220, and (iv) a sequence that is at least 95% identical to SEQ ID NO: 150; (d) (i) a sequence that is at least 95% identical to SEQ ID NO: 142, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 150; (e) (i) a sequence that is at least 95% identical to SEQ ID NO: 217, (ii) a sequence that is at least 95% identical to SEQ ID NO: 189, and (iii) a sequence that is at least 95% identical to SEQ ID NO: 150; (f) (i) a sequence that is at least 95% identical to SEQ ID NO: 196, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 155; (g) (i) a sequence that is at least 95% identical to SEQ ID NO: 194, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 155, or (h) The NARE of claim 61, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 184, and (ii) a sequence that is at least 95% identical to SEQ ID NO:

187.

63. (a) (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 149; (b) (i) SEQ ID NO: 164, (ii) SEQ ID NO: 134, (iii) SEQ ID NO: 135, and (vi) SEQ ID NO: 149; (c) (i) SEQ ID NO: 165, (ii) SEQ ID NO: 154, (iii) SEQ ID NO: 220, and (iv) SEQ ID NO: 150; (d) (i) SEQ ID NO: 142, and (ii) SEQ ID NO: 150; (e) (i) SEQ ID NO: 217, (ii) SEQ ID NO: 189, and (iii) SEQ ID NO: 150; (f) (i) SEQ ID NO: 196, and (ii) SEQ ID NO: 155; (g) (i) SEQ ID NO: 194, and (ii) SEQ ID NO: 155, or (h) The NARE of claim 61 comprising (i) SEQ ID NO: 184, and (ii) SEQ ID NO:

187.

64. A NARE comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, or 74-120.

65. 65. The NARE of claim 64, comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, or 74-120.

66. 65. The NARE of claim 64, comprising any one of SEQ ID NOs: 1-18, 20, 21, 23, 24, 32-49, 51-70, or 74-120.

67. A NARE comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 1-18 or 32.

68. 68. The NARE of claim 67, comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 1-18 or 32.

69. 68. The NARE of claim 67, comprising any one of SEQ ID NOs: 1-18 or 32.

70. An expression construct comprising a NARE according to any one of the preceding claims and an operably linked transgene.

71. 71. The expression construct of claim 70, further comprising a polyadenylation sequence.

72. A vector comprising the expression construct of claim 70.

73. 73. The vector of claim 72, wherein the vector is a non-viral vector.

74. 73. The vector of claim 72, wherein the vector is a viral vector.

75. 75. The vector of claim 74, wherein the vector is an adeno-associated virus (AAV) vector.

76. 71. A vector comprising a nucleic acid sequence comprising: (i) an expression construct according to 70; and (ii) one or more inverted terminal repeats (ITRs).

77. 77. The vector of claim 76, wherein the nucleic acid sequence comprises a 5' ITR and a 3' ITR.

78. 77. The vector of claim 76, wherein the 5' ITR and 3' ITR are derived from adeno-associated virus (AAV) serotype AAV2.

79. A cell comprising an expression construct according to claim 70 or a vector according to any one of claims 72 to 78.

80. 80. The cell of claim 79, wherein the cell is a muscle cell.

81. 79. A pharmaceutical composition comprising: (i) the expression construct of claim 70 or the vector of any one of claims 72 to 78; and (ii) a pharmaceutically acceptable excipient.

82. A method for expressing a transgene in a cell comprising an expression construct according to claim 70 or a vector according to any one of claims 72 to 78.

83. A method for regulating transgene expression in a cell comprising an expression construct according to claim 70 or a vector according to any one of claims 72 to 78.

84. 84. The method of claim 82 or 83, wherein the cell is a muscle cell.

Images