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

Abstract

The present application relates to nucleic acid regulatory elements capable of enhancing constitutive expression of a gene in a variety of tissues or in particular tissues including muscles. The present application also relates to methods employing 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

[0001] Cross - Reference to Related Applications

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

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (SeqList - 162027.54176.xml; size: 290,976 bytes; and creation date: October 10, 2023) are hereby incorporated by reference in their entirety. Technical Field

[0005] This application relates to nucleic acid regulatory elements that are capable of enhancing gene expression in a variety of tissues and particularly in tissues including muscle. This application also relates to methods of using these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. These are particularly useful for applications using gene therapy. Background Art

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

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

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

[0009] Provided herein are nucleic acid regulatory elements (NAREs), including NAREs particularly suitable for expressing an operably linked sequence (e.g., a protein or RNA coding sequence) 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.

[0010] 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 Table 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 Table 6. In embodiments, a polynucleotide is provided that comprises a NARE provided in Table 4 or Table 6.

[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 M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120 (as listed in Table 6). 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 M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120. In embodiments, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120.

[0012] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of: M21, M23 - M28, M32, M34 - M36, M51, M53, M55, M57, M59, M61, and M63 (listed in Table 6). In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of: M21, M23 - M28, M32, M34 - M36, M51, M53, M55, M57, M59, M61, and M63. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M21, M23 - M28, M32, M34 - M36, M51, M53, M55, M57, M59, M61, and M63.

[0013] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of: M21 and M24. In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of: M21 and M24. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M21 and M24.

[0014] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of: M21, M24 - M27, M30, and M32 - M36 (listed in Table 6). In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of: M21, M24 - M27, M30, and M32 - M36. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M21, M24 - M27, M30, and M32 - M36.

[0015] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80% identical to a NARE selected from the group consisting of: M49, M51, M61, M98, M99, and M111 - M115 (listed in Table 6). In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 90% identical to a NARE selected from the group consisting of: M49, M51, M61, M98, M99, and M111 - M115. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M49, M51, M61, M98, M99, and M111 - M115.

[0016] In an embodiment, one or more of the NAREs disclosed herein are operably linked to a transgene.

[0017] In one aspect, a NARE is provided that comprises: (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.

[0018] 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.

[0019] On the one hand, there is provided a NARE, which 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, there is provided a NARE, which 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, there is provided a NARE, which comprises: (i) SEQ ID NO: 164; (ii) SEQ ID NO: 134; (iii) SEQ ID NO: 135; and (vi) SEQ ID NO: 148 or 149.

[0020] On the one hand, there is provided a NARE, which comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NO: 218; and (ii) a sequence that is at least 90% identical to any one of SEQ ID NO: 148 or 149. In some embodiments, there is provided a NARE, which 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 NO: 148 or 149. In some embodiments, there is provided a NARE, which comprises: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 148 or 149.

[0021] On the one hand, there is provided a NARE, which comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NO: 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.

[0022] In some embodiments, a NARE is provided, the NARE comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NO: 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.

[0023] In some embodiments, a NARE is provided, the NARE comprising: (i) any one of SEQ ID NO: 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.

[0024] In one aspect, a NARE is provided, the 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.

[0025] In some embodiments, a NARE is provided, the NARE 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.

[0026] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126 or 213; and (iii) optionally, SEQ ID NO: 200.

[0027] In some embodiments, a NARE is provided, the 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; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 200.

[0028] In some embodiments, provided is a NARE 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.

[0029] In some embodiments, provided is a NARE comprising: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126; and (iii) optionally, SEQ ID NO: 200.

[0030] In one aspect, provided is 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.

[0031] In some embodiments, provided is a NARE 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.

[0032] In some embodiments, provided is a NARE comprising: (i) any one of SEQ ID NOs: 190 - 199; and (ii) SEQ ID NO: 155.

[0033] In one aspect, provided is 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.

[0034] In some embodiments, provided is a NARE 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.

[0035] In some embodiments, provided is a NARE comprising: (i) any one of SEQ ID NOs: 190 - 199; and (ii) any one of SEQ ID NOs: 128 - 130.

[0036] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID 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.

[0037] In some embodiments, a NARE is provided, which 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.

[0038] In some embodiments, a NARE is provided, which 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.

[0039] On the one hand, a NARE is provided, which comprises: (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.

[0040] In some embodiments, a NARE is provided, which 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.

[0041] In some embodiments, a NARE is provided, which 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.

[0042] In some embodiments, a NARE is provided, the NARE 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.

[0043] In some embodiments, a NARE is provided, the NARE 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.

[0044] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 121; and (ii) SEQ ID NOs: 166-169, 171, or 172.

[0045] In some embodiments, a NARE is provided, the NARE 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.

[0046] In some embodiments, a NARE is provided, the NARE 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.

[0047] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 122 or 154; (ii) SEQ ID NO: 121; and (iii) SEQ ID NO: 167.

[0048] In one aspect, a NARE is provided, the 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.

[0049] In some embodiments, a NARE is provided, the NARE 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.

[0050] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 122; (ii) any one of SEQ ID NOs: 173 - 179 or 188; and (iii) optionally, SEQ ID NO: 207.

[0051] In some embodiments, a NARE is provided, the NARE 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.

[0052] In some embodiments, a NARE is provided, the NARE 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.

[0053] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 122; and (ii) any one of SEQ ID NOs: 173 - 179.

[0054] In one aspect, a NARE is provided, the 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.

[0055] In some embodiments, a NARE is provided, the NARE 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.

[0056] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 184; and (ii) any one of SEQ ID NOs: 185 - 187.

[0057] On the one hand, a NARE is provided, which comprises: (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 either SEQ ID NO: 181 or 182.

[0058] In some embodiments, a NARE is provided, which 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 either SEQ ID NO: 181 or 182.

[0059] In some embodiments, a NARE is provided, which comprises: (i) SEQ ID NO: 180; and (ii) either SEQ ID NO: 181 or 182.

[0060] On the one hand, a NARE is provided, which 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 NO: 200, 201, 205 or 208.

[0061] In some embodiments, a NARE is provided, which 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 NO: 200, 201, 205 or 208.

[0062] In some embodiments, a NARE is provided, which comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) optionally, any one of SEQ ID NO: 200, 201, 205 or 208.

[0063] In some embodiments, a NARE is provided, which 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 NO: 200, 201, 205 or 208.

[0064] In some embodiments, a NARE is provided, the NARE 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.

[0065] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) any one of SEQ ID NOs: 200, 201, 205, or 208.

[0066] In one aspect, a NARE is provided, the 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.

[0067] In some embodiments, a NARE is provided, the NARE 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.

[0068] In some embodiments, a NARE is provided, the NARE comprising: (i) SEQ ID NO: 154 or 164; and (ii) any one of SEQ ID NOs: 128 - 130.

[0069] In one aspect, a NARE is provided, the 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.

[0070] In some embodiments, a NARE is provided, the NARE 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.

[0071] In some embodiments, there is provided a NARE comprising: (i) SEQ ID NO: 170; (ii) optionally, SEQ ID NO: 188; and (iii) SEQ ID NO: 203 or 204.

[0072] In one aspect, there is provided a NARE comprising:

[0073] (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;

[0074] (b) (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) SEQ ID NO: 149;

[0075] (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;

[0076] (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;

[0077] (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;

[0078] (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;

[0079] (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

[0080] (h) (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.

[0081] In some embodiments, there is provided a NARE comprising:

[0082] (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;

[0083] (b) (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) SEQ ID NO: 149;

[0084] (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;

[0085] (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;

[0086] (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;

[0087] (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;

[0088] (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

[0089] (h) (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.

[0090] In some embodiments, there is provided a NARE comprising:

[0091] (a) (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 149;

[0092] (b) SEQ ID NO: 164; (ii) SEQ ID NO: 134; (iii) SEQ ID NO: 135; and (vi) SEQ ID NO: 149;

[0093] (c) (i) SEQ ID NO: 165; (ii) SEQ ID NO: 154; (iii) SEQ ID NO: 220; and (iv) SEQ ID NO: 150;

[0094] (d) (i) SEQ ID NO: 142; and (ii) SEQ ID NO: 150;

[0095] (e) (i) SEQ ID NO: 217; (ii) SEQ ID NO: 189; and (iii) SEQ ID NO: 150;

[0096] (f) (i) SEQ ID NO: 196; and (ii) SEQ ID NO: 155;

[0097] (g) (i) SEQ ID NO: 194; and (ii) SEQ ID NO: 155; or

[0098] (h) (i) SEQ ID NO: 184; and (ii) SEQ ID NO: 187.

[0099] On the one hand, a NARE is provided, which comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70 or 74 - 120. In some embodiments, a NARE is provided, which comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70 or 74 - 120. In some embodiments, a NARE is provided, which comprises any one of SEQ ID NO: 1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70 or 74 - 120. In some embodiments, a NARE is provided, which comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 1 - 18 or 32. In some embodiments, a NARE is provided, which comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 1 - 18 or 32. In some embodiments, a NARE is provided, which comprises any one of SEQ ID NO: 1 - 18 or 32.

[0100] On the one hand, an expression construct is provided, which comprises a NARE disclosed herein and an operably linked transgene. In one embodiment, the expression construct further comprises a polyadenylation sequence.

[0101] On the one hand, a vector is provided, which comprises the expression construct disclosed herein. In one embodiment, the vector is a non-viral vector. In one embodiment, the vector is a viral vector. In one embodiment, the vector is an adeno-associated virus (AAV) vector. In one embodiment, the vector comprises a nucleic acid sequence, which comprises (i) the 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.

[0102] On the one hand, a cell is provided, which comprises the expression construct or the vector disclosed herein. In some embodiments, the cell is a muscle cell.

[0103] On the one hand, a pharmaceutical composition is provided, which comprises (i) the expression construct disclosed herein or the vector disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0104] On the one hand, a method for expressing a transgene in a cell comprising the expression construct or the vector disclosed herein is provided. On the one hand, a method for regulating the expression of a transgene in a cell comprising the expression construct or the vector disclosed herein is provided. In some embodiments, the cell is a muscle cell. Description of the Drawings

[0105] Figure 1A and Figure 1B . Dual reporter gene design and measurement. Figure 1a illustrates the design of the dual reporter gene construct. The candidate NARE was cloned upstream (5′) of the mClover3 coding sequence. Each construct contains a constant region, which includes a tdTomato transgene used as a normalization control. Figure 1B Examples of flow cytometry data obtained with the dual reporter gene system are provided, wherein the mClover3 expression in tdTomato+ cells reflects the level of NARE activity.

[0106] Figure 2A 、 Figure 2B 、 Figure 2C and Figure 2D . Generation of a NARE that is highly suitable for gene expression in muscle.Figure 2A : Example of 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 : Evolution of the strong NARE M24, which increases NARE strength up to 7-fold compared to NARE M22 in C2C12 cells transfected in vitro. Figure 2C : NARE M24 is more potent than the reference promoter tMCK in cultured C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes and transduced with AAV8 at 10k MOI. Fluorescence of the iRFP713 reporter was measured on the indicated days. Figure 2D : NARE M24 is stronger than the reference tMCK promoter and shows persistent expression in primary human myotubes. Primary human myoblasts were differentiated into myotubes and transduced with AAV8 at 150k MOI.

[0107] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E and Figure 3F .In vivo expression of NAREs M21 and M24. Figure 3A : AAV8-iRFP713 containing the tMCK promoter or the synthetic M24 NARE was injected intramuscularly into 8-week-old male C57Bl6 mice and imaged using LICOR Pearl. Figure 3B : Representative imaging data showing AAV8-tMCK and AAV8-M24 in non-injected and injected hindlimbs after 7, 10, or 14 days, respectively. Figure 3C : Fluorescence quantification showing that NARE M24 (top line) induced 8-fold higher expression than the reference promoter tMCK (bottom line) (expression was maintained up to 300 days after injection). Figure 3D : AAV8-eGFP was injected into the gastrocnemius muscle of 8-week-old male C57Bl6 mice. After 4 weeks, the muscles were harvested and sectioned to measure native eGFP fluorescence. Figure 3E and Figure 3F : Quantitative ( Figure 3E ) and representative images ( Figure 3F ), scale bar is 100 μm. In mouse gastrocnemius muscle, NAREs M21 and M24 are more potent than the reference promoters CAG or CK8e.

[0108] Figure 4. Potency of selected NAREs in muscle cells. A library of NAREs was generated that contained NAREs from genes highly expressed in human skeletal muscle, and the NAREs were modified with various elements to increase NARE strength. The NAREs were sized in the range of 450 bp to 1.6 kb. Dual reporter gene plasmids with different NARE candidates were transiently transfected into C2C12 cells. The GFP expression level in transfected (RFP+) cells was quantified by flow cytometry.

[0109] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E , Figure 5F , Figure 5G , Figure 5H , Figure 5I , Figure 5J , Figure 5K , Figure 5L , Figures 5M, 5N, 5O, and 5P. Potency of selected NAREs in muscle cells. Figures 9A, 9B, and 9C: Synthetic enhancers were created by shuffling transcription factor (TF) motifs to increase expression in muscle. Figure 5A : Diagram of creating an enhancer located upstream of the 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, CAAT box, AP-1, AP-2) were randomly ligated and inserted upstream of the minimal promoter (C6) and the mClover3 transgene to test enhancer function. Figure 5B : Representative sequencing showed that most synthetic sequences were 200 bp or shorter. Figure 5C : In co-transfected C2C12 cells, the relative expression of synthetic enhancers coupled to promoter C6 was normalized against the parental NARE. In mouse C2C12 muscle cells, the synthetic fragments increased the expression of the minimal NARE by up to 8-fold. Figure 5D : Representative images of NAREs with particularly good performance identified by screening (right) compared to promoter C6 (left) in transfected C2C12 cells. Figure 5E : The synthetic fragments identified above can be used in combination with other NAREs to generate effective and novel NAREs suitable for expression in muscle. The expression of these NAREs in transfected C2C12 cells was measured using a dual reporter assay. Figure 5F : Potency of NARE M51 compared to selected reference promoters in transfected C2C12 cells. Figure 5G: Potency of CK8e-derived NARE M49 in transfected C2C12 cells. Figure 5H : Potency of minMCK-derived NARE M51 in transfected C2C12 cells. Figure 5I : Transduce primary human myotubes with AAV8-miRFP713-WPRE-SV40polyA containing the indicated NARE. Figure 5J : Potency of ACTA1-derived NARE compared to the indicated reference promoter using a dual reporter assay in C2C12 cells. Figure 5K : Potency of hDes-derived NARE compared to the indicated reference promoter using a dual reporter assay in C2C12 cells. Figure 5L : Potency of tMCK-derived NARE compared to the indicated reference promoter using a dual reporter assay in C2C12 myoblasts. Detailed Description

[0110] Nucleic acid regulatory elements (NAREs), including promoters, are essential components of gene therapy, controlling the expression level and expression persistence of therapeutic genes. NAREs can drive cell-specific expression of transgenes independently of, for example, capsid selection. Incorporating stronger NAREs can increase potency and efficacy at lower viral vector doses, potentially reducing safety risks, immune responses, and vector production costs. Additionally, reducing the NARE size while maintaining strength and specificity allows for efficient packaging of larger transgenes or expression cassettes into AAVs. This application provides a library of nucleic acid regulatory elements to improve the safety, efficacy, and persistence of therapeutic transgene expression.

[0111] Although multiple NAREs and particularly promoter sequences have been characterized / optimized through conventional low-throughput analysis (rational design) or newer high-throughput methods such as MPRA, these methods still require expensive and time-consuming in vitro optimization. Here, using an advanced artificial intelligence model, a convolutional neural network (CNN) was repurposed and optimized to predict 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 effective, compact constitutive promoters. Subsequently, the correct spacing between enhancer elements that performed particularly well and promoter sequences was optimized. At the same time, in silico saturation mutagenesis was performed, introducing all possible point mutations within the NARE sequence and selecting those that had the greatest impact on the potency of the NARE. Particularly well-performing NAREs were generated and tested in vitro. While most nucleic acid regulatory elements showed better or equivalent performance compared to the original promoter, the best enhancer elements and point mutations were selected and incorporated in a second round of optimization. After synthesizing and testing this new set in vitro, enhanced nucleic acid regulatory elements were obtained that exhibited increased potency.

[0112] NARE

[0113] As provided herein, NARE may include a promoter and / or enhancer as part of its sequence. Traditionally, a promoter is defined as the DNA region that initiates transcription. A promoter includes specific DNA motifs to which transcription factors (TFs) and their complexes can access. On the other hand, an enhancer is defined as the DNA region that amplifies transcription initiation by directly interacting with the target promoter. Similarly, enhancer sequences far from the target promoter contain DNA motifs that serve as binding sites for TFs and cofactors. Sometimes, the term promoter can be used as a shorthand to refer to a nucleic acid sequence containing multiple regulatory elements. For example, CAG contains the CMV enhancer, the β-actin promoter region, and an intron, but can also be referred to as a promoter. Specifically, the CAG promoter consists of: (1) the cytomegalovirus (CMV) early enhancer element, (2) the promoter, the first exon, and the first intron of the chicken β-actin gene, and (3) the splice acceptor of the rabbit β-globin gene.

[0114] As used herein, the term "NARE" can refer to a promoter as defined in the traditional sense, as well as a combination of nucleic acid regulatory elements that includes a promoter and / or other nucleic acid regulatory elements that regulate the expression of a gene operably linked to such elements. The nucleic acid regulatory elements can be or include, for example, one or more of a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0115] As used herein, a "transgene" refers to, for example, a gene (specifically, the coding sequence of such gene) transferred into one or more cells of an organism using a vector as described herein. A transgene can encode a protein or RNA that is normally expressed in the cells of the target organism, or can encode a protein or RNA from a different organism. A transgene can be integrated into the genome of the target cell, or can exist as part of an extrachromosomal expression construct.

[0116] As used herein, "operatively linked" means that a first molecule is joined to a second molecule, where the molecules are arranged such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. By way of example, if a NARE regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, the NARE is operatively linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are joined such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion, they are operatively linked to each other. Two transcriptional regulatory elements can be operatively linked to each other by a linker nucleic acid (e.g., intervening non-coding nucleic acid) or can be operatively linked to each other in the absence of intervening nucleotides.

[0117] The present disclosure provides engineered regulatory elements that can drive the expression of transgenes in a variety of different tissues including muscle. The NARE engineering platform described herein generates a number of NAREs that can be used to drive the expression of a variety of therapeutic transgenes.

[0118] The present disclosure provides NAREs that are particularly suitable for driving expression in muscle. As used herein, NAREs starting with "M" are particularly useful for the expression of genes in muscle. Muscle NAREs can be muscle-specific, meaning that they show a significant increase or preferential expression of an operatively linked transgene in muscle compared to other tissues.

[0119] Tables 4 and 6 provide NAREs that are particularly suitable for driving expression in muscle.

[0120] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the NAREs provided in Tables 4 and 6. In an embodiment, a polynucleotide is provided that comprises one or more of the NAREs provided in Tables 4 and 6.

[0121] As used herein, the term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each of the sequences that are aligned for purposes of comparison. For example, when a position in the compared nucleotide sequences is occupied by the same base, then the molecules are identical at that position. The degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. Methods and computer programs for determining sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990) and the ALIGN program (version 2.0). The well-known Smith-Waterman algorithm can also be used to determine similarity. The BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0, http: / / www.ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions, and other modifications when comparing sequences.

[0122] 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: M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120 (listed in Table 6). In embodiments, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M1-M18, M20, M21, M23, M24, M32-M49, M51-M70, and M74-M120.

[0123] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of: M21, M23-M28, M32, M34-M36, M51, M53, M55, M57, M59, M61, and M63 (listed in Table 6). In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M21, M23-M28, M32, M34-M36, M51, M53, M55, M57, M59, M61, and M63.

[0124] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of: M21 and M24. In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M21 and M24.

[0125] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of: M21, M24-M27, M30, and M32-M36 (listed in Table 6). In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of: M21, M24-M27, M30, and M32-M36.

[0126] In an embodiment, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of: M49, M51, M61, M98, M99, and M111-M115 (listed in Table 6).

[0127] In an embodiment, a polynucleotide sequence is provided that comprises a NARE selected from the group consisting of M49, M51, M61, M98, M99, and M111 - M115.

[0128] Provided herein is a method for expressing a transgene in a muscle cell, wherein the transgene is operably linked to a NARE that is particularly suitable for driving expression in muscle and is disclosed herein.

[0129] 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 the NARE provided herein. Provided herein is a method for expressing a transgene in a muscle cell, wherein the transgene is operably linked to the NARE provided herein.

[0130] 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 selected from the group consisting of M1 - M18, M20, M21, M23, M24, M32 - M49, M51 - M70, and M74 - M120. Provided herein is a method 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.

[0131] In some embodiments, the NARE comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70, and 74 - 120.

[0132] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NO:1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70, and 74 - 120.

[0133] In some embodiments, the NARE comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-18 and 32.

[0134] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NOs: 1-18 and 32.

[0135] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 134; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135; and (vi) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 148 or 149.

[0136] 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.

[0137] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 134; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135; and (vi) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 148.

[0138] 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.

[0139] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 134; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135; and (vi) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 149.

[0140] 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.

[0141] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 218; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 148 or 149.

[0142] In some embodiments, the NARE comprises: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 148 or 149.

[0143] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 218; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 148.

[0144] In some embodiments, the NARE comprises: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 148.

[0145] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 218; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 149.

[0146] In some embodiments, the NARE comprises: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 149.

[0147] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 133, 142, 165 or 217; (ii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 154 or 189; (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 220; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 150.

[0148] In some embodiments, the NARE comprises: (i) any one of SEQ ID NO: 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.

[0149] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 133; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 150.

[0150] In some embodiments, the NARE comprises: (i) SEQ ID NO: 133; and (ii) SEQ ID NO: 150.

[0151] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 165; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 154; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 220; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 150.

[0152] 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.

[0153] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 142; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 150.

[0154] In some embodiments, the NARE comprises: (i) SEQ ID NO: 142; and (ii) SEQ ID NO: 150.

[0155] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 217; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 189; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 150.

[0156] In some embodiments, the NARE comprises: (i) SEQ ID NO: 217; (ii) SEQ ID NO: 189; and (iii) SEQ ID NO: 150.

[0157] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 156 - 163.

[0158] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) any one of SEQ ID NOs: 156 - 163.

[0159] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 156.

[0160] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 156.

[0161] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 157.

[0162] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 158.

[0163] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 158.

[0164] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 159.

[0165] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 159.

[0166] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 160.

[0167] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 160.

[0168] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 161.

[0169] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 161.

[0170] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162.

[0171] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 162.

[0172] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 132; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 163.

[0173] In some embodiments, the NARE comprises: (i) SEQ ID NO: 132; and (ii) SEQ ID NO: 163.

[0174] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 131; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126 or 213; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 200.

[0175] 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.

[0176] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 131; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 200.

[0177] In some embodiments, the NARE comprises: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126; and (iii) optionally, SEQ ID NO: 200.

[0178] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 131; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 126.

[0179] In some embodiments, the NARE comprises: (i) SEQ ID NO: 131; and (ii) SEQ ID NO: 126.

[0180] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 131; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 126; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 200.

[0181] In some embodiments, the NARE comprises: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126; and (iii) SEQ ID NO: 200.

[0182] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 131; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 213; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 200.

[0183] In some embodiments, the NARE comprises: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 213; and (iii) SEQ ID NO: 200.

[0184] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 190 - 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0185] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 190 - 199; and (ii) SEQ ID NO: 155.

[0186] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 196; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0187] In some embodiments, the NARE comprises: (i) SEQ ID NO: 196; and (ii) SEQ ID NO: 155.

[0188] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0189] In some embodiments, the NARE comprises: (i) SEQ ID NO: 190; and (ii) SEQ ID NO: 155.

[0190] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 191; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0191] In some embodiments, the NARE comprises: (i) SEQ ID NO: 191; and (ii) SEQ ID NO: 155.

[0192] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 192; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0193] In some embodiments, the NARE comprises: (i) SEQ ID NO: 192; and (ii) SEQ ID NO: 155.

[0194] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0195] In some embodiments, the NARE comprises: (i) SEQ ID NO: 193; and (ii) SEQ ID NO: 155.

[0196] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 194; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0197] In some embodiments, the NARE comprises: (i) SEQ ID NO: 194; and (ii) SEQ ID NO: 155.

[0198] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 195; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0199] In some embodiments, the NARE comprises: (i) SEQ ID NO: 195; and (ii) SEQ ID NO: 155.

[0200] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0201] In some embodiments, the NARE comprises: (i) SEQ ID NO: 197; and (ii) SEQ ID NO: 155.

[0202] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 198; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0203] In some embodiments, the NARE comprises: (i) SEQ ID NO: 198; and (ii) SEQ ID NO: 155.

[0204] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0205] In some embodiments, the NARE comprises: (i) SEQ ID NO: 199; and (ii) SEQ ID NO: 155.

[0206] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 190 - 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0207] 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.

[0208] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 196; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0209] In some embodiments, the NARE comprises: (i) SEQ ID NO: 196; and (ii) any one of SEQ ID NOs: 128 - 130.

[0210] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0211] In some embodiments, the NARE comprises: (i) SEQ ID NO: 190; and (ii) any one of SEQ ID NOs: 128 - 130.

[0212] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 191; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0213] In some embodiments, the NARE comprises: (i) SEQ ID NO: 191; and (ii) any one of SEQ ID NOs: 128 - 130.

[0214] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 192; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 128-130.

[0215] In some embodiments, the NARE comprises: (i) SEQ ID NO: 192; and (ii) any one of SEQ ID NOs: 128-130.

[0216] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 193; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 128-130.

[0217] In some embodiments, the NARE comprises: (i) SEQ ID NO: 193; and (ii) any one of SEQ ID NOs: 128-130.

[0218] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 194; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 128-130.

[0219] In some embodiments, the NARE comprises: (i) SEQ ID NO: 194; and (ii) any one of SEQ ID NOs: 128-130.

[0220] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 195; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0221] In some embodiments, the NARE comprises: (i) SEQ ID NO: 195; and (ii) any one of SEQ ID NOs: 128 - 130.

[0222] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0223] In some embodiments, the NARE comprises: (i) SEQ ID NO: 197; and (ii) any one of SEQ ID NOs: 128 - 130.

[0224] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 198; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0225] In some embodiments, the NARE comprises: (i) SEQ ID NO: 198; and (ii) any one of SEQ ID NOs: 128 - 130.

[0226] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0227] In some embodiments, the NARE comprises: (i) SEQ ID NO: 199; and (ii) any one of SEQ ID NOs: 128 - 130.

[0228] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 190 - 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0229] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 190 - 199; and (ii) SEQ ID NO: 130.

[0230] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 196; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0231] In some embodiments, the NARE comprises: (i) SEQ ID NO: 196; and (ii) SEQ ID NO: 130.

[0232] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0233] In some embodiments, the NARE comprises: (i) SEQ ID NO: 190; and (ii) SEQ ID NO: 130.

[0234] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 191; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0235] In some embodiments, the NARE comprises: (i) SEQ ID NO: 191; and (ii) SEQ ID NO: 130.

[0236] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 192; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0237] In some embodiments, the NARE comprises: (i) SEQ ID NO: 192; and (ii) SEQ ID NO: 130.

[0238] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0239] In some embodiments, the NARE comprises: (i) SEQ ID NO: 193; and (ii) SEQ ID NO: 130.

[0240] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 194; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0241] In some embodiments, the NARE comprises: (i) SEQ ID NO: 194; and (ii) SEQ ID NO: 130.

[0242] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 195; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0243] In some embodiments, the NARE comprises: (i) SEQ ID NO: 195; and (ii) SEQ ID NO: 130.

[0244] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0245] In some embodiments, the NARE comprises: (i) SEQ ID NO: 197; and (ii) SEQ ID NO: 130.

[0246] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 198; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0247] In some embodiments, the NARE comprises: (i) SEQ ID NO: 198; and (ii) SEQ ID NO: 130.

[0248] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0249] In some embodiments, the NARE comprises: (i) SEQ ID NO: 199; and (ii) SEQ ID NO: 130.

[0250] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 123 - 125, 127, 136 - 141, 143, 145 - 147, 152, 153, 209 - 212, 219, and 221.

[0251] 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.

[0252] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 137.

[0253] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 137.

[0254] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 138.

[0255] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 138.

[0256] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 219.

[0257] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 219.

[0258] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 212.

[0259] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 212.

[0260] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 211.

[0261] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 211.

[0262] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 143.

[0263] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 143.

[0264] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 221.

[0265] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 221.

[0266] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 123.

[0267] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 123.

[0268] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 127.

[0269] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 127.

[0270] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 139.

[0271] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 139.

[0272] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 136.

[0273] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 136.

[0274] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 141.

[0275] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 141.

[0276] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 209.

[0277] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 209.

[0278] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 124.

[0279] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 124.

[0280] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125.

[0281] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 125.

[0282] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 152.

[0283] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 152.

[0284] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 140.

[0285] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 140.

[0286] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 145.

[0287] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 145.

[0288] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 146.

[0289] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 146.

[0290] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 147.

[0291] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 147.

[0292] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153.

[0293] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 153.

[0294] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210.

[0295] In some embodiments, the NARE comprises: (i) any one of SEQ ID NOs: 128 - 130; and (ii) SEQ ID NO: 210.

[0296] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 123 - 125, 127, 136 - 141, 143, 145 - 147, 152, 153, 209 - 212, 219, or 221.

[0297] 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.

[0298] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:137.

[0299] In some embodiments, the NARE comprises: (i) SEQ ID NO:128; and (ii) SEQ ID NO:137.

[0300] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:138.

[0301] In some embodiments, the NARE comprises: (i) SEQ ID NO:128; and (ii) SEQ ID NO:138.

[0302] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:219.

[0303] In some embodiments, the NARE comprises: (i) SEQ ID NO:128; and (ii) SEQ ID NO:219.

[0304] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 212.

[0305] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 212.

[0306] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211.

[0307] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 211.

[0308] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 143.

[0309] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 143.

[0310] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 221.

[0311] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 221.

[0312] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 123.

[0313] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 123.

[0314] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 127.

[0315] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 127.

[0316] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 139.

[0317] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 139.

[0318] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 136.

[0319] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 136.

[0320] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 141.

[0321] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 141.

[0322] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:209.

[0323] In some embodiments, the NARE comprises: (i) SEQ ID NO:128; and (ii) SEQ ID NO:209.

[0324] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:124.

[0325] In some embodiments, the NARE comprises: (i) SEQ ID NO:128; and (ii) SEQ ID NO:124.

[0326] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:125.

[0327] In some embodiments, the NARE comprises: (i) SEQ ID NO:128; and (ii) SEQ ID NO:125.

[0328] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 152.

[0329] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 152.

[0330] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 140.

[0331] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 140.

[0332] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 145.

[0333] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 145.

[0334] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 146.

[0335] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 146.

[0336] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 147.

[0337] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 147.

[0338] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153.

[0339] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 153.

[0340] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210.

[0341] In some embodiments, the NARE comprises: (i) SEQ ID NO: 128; and (ii) SEQ ID NO: 210.

[0342] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 166 - 169, 171, or 172; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122 or 154

[0343] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 166 - 168, 171, 172; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122 or 154

[0344] 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;

[0345] 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

[0346] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 166 - 169, 171, or 172.

[0347] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 166 - 168, 171, or 172.

[0348] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) any one of SEQ ID NOs: 166 - 169, 171, or 172.

[0349] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) any one of SEQ ID NOs: 166 - 168, 171, or 172.

[0350] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122 or 154; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 121; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 167.

[0351] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122 or 154; (ii) SEQ ID NO: 121; and (iii) SEQ ID NO: 167.

[0352] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 166.

[0353] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 166.

[0354] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 167.

[0355] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 167.

[0356] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168 or 169.

[0357] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 168 or 169.

[0358] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168.

[0359] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 168.

[0360] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 171.

[0361] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 171.

[0362] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 167.

[0363] In some embodiments, the NARE comprises: (i) SEQ ID NO: 154; (ii) SEQ ID NO: 121; and (iii) SEQ ID NO: 167.

[0364] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 167.

[0365] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; (ii) SEQ ID NO: 121; and (iii) SEQ ID NO: 167.

[0366] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 172.

[0367] In some embodiments, the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NO: 172.

[0368] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 122; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 173 - 179 or 188, and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 207.

[0369] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 122; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 173, 174, 176, 178, 179 or 188, and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 207.

[0370] 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.

[0371] 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.

[0372] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 173 - 179.

[0373] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 173, 174, 176, 178, or 179.

[0374] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; and (ii) any one of SEQ ID NOs: 173 - 179.

[0375] 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.

[0376] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 173.

[0377] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; and (ii) SEQ ID NO: 173.

[0378] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174 or 175.

[0379] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; and (ii) SEQ ID NO: 174 or 175.

[0380] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 174.

[0381] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; and (ii) SEQ ID NO: 174.

[0382] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 176 or 177.

[0383] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; and (ii) SEQ ID NO: 176 or 177.

[0384] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:176.

[0385] In some embodiments, the NARE comprises: (i) SEQ ID NO:122; and (ii) SEQ ID NO:176.

[0386] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:178.

[0387] In some embodiments, the NARE comprises: (i) SEQ ID NO:122; and (ii) SEQ ID NO:178.

[0388] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:122; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:179.

[0389] In some embodiments, the NARE comprises: (i) SEQ ID NO:122; and (ii) SEQ ID NO:179.

[0390] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 188; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207.

[0391] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; (ii) SEQ ID NO: 188; and (iii) SEQ ID NO: 207.

[0392] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 184; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 185 - 187.

[0393] In some embodiments, the NARE comprises: (i) SEQ ID NO: 184; and (ii) any one of SEQ ID NOs: 185 - 187.

[0394] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 184; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 185.

[0395] In some embodiments, the NARE comprises: (i) SEQ ID NO: 184; and (ii) SEQ ID NO: 185.

[0396] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 184; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186.

[0397] In some embodiments, the NARE comprises: (i) SEQ ID NO: 184; and (ii) SEQ ID NO: 186.

[0398] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 184; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 187.

[0399] In some embodiments, the NARE comprises: (i) SEQ ID NO: 184; and (ii) SEQ ID NO: 187.

[0400] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 180; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 181 or 182.

[0401] In some embodiments, the NARE comprises: (i) SEQ ID NO: 180; and (ii) SEQ ID NO: 181 or 182.

[0402] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 180; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 181.

[0403] In some embodiments, the NARE comprises: (i) SEQ ID NO: 180; and (ii) SEQ ID NO: 181.

[0404] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 180; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182.

[0405] In some embodiments, the NARE comprises: (i) SEQ ID NO: 180; and (ii) SEQ ID NO: 182.

[0406] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 200, 201, 205, or 208.

[0407] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) optionally, any one of SEQ ID NO: 200, 201, 205 or 208.

[0408] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 200, 201, 205 or 208.

[0409] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) any one of SEQ ID NO: 200, 201, 205 or 208.

[0410] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 183; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 182.

[0411] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; and (ii) SEQ ID NO: 182.

[0412] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 200.

[0413] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205.

[0414] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) SEQ ID NO: 205.

[0415] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0416] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) SEQ ID NO: 208.

[0417] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 201.

[0418] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) SEQ ID NO: 201.

[0419] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 170; (ii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 188; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 203 or 204.

[0420] 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.

[0421] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 170; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 203.

[0422] In some embodiments, the NARE comprises: (i) SEQ ID NO: 170; and (ii) SEQ ID NO: 203.

[0423] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 188; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 204.

[0424] In some embodiments, the NARE comprises: (i) SEQ ID NO: 122; (ii) SEQ ID NO: 188; and (iii) SEQ ID NO: 204.

[0425] In some embodiments, the NARE comprises: (i) at least one copy of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154 or 164; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 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.

[0426] In some embodiments, the NARE comprises: (i) at least one copy of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130. In some embodiments, the NARE comprises: (i) a first sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; (ii) a second sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0427] 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.

[0428] In some embodiments, the NARE comprises: (i) at least one copy of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128. In some embodiments, the NARE comprises: (i) a first sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; (ii) a second sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128.

[0429] 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.

[0430] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128 - 130.

[0431] In some embodiments, the NARE comprises: (i) SEQ ID NO: 164; and (ii) any one of SEQ ID NOs: 128 - 130.

[0432] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 164; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 129.

[0433] In some embodiments, the NARE comprises: (i) SEQ ID NO: 164; and (ii) SEQ ID NO: 129.

[0434] In embodiments, the NARE comprises an intron or a portion of an intron. The intron or portion of the intron may be selected from: ACTA1 intron, ACTA1e intron, ACTC1 intron, ACTC1.3 intron, ACTC1e intron, ACTC1e-ACTC1p intron, ALDOA intron, APOC intron, APOC1 intron, ATF5 intron, CAMK2A intron, CBA intron, chimeric intron, CMV-rabbit β-globin intron, CRYAB intron, DES.4 intron, EEF1A1 intron, EEF1B2 intron, EF1a intron, FHL1 intron, FLOT1 intron, FXYD1 intron, GFAP intron, HBB intron, hCPE intron, hEf1a2-intron, HPD intron, IFI27L2 intron, murine IgG chimeric intron, MVM intron, MVMi-AATp intron, MVMi-SynE-mTTRp intron, rabbit β-globin intron, RBP4 intron, RPL26 intron, RPL27 intron, S100A6 intron, sEEF1A1 intron, SV40 intron, TMSB10 intron, and UCHL1 intron.

[0435] In embodiments, the NARE comprises an enhancer or a portion of an enhancer. The enhancer or portion of the enhancer may be selected from: ACTA1 enhancer, ACTC1 enhancer, CKM enhancer, CMV enhancer, CMV enhancer, MCK enhancer, mDES enhancer, mDES.1 enhancer, minCKM enhancer, minCKM2 enhancer, minDes enhancer, NRGN enhancer 1.1, NRGN enhancer 1.2, NRGN enhancer 2.1, NRGN enhancer 2.2, SV40 enhancer, and SV40 enhancer (SV40e).

[0436] In an embodiment, the NARE comprises a UTR or a portion of a UTR. The UTR or portion of the UTR can be selected from: CTNNB1 UTR, hEf1a2-utr1, hEf1a2-utr2, hNSE utr1, hNSE utr2, hSyn1_utr1, hSyn1_utr2, HTLV 5'UTR, HTLV 5'UTR, L21 UTR, TMSB10_utr1, and TMSB10_utr2.

[0437] Also provided herein are NAREs for use as a reference or control. Some NAREs provide constitutive expression of an operably linked transgene, i.e., the expression of the transgene is maintained at a constant level. Constitutive NAREs can drive the expression of an operably linked transgene in a variety of cell types and tissues. As used herein, a NARE starting with "C" is a constitutive NARE. The constitutive NAREs disclosed herein can be used for gene expression in muscle.

[0438] NARE modification

[0439] Those skilled in the art will understand that certain modifications can be made to the NAREs disclosed herein without eliminating the ability of the NAREs to drive gene expression in the desired tissue / cell type. Various in vitro and in vivo methods for confirming that a modified NARE is still capable of driving gene expression are known in the art, including but not limited to the methods used herein. By way of example, the strength of a NARE can be assessed by operably linking the NARE to a transgene encoding a protein and measuring transgene expression, such as by detecting the mRNA encoding the protein, or by measuring the presence or activity of the protein, such as by ELISA, Western Blot, fluorescence, enzymatic activity of the protein, etc.

[0440] The present disclosure provides NAREs comprising one or more components. The present disclosure provides NAREs comprising one or more sequence components (or sequence variants thereof) listed in any of Tables 4-6. The present disclosure provides NAREs comprising one or more sequence components (or sequence variants thereof) listed in Table 4. The present disclosure provides NAREs comprising one or more components (or sequence variants thereof) of the full-length NAREs provided in Table 6. By way of non-limiting example only, a NARE is provided that comprises one or more components (or sequence variants thereof) of NAREM37 (CK8emd2): (1) CK8e.1; and (2) md2. In an embodiment, a NARE is provided that comprises one or more components (or sequence variants thereof) of the full-length NAREs provided in Table 4 or Table 6, whereby the components (or sequence variants thereof) are arranged 5′ to 3′ as shown in Table 6. In an embodiment, a NARE is provided that comprises one or more components (or sequence variants thereof) of the full-length NAREs provided in Table 6, whereby the components (or sequence variants thereof) are arranged in a different order 5′ to 3′ than shown in Table 6.

[0441] In an embodiment, in the NAREs of Table 4 or Table 6, one or more components have been replaced with different sequences, including components (or sequence variants thereof) from different NAREs of Table 4 or Table 6. In addition, NAREs are contemplated that comprise components (or sequence variants thereof) derived from two or more NAREs disclosed in Tables 4-6.

[0442] In addition, NARE variants are contemplated that include sequences other than the elements of the NAREs disclosed herein (e.g., added to either end or inserted within the NARE sequence). Also, NARE variants are provided where certain sequences have been removed from the NAREs disclosed herein (e.g., as terminal or internal deletions). In an embodiment, a NARE is provided that comprises one or more components without any additional nucleic acid sequence joining the components of the NARE. In an embodiment, a NARE is provided that comprises one or more components whereby the individual components are linked by additional nucleic acid sequences. These additional nucleic acid sequences may or may not be related to the expression of an operably linked transgene. In an embodiment, some components are directly linked while other components are linked to other components by intervening sequences. Thus, variants of the NAREs disclosed in Table 4 or Table 6 are contemplated where additional sequences are added between different components. Further variants of the NAREs disclosed in Table 4 or Table 6 are contemplated that disclose the same components of a given NARE in Table 4 or Table 6 but differ in the sequences linking the individual components.

[0443] Nucleic Acid Constructs and Vectors

[0444] On the one hand, nucleic acid constructs and vectors and their use for introducing transgenes or expression constructs into cells are provided. The 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 capable of being transcribed in a cell. Methods for constructing expression constructs and plasmids by standard recombinant techniques are known in the art.

[0445] In some embodiments, the vector contains a recombinant DNA construct, which includes additional DNA elements, including DNA segments that provide DNA replication in a host cell and expression of a target gene at an appropriate level in a target cell. As used herein, "vector" means an agent that contains a polynucleotide to be delivered to a host cell in vitro, ex vivo, or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA microcircles, or viruses (including virus-derived sequences). A vector can also refer to a viral particle that contains a nucleic acid to be delivered to a host cell in vitro, ex vivo, or in vivo. In some embodiments, a vector refers to a viral particle that contains a recombinant viral genome, wherein the viral genome contains one or more ITRs and a transgene.

[0446] In one embodiment, the vector is a viral vector or a combination of multiple viral vectors. On the one hand, a vector is provided that contains any nucleic acid construct disclosed herein.

[0447] Nucleic acid constructs and vectors are provided herein, which contain the NAREs 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 promoters, enhancers, translation initiation signals, introns, and / or splicing enhancers. 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 generate more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to produce a protein containing more than one polypeptide chain, or to express two different proteins in the same cell 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' to the transgene.

[0448] Viral vector

[0449] Viral vectors for expressing a target gene in a target cell, tissue, or organism are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxviral vectors, baculoviral vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric viruses, mosaic viruses, or pseudotyped viruses and / or viruses containing exogenous proteins, synthetic polymers, nanoparticles, or small molecules).

[0450] AAV vector

[0451] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires a helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames that encode the Rep protein and the Cap protein, respectively. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. The functions of these proteins are mainly to regulate AAV replication and rescue AAV and integrate it into the chromosome of the host cell. The Cap reading frame encodes three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the viral particle capsid. More than 80% of the total protein in the AAV viral particle consists of VP3. Flanking the rep and cap open reading frames at the 5' and 3' ends are inverted terminal repeats (ITRs) approximately 145 bp in length. The two ITRs are the only cis-elements required for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter gene transgene.

[0452] 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 completely or partially deleted, preferably the Rep and / or Cap genes, but retain the functional flanking ITR sequences.

[0453] In some embodiments, the viral vector is an rAAV viral particle that contains an rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome contains the nucleic acid construct disclosed herein.

[0454] In some embodiments, the viral vectors disclosed herein comprise a nucleic acid that includes AAV 5′ ITR and 3′ ITR located 5′ and 3′, respectively, of a sequence encoding a transgene. In an embodiment, the transgene is UPF1, BDNF, or ATP7B. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5′ ITR and 3′ ITR sequences arranged in tandem, such as 5′ to 3′ or head-to-tail or in another alternative configuration. In other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITR, or to have 5′ ITRs (or, conversely, 3′ ITRs) located both 5′ and 3′ of the transgene. The ITR sequences may be located immediately upstream and / or downstream of a heterologous molecule, or intervening sequences may be present. The ITR need not be a wild-type nucleotide sequence and may be altered (e.g., by insertion, deletion, or substitution of nucleotides) so long as the sequence provides for functional rescue, replication, and packaging. The ITR may be selected from AAV2 or other AAV serotypes as described herein.

[0455] In some embodiments, a vector comprising a nucleic acid sequence is provided, the nucleic acid sequence comprising (i) a nucleic acid construct as 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 3′ ITR are derived from adeno-associated virus (AAV) serotype AAV2.

[0456] In some embodiments, the viral vector is an AAV vector, such as AAV1 (i.e., an AAV that contains AAV1 ITR and AAV1 capsid proteins), AAV2 (i.e., an AAV that contains AAV2 ITR and AAV2 capsid proteins), AAV3 (i.e., an AAV that contains AAV3 ITR and AAV3 capsid proteins), AAV4 (i.e., an AAV that contains AAV4 ITR and AAV4 capsid proteins), AAV5 (i.e., an AAV that contains AAV5 ITR and AAV5 capsid proteins), AAV6 (i.e., an AAV that contains AAV6 ITR and AAV6 capsid proteins), AAV7 (i.e., an AAV that contains AAV7 ITR and AAV7 capsid proteins), AAV8 (i.e., an AAV that contains AAV8 ITR and AAV8 capsid proteins), AAV9 (i.e., an AAV that contains AAV9 ITR and AAV9 capsid proteins), AAVrh74 (i.e., an AAV that contains AAVrh74 ITR and AAVrh74 capsid proteins), AAVrh.8 (i.e., an AAV that contains AAVrh.8 ITR and AAVrh.8 capsid proteins), or AAVrh.10 (i.e., an AAV that contains AAVrh.10 ITR and AAVrh.10 capsid proteins).

[0457] In some embodiments, the viral vector is a pseudotyped AAV vector that contains 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 that contains AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV that contains AAV2 ITRs and AAV10 capsid proteins).

[0458] In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV that contains AAV2 ITRs and AAV7m8 capsid proteins).

[0459] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein of a chimeric body containing one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In an embodiment, the capsid is a variant AAV capsid, such as AAV2 variant rAAV2-retro (SEQ ID NO:44, from WO 2017 / 218842, incorporated herein by reference).

[0460] 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 in ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively. In some embodiments, the AAV vector contains AAVrh.10 and rAAV2-retro capsid proteins in ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively.

[0461] 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, the ratio of (1) the AAV vector comprising rAAV2-retro to (2) the AAV vector comprising AAVrh.10 is 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 ratio of (1) the AAV vector comprising AAVrh.10 to (2) the AAV vector comprising rAAV2-retro is 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.

[0462] Other viral vectors

[0463] 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-defective by deletions in the E1 and E3 regions. A transcription cassette can be inserted into the E1 region, thereby generating a recombinant E1 / E3-deleted AV vector. Adenovirus vectors also include helper-dependent high-capacity adenovirus vectors (also referred to as high-capacity "gutless" or "gutted" vectors), which do not contain viral coding sequences. These vectors contain cis-acting elements required for viral DNA replication and packaging, mainly inverted terminal repeats (ITRs) and a packaging signal (CY). These helper-dependent AV vector genomes have the potential to carry exogenous DNA ranging from a few hundred base pairs to approximately 36 kb.

[0464] Alternatively, other systems such as lentiviral vectors can be used. Lentivirus-based systems can transduce both non-dividing and dividing cells, making them useful for applications targeting non-dividing cells such as those in the CNS. Lentiviral vectors are derived from the human immunodeficiency virus and, like the virus, integrate into the host genome, thereby providing the potential for very long-term gene expression.

[0465] Cationic lipids, polymers, or both can also be used as carriers to introduce polynucleotides (including plasmids, YACs, minichromosomes, and microcircles) carrying a target gene containing an expression cassette into cells or organisms via a non-viral vector system. Conjugated poly-L-lysine (PLL) polymers and polyethyleneimine (PEI) polymer systems can also be used to deliver the vector to cells. Other methods for delivering the vector to cells include hydrodynamic injection and electroporation, as well as the use of ultrasound, both for cell culture and for organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012; 1:27), which is incorporated herein by reference.

[0466] rAAV virion production

[0467] The rAAV viral particles disclosed herein can be constructed and produced using the materials and methods described herein and those known to those of skill in the art. Such engineering methods for constructing any embodiment of the present disclosure are known to those skilled in nucleic acid manipulation and include genetic engineering, recombineering, and synthetic techniques. See, for example, Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989); and International Patent Publication No. WO 95 / 13598. In addition, methods suitable for generating rAAV cassettes in adenoviral capsids have been described in U.S. Patent Nos. 5,856,152 and 5,871,982.

[0468] 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 and helper genes necessary for AAV production are used. The AAV rep and cap sequences are obtained from AAV sources identified herein. The AAV rep and cap sequences can be introduced into host cells in any manner known to those skilled in the art, including but not limited to transfection, electroporation, liposome delivery, membrane fusion techniques, high-velocity DNA-coated microprojectiles, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into host cells by one or more nucleic acid molecules and stably exist in the cells as episomes. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cells. Another embodiment has rep and cap sequences that are transiently expressed in host cells. For example, the nucleic acid molecule that can be used for such transfection contains, from 5' to 3', a promoter, an optional spacer inserted between the start site of the promoter and the rep gene sequence, the AAV rep gene sequence, and the AAV cap gene sequence.

[0469] The rep and cap sequences, along with their expression control sequences, can be provided on a single vector, or each sequence can be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences can be provided on a vector containing other DNA sequences to be introduced into host cells. Preferably, the promoter used for such constructs can be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins can be in any form that transfers these components to host cells. Ideally, such a molecule is in plasmid form, which can contain other non-viral sequences, such as those of marker genes. Such a molecule does not contain AAV ITR, and generally does not contain AAV packaging sequences. To avoid the occurrence of homologous recombination, other viral sequences, especially those of adenoviruses, are avoided in such plasmids. The plasmid is ideally constructed such that it can be stably transfected into cells.

[0470] Although the molecule providing the rep and cap can be transiently transfected into host cells, it is preferred to stably transform host cells with sequences necessary for expressing functional rep / cap proteins in host cells, such as as episomes or by integration into the chromosomes of host cells. Depending on the promoter controlling the expression of such stable transfection in host cells, the rep / cap proteins can be transiently expressed (e.g., by using an inducible promoter).

[0471] The methods for constructing the embodiments of the present disclosure are conventional genetic engineering or recombinant engineering techniques, such as those described in the above references. For example, rAAV can be produced using the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.) with a triple transfection method according to the manufacturer's instructions. See also Herzog et al., 1999, Nature Medic., 5(1):56 - 63. For the methods used in the following examples, a plasmid with a transgene, a helper plasmid containing AAV rep and cap, and a plasmid providing adenovirus helper functions of E2A, E4Orf6, and VA are employed. Although this specification provides illustrative examples of specific constructs, using the information provided herein, those skilled in the art can select and design other suitable constructs using the selection of spacers, promoters, and other elements (including at least one translation initiation and termination signal) and optionally adding polyadenylation sites.

[0472] rAAV viral particles can be produced by culturing host cells containing the rAAV virus as described herein, the host cells containing the rAAV genome to be packaged into the rAAV viral particles, the AAV rep sequence and the AAV cap sequence under the control of regulatory sequences directing expression. Suitable viral helper genes (such as adenovirus E2A, E4Orf6, and VA, and other possible helper genes) can be provided to the culture in a variety of ways known in the art, preferably on a separate plasmid. Thereafter, in the absence of contaminating helper virus or wild - type AAV, the recombinant AAV viral particles directing the expression of the transgene are isolated from the cells or cell culture.

[0473] The expression of the transgene can be measured in ways known in the art. For example, target cells can be infected in vitro, and the copy number of the transgene in the cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression levels can be monitored by Northern blotting or quantitative reverse transcriptase (RT) - PCR; and protein expression levels can be monitored by Western blotting, immunohistochemistry, enzyme - linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by specific methods detailed in the examples below.

[0474] Cell

[0475] In an embodiment, the nucleic acid constructs and vectors disclosed herein are used to deliver the NARE operably linked to a transgene to a cell. Cells containing the NARE, nucleic acid construct, or vector disclosed herein are provided. In an embodiment, the cell is a muscle cell. The cell can be a mammalian cell. The cell can be a human cell. The cell may be isolated.

[0476] Drug composition

[0477] The present disclosure provides a drug composition comprising the nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.

[0478] Preferably, the nucleic acid construct or vector disclosed herein is assayed for contamination by conventional methods and then formulated into a drug composition suitable for storage and / or administration to a patient.

[0479] The formulation of the nucleic acid construct or vector disclosed herein involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly a vehicle or carrier suitable for subretinal injection, such as buffered saline or other buffers (e.g., HEPES), to maintain the pH at an appropriate physiological level. The nucleic acid construct or vector disclosed herein can be formulated into a drug composition. In addition to the carrier, these compositions can also contain pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can be determined by a skilled person according to the route of administration. The drug composition is generally in liquid form. Liquid drug compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, which is incorporated herein by reference. It may include saline solutions, magnesium chloride, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, surfactants such as Pluronic acid (PF68) 0.001% can be used. In some cases, Ringer's injection solution, lactated Ringer's injection solution, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.

[0480] A pharmaceutical composition comprising the nucleic acid construct or vector disclosed herein can be formulated with one or more pharmaceutically acceptable excipients, which can be pharmaceutically acceptable materials, compositions or vehicles such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate or zinc stearate or stearic acid), solvents or encapsulating materials involved in carrying or transporting a therapeutic compound, bulking agents, salts, surfactants and / or preservatives for administration to a subject. Some examples of materials that can be used as pharmaceutically acceptable excipients include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations.

[0481] A bulking agent is a compound that increases the mass of a pharmaceutical formulation and contributes to the physical structure of a formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol and sorbitol.

[0482] The use of a surfactant can reduce the aggregation of the reconstituted protein and / or reduce the formation of microparticles in the reconstituted formulation. The amount of surfactant added should be such that it reduces the aggregation of the reconstituted protein and minimizes the formation of microparticles after reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or sodium methyl oleoyl taurate; and copolymers of polyethylene glycol, polypropylene glycol and ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.).

[0483] Preservatives can be used in the formulations of the present invention. Suitable preservatives for the formulations of the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl-dimethyl ammonium chlorides, wherein the alkyl is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols (such as phenol, butanol, and benzyl alcohol), alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Edition, Mack Publishing Company, Easton, Pa., (1995).

[0484] For delayed release, the nucleic acid constructs or vectors disclosed herein can be included in pharmaceutical compositions formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

[0485] If the vector is intended for long-term storage, it can be frozen in the presence of glycerol.

[0486] Method

[0487] Methods for inducing the expression of a transgene in cells and / or tissues are provided herein. A method for inducing transgene expression is provided, the method comprising providing a cell comprising a nucleic acid construct comprising a NARE disclosed herein operably linked to a transgene, and culturing the cell under conditions that permit transgene expression. In an embodiment, the cell is a muscle cell. Methods for inducing transgene expression in vivo are provided herein. Methods for inducing transgene expression in vitro are provided herein. Methods for inducing transgene expression ex vivo are provided herein.

[0488] Methods for treating a disease in a subject in need thereof using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein are provided.

[0489] In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals include, but are not limited to, human or non-human mammals, such as cows, equines, canines, sheep, or felines, etc. An individual and a patient are also subjects herein.

[0490] As used herein, the term "treat / treated / treating / treatment" refers to a therapeutic treatment in which the goal is to slow down (alleviate) an undesired physiological disorder, condition or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of a disorder, condition or disease; stabilization (i.e., non-worsening) of the state of a disorder, condition or disease; delay in the onset or slowdown in the progression of a disorder, condition or disease; improvement in one or more symptoms of the state of a disorder, condition or disease; and remission (whether partial or total) or enhancement or improvement of a disorder, condition or disease. Treatment includes eliciting a clinically significant response without excessive side effects. Treatment also includes prolonging survival as compared to the expected survival in the absence of treatment. The terms "prevent / prevention" etc. refer to acting before the onset of an apparent disease or condition to prevent the development of the disease or condition or to minimize the extent of the disease or condition, or to slow its progression.

[0491] In some embodiments, treatment refers to increasing survival (e.g., survival time). For example, treatment can result in an increase in the life expectancy of a patient. In some embodiments, treatment results in an increase in the life expectancy of a patient by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more as compared to the average life expectancy of one or more untreated control individuals with ALS. In some embodiments, treatment results in an increase in the life expectancy of a patient by more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or longer as compared to the average life expectancy of one or more untreated diseased control individuals. In some embodiments, treatment results in long-term survival of a patient. As used herein, the term "long-term survival" refers to a survival time or life expectancy of more than about 40 years, 45 years, 50 years, 55 years, 60 years or longer.

[0492] Administration methods include, but are not limited to, intracisternal, intraventricular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intrathecal, intravaginal, transdermal, rectal, by inhalation or topically (especially to the ear, nose, eye or skin). The mode of administration is at the discretion of the practitioner.

[0493] In some cases, the nucleic acid constructs or vectors described herein are administered locally. By way of example, this can be achieved by local infusion during surgery, topical application (e.g., in a cream or lotion), by injection, via a catheter, via a suppository or enema or via an implant, which is a porous, non-porous or gel-like material, including membranes, such as silicone rubber membranes or fibers. In some cases, the nucleic acid constructs or vectors described herein are introduced into the central nervous system, the circulatory system or the gastrointestinal tract by any suitable route, including intraventricular injection, intrathecal injection, paravertebral injection, epidural injection, enema and injection adjacent to a peripheral nerve.

[0494] The compositions described herein can be administered in a single dose or in multiple doses. Such compositions can be administered at regular time intervals depending on the nature, severity and extent of the disorder of the subject. In some embodiments, a therapeutically effective amount of the nucleic acid construct or vector is administered intrathecally at regular time intervals (e.g., once a year, once every six months, once every five months, once every three months, bi-monthly (once every two months), monthly (monthly / once every month), bi-weekly (once every two weeks) or weekly).

[0495] The amount of the nucleic acid construct or vector described herein effective for treating a disease can be determined using standard clinical techniques known to those of skill in the art. In addition, in vitro or in vivo assays can optionally be used to assist in identifying the optimal dosage range. The precise dose to be employed will also depend on the route of administration, the disorder, the severity of the disorder being treated and various physical factors associated with the individual being treated, and can be determined at the discretion of the healthcare practitioner.

[0496] An effective amount of rAAV carrying a nucleic acid sequence encoding a transgene (including, but not limited to, UPF1, ATP7B and BDNF) under the control of a NARE can be, for example, in the range of about 1×10 9 to about 1x10 14 rAAV genome particles (vg) / kg body weight. "Genome particles" are defined herein as AAV capsids containing single-stranded DNA molecules, which can be quantified by sequence-specific methods such as qPCR or ddPCR. In some embodiments, rAAV is at about 1×10 12 to about 1x 10 13rAAV is administered at vg / kg body weight. In some embodiments, rAAV is administered at about 5 x 10 11 to about 5 x 10 12 vg / mL cerebrospinal fluid (CSF) volume. In some embodiments, rAAV is administered at a total of about 7.5 x 10 13 to 7.5 x 10 14 vg per patient.

[0497] In some embodiments, rAAV is administered to an animal at about 1×10 11 to about 1 x 10 14 rAAV genome particles (vg) / kg body weight.

[0498] In some embodiments, rAAV genome particles are provided in a volume between about 20 μL and about 50 mL. In some embodiments, rAAV genome particles are provided in a volume between about 30 μL and about 30 mL. In some embodiments, rAAV genome particles are provided in a volume of about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 μL. In some embodiments, 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.

[0499] The attending physician may also select other doses within these ranges. It should be understood that for any particular subject, the specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the nucleic acid construct or vector, and the dosage ranges described herein are only exemplary and are not intended to limit the scope or practice of the claimed invention.

[0500] The nucleic acid constructs or vectors disclosed herein can also be advantageously provided ex vivo to cells, and the live cells are subsequently administered to a subject. Methods of treating diseases by implanting cells that have been modified to express a recombinant protein are also well known. See, for example, U.S. Patent No. 5,399,346, which discloses methods for introducing nucleic acids into primary human cells for introduction into the human body. Although human cells are preferably used for ex vivo therapy in some embodiments, other cells (such as bacterial cells) can also be implanted into the vasculature of a subject to continuously release a therapeutic agent. See, for example, U.S. Patent Nos. 4,309,776 and 5,704,910.

[0501] 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 herein by reference in its entirety.

[0502] It should be understood that the present invention is not limited to the specific molecules, compositions, methods, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein may be used to practice or test embodiments of the present invention. It should also be understood that the disclosure of the present invention in this specification includes all possible combinations of such specific features. For example, where a specific feature is disclosed in the context of a particular aspect or embodiment of the present invention or a particular claim, that feature may also be used in combination with and / or in the context of other particular aspects and embodiments of the present invention to the extent possible, and is generally used in the present invention.

[0503] Where a method involving two or more defined steps is mentioned herein, the defined steps may be performed in any order or simultaneously (unless the context excludes such possibilities), and the method may include one or more other steps performed before any defined step, between two defined steps, or after all defined steps (unless the context excludes those possibilities).

[0504] All other cited patents and applications are incorporated herein by reference in their entirety. Additionally, when the definition or use of a term in a reference incorporated herein by reference is inconsistent with or contradicts the definition of the term provided herein, the definition of the term provided herein shall control and the definition of the term in the reference shall not apply.

[0505] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be construed as limiting or defining the entire scope of the present invention.

[0506] Examples

[0507] Example 1: NARE Design

[0508] Materials and Methods

[0509] Potential NARE candidates were identified from genes highly expressed in human target tissues (muscle). Subsequently, promoter regions were defined or cis-regulatory elements were identified based on chromatin marks, accessibility, conservation, and other genome-wide datasets.

[0510] Incorporation of the ten-motif element (MTE) / downstream promoter element (DPE) modification was used to increase transcription, and various cellular, viral, or synthetic 5′ UTR motifs were incorporated to increase transcription / translation. The choice of introns was varied, for example, to include short cellular introns that naturally have unique independent transcription start sites, indicating the presence of a promoter within this particular intron. Additionally, introns were inserted at different positions within the sequence, and / or the introns were truncated while maintaining splicing elements. Furthermore, individual or multiple point mutations were introduced to increase expression or prevent inactivation. Additionally, different transcriptional enhancers were introduced upstream of the core promoter. In some cases, transcriptional motif insertion, replacement, and / or shuffling were employed, and CpG sequences were removed to reduce immunogenicity while maintaining promoter activity.

[0511] Gene regulatory elements were also optimized by: (i) identifying, shuffling, and cloning highly expressed transcription factor binding sites (TFBS) upstream of the core promoter; (ii) using artificial intelligence (AI) algorithms to iteratively mutate and score the promoter.

[0512] Furthermore, nested NAREs ( Figure 2A ) were created. One or more promoters were cloned into the intron of one of the existing promoters while ensuring that the placement did not create any unintended translation start sites. Dual, triple, and quadruple promoters were created in this way, which can increase NARE strength while minimizing the total length. For example, in Figure 2A , tMCK was modified by adding the MTE / DPE modification. tMCK is a muscle creatine kinase (MCK) promoter that was shortened by triple tandem ligation of the MCK enhancer (206 bp) to its 87-bp basal promoter, thereby generating the tMCK (720 bp) NARE. A synthetic HBG intron variant containing the enhancer (SIE) and the modified human Desmin promoter was inserted downstream. This nested promoter strategy can increase transcriptional output through multiple promoters and avoid or minimize expression loss due to promoter inactivation. The intron position allows for the generation and processing of multiple transcripts and prevents a potential adverse effect of the second promoter sequence on the translation rate as it will be spliced out in some transcripts. Additionally, this strategy allows targeting of multiple cell types using a single complex promoter and can confer tissue specificity to a normal constitutive promoter.

[0513] Generation and screening of synthetic cis-regulatory elements (CREs) by transcription factor shuffling

[0514] Print the binding site sequences of tissue-specific and highly expressed transcription factors of a given tissue as a pool of single-stranded oligonucleotides ( Figure 5A ). Anneal the oligonucleotides into double-stranded oligonucleotides and randomly ligate them together overnight. Then, ligate the fragments into an AAV plasmid upstream of a small synthetic JeT promoter driving the expression of mClover3-WPRE3-synPolyA. Transform this pooled library into cells and perform minipreps on individual colonies to obtain sufficient DNA for transient transfection experiments.

[0515] Next, transiently co-transfect the cells of interest with the TFBS candidate plasmid and an mCherry plasmid as a normalization control. After 48 hours, perform mClover3 and mCherry fluorescence imaging on the cells as described in Example 2. Automatically analyze the images using CellProfiler to identify red fluorescent protein-positive cells and measure the green fluorescent protein intensity of these cells. In this screen, use the GFP intensity of the candidate TFBS NARE relative to the JeT promoter alone to identify positive hits. Then, subclone the positive hits into a dual reporter assay system to verify the screening results for JeT or other basal NAREs.

[0516] Example 2: Dual reporter protein expression assay

[0517] To test the ability of NARE candidates to drive gene expression, a dual reporter gene assay using flow cytometry was utilized. Figure 1 provides a diagram of the dual reporter gene expression construct and assay. Clone different synthetic candidate NARE sequences upstream of a transgene encoding mClover3, a green / yellow fluorescent protein, which is in turn upstream of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE250) and a synthetic polyadenylation (polyA) signal. mClover3 fluorescence serves as a measure of NARE strength. The plasmid also contains a separate expression cassette (5' to 3': SV40 promoter-tdTomato, a red fluorescent protein-SV40 polyA), which is the same in all NARE test constructs (Figure 1). tdTomato fluorescence serves as an internal normalization control that can account for variations in transfection efficiency.

[0518] Using the Turbofect transfection reagent, various cell lines were transiently transfected with a dual reporter gene plasmid. After 48 hours, the cells were trypsinized and resuspended for flow cytometry. The raw data was processed, and only live single cells were selected for analysis. A sample containing untransfected cells was used to set the gating for the tdTomato signal. Subsequently, this gating was applied to all live single cells to identify tdTomato-positive (and thus transfected) cells. Then, mClover3 fluorescence was determined for the tdTomato-positive population. Since transfection varied slightly between different experiments, the relative NARE potency was calculated by the ratio of mClover3 fluorescence to tdTomato fluorescence. The synthetic candidate NAREs were tested together with known constitutive and tissue-specific reference promoters such as CAG, CMV, or AAT.

[0519] Example 3: Intensities of Different NAREs in Muscle Cells (In Vitro)

[0520] Materials and Methods

[0521] Myotube model

[0522] Three myotube cell models were used to test the intensities of the selected muscle-specific NAREs. One model was the C2C12 immortalized cell line. C2C12 cells are murine skeletal muscle myoblasts derived from satellite cells of the thigh muscle of two-month-old female C3H mice. The C2C12 cells were seeded onto collagen I-coated vessels and grown in expansion medium: DMEM (high glucose, without sodium pyruvate) + 10% fetal bovine serum + 2 mM L-glutamine + 100 units / mL penicillin and 100 μg / mL streptomycin. After reaching confluence, the C2C12 cells were switched to differentiation medium: DMEM (high glucose, without sodium pyruvate) + 2% horse serum + 2 mM L-glutamine + 1 μM insulin + 100 units / mL penicillin and 100 μg / mL streptomycin. The C2C12 cells were cultured in the differentiation medium for approximately 7 days to form elongated myotubes, which is the result of myoblast fusion during myogenesis.

[0523] The second model used primary human skeletal myoblasts (Thermo Fisher Scientific), which were derived from a human donor batch of quadriceps biopsies. The primary human skeletal myoblasts were seeded onto collagen I-coated vessels and thawed in differentiation medium: DMEM (high glucose, without sodium pyruvate) + 2% horse serum + 2 mM L-glutamine + 1 μM insulin + 100 units / mL penicillin and 100 μg / mL streptomycin. The primary human skeletal myoblasts were cultured in the differentiation medium for 2 - 3 days for rapid differentiation.

[0524] Alternatively, a third cell model (i.e., human induced pluripotent stem cell-derived myoblasts) can be used to test the NARE strength in myotubes. The hiPSC-derived myoblasts were seeded on collagen I-coated vessels and grown in skeletal myoblast medium (AMSBIO). After reaching confluence, the hiPSC-derived myoblasts were media-changed with myotube medium or myotube fusion medium (AMSBIO).

[0525] After successful differentiation of myoblasts into myotubes, the cells were AAV-transduced with synthetic candidate NAREs to drive the expression of fluorescent proteins (such as mClover3 and mIRFP713). The candidate NAREs were tested together with known constitutive and tissue-specific reference promoters (such as CAG, CMV, tMCK, and creatine kinase 8 promoter (CK8e)). With the same multiplicity of infection in triplicate samples, the AAV candidates were diluted in the differentiation medium to replace the medium containing adherent myotubes. After incubation at 37 °C and 5% CO2 for 4 hours, differentiation medium supplemented with 200 nM doxorubicin was added, with the same volume as the AAV-containing medium, such that the final concentration of doxorubicin was 100 nM, as an agent 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 fluorescent protein expression of myotubes was detected by an Agilent / BioTek Cytation 5 cell imaging multimode reader. The fluorescence intensity of myotubes was measured as an index of NARE strength, where the synthetic candidate NAREs were compared among known reference promoters.

[0526] Results

[0527] A library of NAREs suitable for gene expression in muscle was developed.

[0528] Some 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 gene plasmids with different NARE candidates were transiently transfected into C2C12 cells. The GFP expression level in transfected (RFP+) cells was quantified by flow cytometry. Figure 4 The relative expression compared to the CAG promoter is shown.

[0529] Additional NAREs were also identified using a nested promoter design (for examples of nested promoter design, see Figure 2A and for the evolution of NARE M24, see Figure 2B)。In cultured myotubes generated using C2C12 cells ( Figure 2C ) and primary human myotubes ( Figure 2D ), M24 is a stronger NARE than tMCK.

[0530] NAREs are also generated by shuffling transcription factor motifs to create synthetic enhancers ( Figure 5A ). Most of the synthetic sequences generated are 200 bp or less ( Figure 5B ). The expression of a transgene encoding GFP under the control of a synthetic enhancer coupled to promoter C6 in C2C12 was normalized against the expression of a transgene encoding GFP under the control of a separate NARE ( Figure 5C and Figure 5D ). The selected hits (MTF1291, MTF1173, MTF1186, MTF1197, MTF1209, MTF1211, MTF1242, MTF1295, MTF1304, MTF1308) were cloned into the minCK promoter (M51, M53, M55, M57, M59, M61, M63, M65, M67, M69), or upstream of the Ck8e promoter (M52, M54, M56, M58, M60, M62, M64, M66, M68, M70).

[0531] Figure 5E and Figure 5F show the potency of the selected NAREs in C2C12 cells.

[0532] Figure 5G shows the potency of the CK8e-derived NARE M49 (which is only 616 bp) in C2C12 cells compared to the parental promoter CK8e (M31).

[0533] Figure 5H shows the potency of the minMCK-derived NARE M51 (a 366 bp NARE that includes an additional enhancer compared to the parental promoter minMCK (M50)) compared to M50 in C2C12 cells. M51 is 1.5-fold stronger than the 443 bp CK8e promoter.

[0534] Table 1 shows the potency of the selected NAREs in C2C12 cells. Figure 5I shows the potency of the selected NAREs in primary human myotubes.

[0535] Table 1. Potency of the indicated NAREs in C2C12 myoblasts. Plasmid transfection.

[0536] NARE Description Size (bp) Potency relative to CK8e M31 CK8e 443 1 M49 CK8e variant 616 1.9 M51 MTFBS promoter 366 1.5 M52 MTFBS promoter 672 1.4 M59 MTFBS promoter 267 0.9 M61 MTFBS promoter 688 1.0 M63 MTFBS promoter 264 0.8 M71 hCK 614 0.4 M73 MHCK7 770 0.3

[0537] Figure 5JShows the potency of selected human skeletal α - actin gene (ACTA1) - derived NAREs in C2C12 cells. The human desmin (hDes) promoter (M19), tMCK promoter (including triple tandem copies of the mouse MCK enhancer and mouse MCK basal promoter) (M22), CK8e (M31), hCK promoter (M71), MHCK7 promoter (M73) were used as controls. M98 and M99 are more potent than the hDes, tMCK, hCK M24, and MHCK7 promoters. Table 2 outlines the potency of the selected NAREs.

[0538] Table 2 Potency of super - compact NAREs in C2C12 myoblasts. Plasmid transfection. tMCK is 731 bp; MHCK7 is 770 bp; hCK is 661 bp; CK8e is 466 bp.

[0539]

[0540]

[0541] Figure 5K Shows the potency of selected hDes - derived NAREs in C2C12 cells. M111 (234 bp) is stronger than all muscle - specific NAREs tested.

[0542] Figure 5L Shows the potency of selected tMCK - derived NAREs in C2C12 cells.

[0543] Example 4: Strength of different NAREs in muscle cells (in vivo)

[0544] Materials and methods

[0545] Intramuscular injection

[0546] Male C57BL6 mice at 6 - 8 weeks of age were housed in standard cages with free access to water and switched from standard chow to a purified diet without alfalfa to minimize background autofluorescence. The mice were anesthetized with 2 - 3% isoflurane and 1.5 LPM oxygen. One leg of each mouse was shaved to remove all hair, cleaned with alcohol wipes, and the gastrocnemius muscle was targeted using a 27G needle, carefully avoiding any blood vessels. The mice were allowed to recover in a clean, warm cage and returned to their home cages after they were awake and mobile.

[0547] Intravenous injection

[0548] Male C57BL6 mice at 6 - 8 weeks of age were housed in standard cages with free access to water and switched from standard chow to a purified diet without alfalfa to minimize background autofluorescence. The mice were restrained and placed under a warming lamp to increase vasodilation, and a 27G needle was used to target either the tail vein. The mice were allowed to recover in a clean, warm cage and returned to their home cages after ensuring they were not bleeding.

[0549] Bioluminescence imaging protocol

[0550] The mice were anesthetized with 2 - 3% isoflurane and 1.5 LPM oxygen and imaged using the LICOR Pearl system in the white channel and 700 nm channel. The animals were imaged once a week for 5 weeks, then once every two months for up to 12 months. The mice were allowed to recover in a clean, warm cage and returned to their home cages after they were awake and mobile. Data analysis was performed using LICOR Image Studio software: Regions of interest (ROIs) were selected to background normalize the parts of the animal without fluorescence. ROIs covering the torso or legs were drawn and normalized against background ROIs. The same regions were used for the animals at each time point to minimize artifacts in data collection. Absolute fluorescence was recorded at each imaging session.

[0551] eGFP / mClover3 imaging of IM injection

[0552] Four weeks after injection, the animals were euthanized, the gastrocnemius muscle was dissected, and fixed by immersion overnight at 4°C in PBS containing 4% paraformaldehyde. The next morning, the muscle was changed to PBS containing 15% sucrose and incubated overnight at 4°C. The muscle was changed to PBS containing 30% sucrose and incubated overnight at 4°C. The muscle was then covered with OCT and allowed to equilibrate for 1 hour at RT in a freezing mold. The samples were frozen on dry ice and then stored at -80°C until used for histological analysis with a cryostat for serial sectioning. Serial 25 - µm thick sections were collected at 600 - µm intervals to obtain a complete representation of the expression in the muscle. After sectioning with n = 3 per group, the slides were air - dried for 10 minutes to complete the mounting process. The slides were then washed 3 times with PBS, mounted with Prolong antifade reagent containing NucBlue, and imaged on a BioTek imager to create a tiled scan of all sections.

[0553] eGFP / mClover3 imaging of IV injection

[0554] Four weeks after injection, the animals were euthanized and successively perfused with PBS and PBS containing 4% paraformaldehyde. The liver lobes were dissected and further fixed in PBS containing 4% paraformaldehyde at room temperature for 1 hour. Then the liver lobes were transferred to PBS containing 15% sucrose and incubated overnight at 4°C. The next morning, the muscle was transferred to PBS containing 30% sucrose and incubated overnight at 4°C. Then the liver lobes were covered with OCT and equilibrated at RT in a freezing mold for 1 hour. The samples were frozen on dry ice and then stored at -80°C until serial sections were cut using a cryostat for histology. Serial sections 25 μm thick were collected at 1000 μm intervals to obtain a complete representation of the expression in the liver. After sectioning with n = 3 for each group, the slides were air-dried for 10 minutes to complete the mounting process. Then the slides were washed 3 times with PBS and mounted with Prolong antifade reagent containing NucBlue, and imaged on a BioTek imager to create a tiled scan of all sections.

[0555] Western blot tissue preparation

[0556] Four weeks after injection, the mice were euthanized and the relevant tissues were dissected and snap-frozen in liquid nitrogen. Then the frozen tissues were cut and 10 mg chunks were dissolved in RIPA buffer and sonicated. The lysates were briefly centrifuged to remove insoluble materials, and the protein concentration was determined using a BCA protein assay. Then, the proteins were diluted and normalized to 1 μg / μl using 4X LDS, 10X DTT, and lysis buffer. The samples were denatured at 70 - 80°C for 10 min and stored at -20°C. Then, the samples were analyzed by Western blotting using a LICOR Odyssey imager.

[0557] Results

[0558] AAV8-iRFP713 under tMCK or M24 NARE control was intramuscularly injected into 8-week-old male C57Bl6 mice, and imaging was performed using a LICOR Pearl ( Figure 3A and Figure 3B ). iRFP713 is a near-infrared fluorescent protein. The expression of NARE M24 was 8-fold higher than that of tMCK and was maintained for up to 300 days after injection ( Figure 3C ).

[0559] In another experiment, AAV8-eGFP expressing NARE CMV, CAG, tMCK, CK8e, M21, and M24 was injected into the gastrocnemius muscles of 8-week-old male C57Bl6 mice ( Figure 3D)。Four weeks later, the muscles were harvested and sectioned to measure native eGFP fluorescence. In the gastrocnemius muscles of mice, compared with CAG or CK8e, NARE M21 and M24 showed increased NARE intensity ( Figure 3E and Figure 3F , Table 3).

[0560] Table 3. Figure 3E Description of the expression constructs shown in. WPRE 250 activity is equal to WPRE.

[0561] NARE ORF CMV <![CDATA[mClover3-WPRE 250 -sPolyA]]> CAG <![CDATA[eGFP-WPRE 250 -bGHpolyA]]> tMCK mClover3-WPRE-SV40polyA CK8e mClover3-WPRE-SV40polyA M21 mClover3-WPRE-SV40polyA M24 mClover3-WPRE-SV40polyA

[0562] An overview of the NARE sequences can be seen in Tables 4 - 6.

[0563] Table 4. NARE sequences.

[0564]

[0565]

[0566] Table 5. NARE components.

[0567]

[0568]

[0569] Table 6. NARE and NARE components. SN = SEQ ID NO.

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

Claims

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

4. A NARE, the 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. The NARE according to claim 4, the NARE 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. The NARE according to claim 4, the NARE 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, the 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. The NARE according to claim 7, the NARE 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. The NARE according to claim 7, the NARE comprising: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 148 or 149.

10. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to any one of SEQ ID NO: 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. The NARE according to claim 10, the NARE comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NO: 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. The NARE according to claim 10, the NARE comprising: (i) any one of SEQ ID NO: 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, the 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. The NARE according to claim 13, the NARE 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. The NARE according to claim 13, the NARE comprising: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126 or 213; and (iii) optionally, SEQ ID NO:

200.

16. The NARE according to claim 13, the 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; and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO:

200.

17. The NARE according to claim 13, wherein the NARE 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.

18. The NARE according to claim 13, wherein the NARE comprises: (i) SEQ ID NO: 131; (ii) SEQ ID NO: 126; and (iii) optionally, SEQ ID NO:

200.

19. A NARE, wherein the NARE comprises: (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. The NARE according to claim 19, wherein the NARE 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.

21. The NARE according to claim 19, wherein the NARE comprises: (i) any one of SEQ ID NOs: 190 - 199; and (ii) SEQ ID NO:

155.

22. A NARE, wherein the NARE comprises: (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. The NARE according to claim 22, wherein the NARE 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.

24. The NARE according to claim 22, wherein the NARE comprises: (i) any one of SEQ ID NOs: 190 - 199; and (ii) any one of SEQ ID NOs: 128 - 130.

25. A NARE, wherein the NARE comprises: (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. The NARE as claimed in claim 25, wherein the NARE 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.

27. The NARE as claimed in claim 25, wherein 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.

28. A NARE, wherein the NARE comprises: (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. The NARE as claimed in claim 28, wherein the NARE 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.

30. The NARE as claimed in claim 28, wherein the NARE 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.

31. The NARE as claimed in claim 28, wherein the NARE 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.

32. The NARE as claimed in claim 28, wherein the NARE 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.

33. The NARE as claimed in claim 28, wherein the NARE comprises: (i) SEQ ID NO: 121; and (ii) SEQ ID NOs: 166 - 169, 171 or 172.

34. The NARE according to claim 28, wherein the NARE 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.

35. The NARE according to claim 28, wherein the NARE 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.

36. The NARE according to claim 28, wherein the NARE comprises: (i) SEQ ID NO: 122 or 154; (ii) SEQ ID NO: 121; and (iii) SEQ ID NO:

167.

37. A NARE, wherein the NARE comprises: (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. The NARE according to claim 37, wherein the NARE 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.

39. The NARE according to claim 37, wherein 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.

40. The NARE according to claim 37, wherein the NARE 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.

41. The NARE according to claim 37, wherein the NARE 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.

42. The NARE according to claim 37, wherein the NARE comprises: (i) SEQ ID NO: 122; and (ii) any one of SEQ ID NOs: 173 - 179.

43. A NARE, wherein the NARE comprises: (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. The NARE according to claim 43, wherein the NARE comprises: (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. The NARE according to claim 43, wherein the NARE comprises: (i) SEQ ID NO: 184; and (ii) any one of SEQ ID NOs: 185 - 187.

46. A NARE, wherein the NARE comprises: (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. The NARE according to claim 46, wherein the NARE 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.

48. The NARE according to claim 46, wherein the NARE comprises: (i) SEQ ID NO: 180; and (ii) any one of SEQ ID NOs: 181 or 182.

49. A NARE, wherein the NARE 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.

50. The NARE according to claim 49, wherein the NARE 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.

51. The NARE according to claim 49, wherein 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.

52. The NARE according to claim 49, wherein the NARE 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.

53. The NARE as claimed in claim 49, wherein the NARE 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 NO: 200, 201, 205 or 208.

54. The NARE as claimed in claim 49, wherein the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 182; and (iii) any one of SEQ ID NO: 200, 201, 205 or 208.

55. A NARE, wherein the NARE comprises: (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 NO: 128 - 130.

56. The NARE as claimed in claim 55, wherein the NARE 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 NO: 128 - 130.

57. The NARE as claimed in claim 55, wherein the NARE comprises: (i) SEQ ID NO: 154 or 164; and (ii) any one of SEQ ID NO: 128 - 130.

58. A NARE, wherein the NARE comprises: (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. The NARE as claimed in claim 58, wherein the NARE 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.

60. The NARE as claimed in claim 58, wherein the NARE comprises: (i) SEQ ID NO: 170; (ii) optionally, SEQ ID NO: 188; and (iii) SEQ ID NO: 203 or 204.

61. A NARE, wherein the NARE comprises: (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 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) 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) (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. The NARE according to claim 61, wherein the NARE comprises: (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 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) 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) (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. The NARE according to claim 61, wherein the NARE comprises: (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) (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 NO: 1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70 or 74 - 120.

65. The NARE according to claim 64, wherein the NARE comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70 or 74 - 120.

66. The NARE according to claim 64, wherein the NARE comprises any one of SEQ ID NO: 1 - 18, 20, 21, 23, 24, 32 - 49, 51 - 70 or 74 - 120.

67. A NARE, wherein the NARE comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 1-18 or 32.

68. The NARE according to claim 67, wherein the NARE comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 1-18 or 32.

69. The NARE according to claim 67, wherein the NARE comprises any one of SEQ ID NO: 1-18 or 32.

70. An expression construct, wherein the expression construct comprises the NARE according to any one of the preceding claims and a transgene operably linked thereto.

71. The expression construct according to claim 70, wherein the expression construct further comprises a polyadenylation sequence.

72. A vector, wherein the vector comprises the expression construct according to claim 70.

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

74. The vector according to claim 72, wherein the vector is a viral vector.

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

76. A vector comprising a nucleic acid sequence, wherein the nucleic acid sequence comprises (i) the expression construct according to claim 70, and (ii) one or more inverted terminal repeats (ITRs).

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

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

79. A cell, wherein the cell comprises the expression construct according to claim 70 or the vector according to any one of claims 72-78.

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

81. A pharmaceutical composition, wherein the pharmaceutical composition comprises: (i) the expression construct according to claim 70 or the vector according to any one of claims 72-78, and (ii) a pharmaceutically acceptable excipient.

82. A method for expressing a transgene in a cell, wherein the cell comprises the expression construct according to claim 70 or the vector according to any one of claims 72-78.

83. A method for regulating transgene expression in a cell, wherein the cell comprises the expression construct according to claim 70 or the vector according to any one of claims 72-78.

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

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