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

Abstract

This application relates to nucleic acid regulatory elements (NAREs) that can enhance gene expression in the liver. This application further relates to methods utilizing NAREs and uses of NAREs. Expression cassettes and vectors containing NAREs are also disclosed. These are particularly useful for applications using gene therapy.

Description

[Technical Field]

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

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (SeqList-162027-54276.xml, size: 359,832 bytes, and creation date: October 6, 2023) are incorporated herein by reference in their entirety.

[0003] This application relates to nucleic acid regulatory elements that can enhance gene expression in various tissues, or tissues, including liver tissue in particular. This application also relates to methods that utilize these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. These are particularly useful for applications using gene therapy. [Background technology]

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

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

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

[0007] Provided herein are nucleic acid regulatory elements (NAREs) comprising NAREs that are particularly suitable for expression of operably linked sequences (e.g., protein- or RNA-coding sequences) in the liver. Methods for utilizing NAREs and uses of NAREs are also provided herein. For example, provided herein are methods for expressing transgenes operably linked to one or more of the nucleic acid regulatory elements disclosed herein. Also provided are expression cassettes and vectors containing NAREs, and cells comprising such expression cassettes and vectors.

[0008] Provided herein are polynucleotide sequences comprising a sequence at least 80% identical to a NARE selected from the group consisting of L2-L7, L9, L10, L10_v2, L11-L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16-L62, L64, L66, L69-L78, L78b, and L79-L140 (see Table 7). Provided herein are polynucleotide sequences comprising a sequence at least 90% identical to a NARE selected from the group consisting of L2 to L7, L9, L10, L10_v2, L11 to L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16 to L62, L64, L66, L69 to L78, L78b, and L79 to L140. Provided herein are polynucleotide sequences comprising a NARE selected from the group consisting of L2 to L7, L9, L10, L10_v2, L11 to L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16 to L62, L64, L66, L69 to L78, L78b, and L79 to L140.

[0009] Provided herein are polynucleotides comprising a sequence at least 80% identical to a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89. Provided herein are polynucleotides comprising a sequence at least 90% identical to a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89. Provided herein are polynucleotides comprising a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89.

[0010] Provided herein is a polynucleotide comprising a sequence at least 80% identical to a NARE selected from the group consisting of L45, L123, L132, and L133. Provided herein is a polynucleotide comprising a sequence at least 90% identical to a NARE selected from the group consisting of L45, L123, L132, and L133. Provided herein is a polynucleotide comprising a NARE selected from the group consisting of L45, L123, L132, and L133.

[0011] In embodiments, there is provided a polynucleotide sequence comprising a sequence that is at least 80% identical to a NARE sequence provided in Table 7. In embodiments, there is provided a polynucleotide sequence comprising a sequence that is at least 90% identical to a NARE sequence provided in Table 7. In embodiments, there is provided a polynucleotide comprising a NARE sequence provided in Table 7.

[0012] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249. In some embodiments, the NARE comprises (i) SEQ ID NO: 74, and (ii) any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249.

[0013] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 151 or SEQ ID NO: 155. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 151 or SEQ ID NO: 155. In some embodiments, the NARE comprises (i) SEQ ID NO: 150, and (ii) SEQ ID NO: 151, or SEQ ID NO: 155.

[0014] In one aspect, a NARE is provided that comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, or 158; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 169, 172, or 173. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, or 158, (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174, and (iii) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 169, 172, or 173. In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 154, 157, or 158, (ii) any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174, and (iii) optionally, any one of SEQ ID NOs: 169, 172, or 173.

[0015] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 153, (ii) a sequence that is at least 90% identical to SEQ ID NO: 171, and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, 158, or 176. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 153, (ii) a sequence that is at least 95% identical to SEQ ID NO: 171, and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, 158, or 176. In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 171, and (iii) any one of SEQ ID NOs: 154, 157, 158, or 176.

[0016] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 153, (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250, and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 153, (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250, and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171. In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250, and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171.

[0017] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 155, (ii) a sequence that is at least 90% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 175 or 177-187. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 153, (ii) a sequence that is at least 95% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 175 or 177-187. In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 176, and (iii) any one of SEQ ID NOs: 175 or 177-187.

[0018] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 190 or SEQ ID NO: 191, (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 193, 194, or 195, and (iii) optionally, a sequence that is at least 90% identical to SEQ ID NO: 162, (iv) optionally, a sequence that is at least 90% identical to SEQ ID NO: 168, and (v) optionally, SEQ ID NO: 169. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 190 or SEQ ID NO: 191, (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 193, 194, or 195, and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 162, (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 168, and (v) optionally, SEQ ID NO: 169. In some embodiments, the NARE comprises (i) SEQ ID NO: 190 or SEQ ID NO: 191, (ii) any one of SEQ ID NOs: 193, 194, or 195, (iii) optionally, SEQ ID NO: 162, (iv) optionally, SEQ ID NO: 168, and (v) optionally, SEQ ID NO: 169.

[0019] In one aspect, a NARE is provided that comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 168; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 197, 202, or 203; (iv) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206; (v) optionally, a sequence that is at least 90% identical to SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 166, 193, or 194. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 168, (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 197, 202, or 203, (iv) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206, (v) optionally, a sequence that is at least 95% identical to SEQ ID NO: 155 or SEQ ID NO: 162, and (vi) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 166, 193, or 194. In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) any one of SEQ ID NOs: 197, 202, or 203, (iv) optionally, any one of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206, (v) optionally, SEQ ID NO: 155 or SEQ ID NO: 162, and (vi) optionally, any one of SEQ ID NOs: 166, 193, or 194.

[0020] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 192, (ii) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO: 194, and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 201-203. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 192, (ii) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO: 194, and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 201-203. In some embodiments, the NARE comprises (i) SEQ ID NO: 192, (ii) SEQ ID NO: 193 or SEQ ID NO: 194, and (iii) any one of SEQ ID NOs: 201-203.

[0021] In one aspect, a NARE is provided that comprises: (i) a sequence that is at least 90% identical to SEQ ID NO: 155, (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238, (iii) optionally, a sequence that is 90% identical to SEQ ID NO: 168, (iv) optionally, a sequence that is at least 90% identical to SEQ ID NO: 169, and (v) optionally, a sequence that is at least 90% identical to SEQ ID NO: 197. In some embodiments, the NARE comprises: (i) a sequence that is at least 95% identical to SEQ ID NO: 155, (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238, (iii) optionally, a sequence that is 90% identical to SEQ ID NO: 168, (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 169, and (v) optionally, a sequence that is at least 95% identical to SEQ ID NO: 197. In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) a sequence at least 95% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238, (iii) optionally, a sequence that is 90% identical to SEQ ID NO: 168, (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 169, and (v) optionally, a sequence that is at least 95% identical to SEQ ID NO: 197.

[0022] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 248, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 239-247. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 248, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 239-247. In some embodiments, the NARE comprises (i) SEQ ID NO: 248, and (ii) any one of SEQ ID NOs: 239-247. In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 208, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 209-214.

[0023] In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 208, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 209-214. In some embodiments, the NARE comprises (i) SEQ ID NO: 208, and (ii) any one of SEQ ID NOs: 209-214. In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO: 194, (ii) a sequence that is at least 90% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 90% identical to SEQ ID NOs: 207 or any one of 209-215.

[0024] In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO: 194, (ii) a sequence that is at least 95% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 207 or 209-215. In some embodiments, the NARE comprises (i) SEQ ID NO: 193 or SEQ ID NO: 194, (ii) SEQ ID NO: 215, and (iii) any one of SEQ ID NOs: 207 or 209-215. In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 155, (ii) a sequence that is at least 90% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 207 or 209-215.

[0025] In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 155, (ii) a sequence that is at least 95% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 95% identical to SEQ ID NO: 207 or any one of 209-215. In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) any one of SEQ ID NO: 207 or 209-215.

[0026] In one aspect, a) (i) a sequence that is at least 90% identical to SEQ ID NO: 53; b) (i) a sequence that is at least 90% identical to SEQ ID NO: 195, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 202; c) (i) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 168, (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 169, (iii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 197, and (iv) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 203. d) (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 220; e) a sequence that is at least 90% identical to (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 229, or f) A NARE is provided, comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 230.

[0027] In some embodiments, the NARE is a) (i) a sequence that is at least 95% identical to SEQ ID NO: 53; b) (i) a sequence that is at least 95% identical to SEQ ID NO: 195, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 202; c) (i) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 168, (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 169, (iii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 197, and (iv) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 203. d) (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 220; e) (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 229, or f) (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 230.

[0028] In some embodiments, the NARE is a) (i) SEQ ID NO: 53; b) (i) SEQ ID NO: 195, and (ii) SEQ ID NO: 202; c) (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, and (iv) SEQ ID NO: 203; d) (i) SEQ ID NO: 74, and (ii) SEQ ID NO: 220; e) (i) SEQ ID NO: 74, and (ii) SEQ ID NO: 229, or f) comprising (i) SEQ ID NO: 74, and (ii) SEQ ID NO: 230.

[0029] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 166, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, or 158. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 166, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, or 158. In some embodiments, the NARE comprises (i) SEQ ID NO: 166, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0030] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 165, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, or 158. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 165, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, or 158. In some embodiments, the NARE comprises (i) SEQ ID NO: 165, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0031] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 199, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO: 194. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 199, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO: 194. In some embodiments, the NARE comprises (i) SEQ ID NO: 199, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0032] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 164, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 200. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 164, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 200. In some embodiments, the NARE comprises (i) SEQ ID NO: 164, and (ii) SEQ ID NO: 200.

[0033] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 189, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO: 194. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 189, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO: 194. In some embodiments, the NARE comprises (i) SEQ ID NO: 189, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0034] In one aspect, a NARE is provided that comprises (i) a sequence that is at least 90% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 195. In some embodiments, the NARE comprises (i) a sequence that is at least 95% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 195. In some embodiments, the NARE comprises (i) SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) SEQ ID NO: 195.

[0035] In one aspect, a NARE is provided that comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 3-8, 11-69, 71-73, or 76-149. In some embodiments, the NARE comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 3-8, 11-69, 71-73, or 76-149. In some embodiments, the NARE comprises any one of SEQ ID NOs: 3-8, 11-69, 71-73, or 76-149. In some embodiments, the NARE comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 53, 80, 81, 132, 141, or 142. In some embodiments, the NARE comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 53, 80, 81, 132, 141, or 142. In some embodiments, the NARE comprises any one of SEQ ID NOs: 53, 80, 81, 132, 141, or 142.

[0036] In one aspect, an expression construct is provided comprising a NARE disclosed herein and an operably linked transgene. In some embodiments, the expression construct further comprises a polyadenylation sequence.

[0037] In one aspect, a vector is provided comprising an expression construct disclosed herein. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector comprises a nucleic acid sequence comprising: (i) an expression construct comprising a NARE disclosed herein and an operably linked transgene; and (ii) one or more inverted terminal repeats (ITRs). In some embodiments, the vector comprises: (i) an expression construct disclosed herein; and (ii) a nucleic acid sequence comprising one or more inverted terminal repeats (ITRs). In some embodiments, the 5'ITR and 3'ITR are derived from adeno-associated virus (AAV) serotype AAV2.

[0038] In one aspect, a cell is provided comprising the expression construct disclosed herein or the vector disclosed herein. In one embodiment, the cell is a liver cell.

[0039] In one aspect, a pharmaceutical composition is provided that includes (i) an expression construct disclosed herein or a vector disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0040] In one aspect, a method is provided for expressing a transgene in a cell comprising an expression construct disclosed herein or a vector disclosed herein. In one aspect, a method is provided for regulating transgene expression in a cell comprising an expression construct disclosed herein or a vector disclosed herein. In one embodiment, the cell is a liver cell.

[0041] In one aspect, provided is a method of treating Wilson's disease in a subject in need thereof, the method comprising administering to the subject an expression construct disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein. In one embodiment, the subject has a mutation in the ATP7B gene. In one embodiment, the subject is human. [Brief explanation of the drawings]

[0042] [Figure 1] This diagram shows a bioinformatics pipeline for the design and optimization of a given promoter sequence using AI. The pipeline is divided into two parallel approaches: (i) promoter enhancement, in which all known / annotated enhancer fragments were collected from the ENCODE database and inserted upstream of the promoter sequence; and (ii) saturation mutagenesis, in which all possible point mutations (no indels) were generated in silico for each base pair of the original input promoter sequence. The best candidates from both approaches were then tested in vitro in the desired cell line and their activity recorded by fluorescence. The best in vitro performers from both strategies were then combined by rational design in a final in vitro screen.

[0043] [Figure 2] Dual reporter design and measurements. A shows the design of the dual reporter constructs. A candidate NARE was cloned upstream (5') of the mClover3 coding sequence. Each construct contains a constant region containing the tdTomato transgene, which is used as a normalization control. B provides example flow cytometry data obtained with the dual reporter system, in which mClover3 expression in tdTomato+ cells reflects the level of promoter activity.

[0044] [Figure 3A] Efficacy of selected NAREs in liver cells in vitro. NAREs specifically suited for expression in the liver were rationally designed by utilizing established or predicted liver-specific core NAREs and enhancers and incorporating other novel changes based on various bioinformatics approaches. Expression was assessed using a dual reporter assay. Efficacy of the indicated NAREs compared to the reference promoters CAG, CMV, and AAT in Huh7 cells.

[0045] [Figure 3B] Efficacy of selected NAREs in liver cells in vitro. NAREs specifically suited for liver expression were rationally designed by utilizing established or predicted liver-specific core NAREs and enhancers and incorporating other novel changes based on various bioinformatics approaches. Expression was assessed using a dual reporter assay. Tissue specificity for selected NAREs (relative expression in HepG2 liver cells vs. HEK293T cells).

[0046] [Figure 3C] Efficacy of selected NAREs in liver cells in vitro. NAREs specifically suited for expression in the liver were rationally designed by utilizing established or predicted liver-specific core NAREs and enhancers and incorporating other novel changes based on various bioinformatics approaches. Expression was assessed using a dual reporter assay. Efficacy of the indicated NAREs in the human liver cell line Huh7 cells compared to the AAT promoter (L1, 305 bp).

[0047] [Figure 3D] Efficacy of selected NAREs in liver cells in vitro. NAREs specifically suited for liver expression were rationally designed by utilizing established or predicted liver-specific core NAREs and enhancers and incorporating other novel changes based on various bioinformatics approaches. Expression was assessed using a dual reporter assay. Brightfield and mClover3 fluorescence of NARE L45 compared to the AAT promoter (L1). While similar cell numbers are present in the brightfield image, stronger mClover3 fluorescence is observed with L45. NARE L45 drove higher mClover3 expression than AAT after 48 hours of transfection in Huh7 cells.

[0048] [Figure 4A]Potency of selected NAREs in liver cells in vitro. Optimization of NARE L45 resulted in new NAREs that were up to 9-fold more potent than the parent (L45). NARE modifications included enhancers, cis-regulatory elements, introns, or other bioinformatically identified sequences predicted to enhance activity. Expression of L45 and modified NAREs against CAG in Huh7 cells as determined using a dual reporter assay.

[0049] [Figure 4B] Potency of selected NAREs in liver cells in vitro. Optimization of NARE L45 resulted in new NAREs up to 9-fold stronger than the parent (L45). NARE modifications included enhancers, cis-regulatory elements, introns, or other bioinformatically identified sequences predicted to enhance activity. Expression levels of 12 liver-specific NAREs were up to 4-fold stronger than the CAG reference promoter and stronger than current clinically used promoters (AAT, LP1, TBG). Dual reporter plasmids were transiently transfected into a human liver cell line (Huh7), and expression of the mClover3 transgene was assayed by flow cytometry.

[0050] [Figure 4C] Potency of selected NAREs in liver cells in vitro. Potency of indicated NAREs in Huh7 cells. DETAILED DESCRIPTION OF THE INVENTION

[0051] Nucleic acid regulatory elements (NAREs), including promoters, are essential components of gene therapy that control the expression level and durability of therapeutic genes. NAREs can, for example, drive cell-specific expression of transgenes independently of capsid selection. Incorporation of stronger NAREs can increase potency and efficacy at lower viral vector doses, thereby potentially reducing safety risks, immune responses, and the cost of vector production. Furthermore, reducing NARE size while maintaining strength and specificity allows for the efficient packaging of larger transgenes or expression cassettes into AAVs. This application provides a library of NAREs for improving the safety, efficacy, and durability of therapeutic transgene expression.

[0052] Many NAREs, and in particular promoter sequences, have been characterized and optimized through traditional low-throughput analysis (rational design) or newer high-throughput methodologies (e.g., MPRA), but these approaches still require expensive and time-consuming in vitro optimization. Here, we used an advanced artificial intelligence model to repurpose convolutional neural networks (CNNs) and optimize them to predict promoter efficacy. A library of NAREs was computationally constructed by cloning all known enhancer elements reported in the ENCODE database upstream of strong, small, constitutive promoters. The precise spacing between the enhancer elements and the promoter sequence that performed particularly well was then optimized. In parallel, in silico saturation mutagenesis was performed, whereby all possible point mutations within the promoter sequence were introduced and those with the best effect on promoter efficacy were selected. Specifically, well-performing elements were produced and tested in vitro. While most NAREs performed better or equivalently to the original promoter, the best enhancer elements and point mutations were selected and combined in a second optimization round. After synthesis and testing of this new set in vitro, enhanced NAREs were obtained that exhibited increased potency.

[0053] NARE Provided herein are NAREs that may contain promoters and / or enhancers as part of their sequences. Conventionally, a promoter is defined as a DNA region where transcription is initiated. A promoter contains specific DNA motifs that can be accessed by transcription factors (TFs) and their complexes. On the other hand, an enhancer is defined as a DNA region that amplifies transcription initiation by directly interacting with a target promoter. Similarly, enhancer sequences distal to a target promoter contain DNA motifs that act as binding sites for TFs and cofactors. The term promoter can sometimes be used as a shortened form to refer to a nucleic acid sequence containing multiple regulatory elements. For example, the regulatory promoter CAG contains a CMV enhancer, a β-actin promoter region, and an intron, but can also be referred to as a promoter. Specifically, the CAG promoter consists of (1) a cytomegalovirus (CMV) early enhancer element, (2) the promoter, the first exon and first intron of the chicken β-actin gene, and (3) the splice acceptor of the rabbit β-globin gene. As used herein, the term "nucleic acid regulatory element" can refer to a promoter, defined in the conventional sense, as well as a combination of elements that include a promoter and / or other nucleic acid regulatory elements that regulate expression of a gene operably linked to the element(s). A nucleic acid regulatory element can be, or can include, for example, a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0054] As used herein, the term "NARE" can refer to a promoter, as defined in the conventional sense, as well as a combination of nucleic acid regulatory elements, including promoters and / or other nucleic acid regulatory elements, that regulate expression of a gene operably linked to the element(s). A nucleic acid regulatory element can be, or can include, for example, a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0055] As used herein, "transgene" refers to a gene (particularly the coding sequence of a gene) that is transferred into one or more cells of an organism, for example, using a vector described herein. A transgene can encode a protein or RNA that is normally expressed in the cells of the target organism, or it can encode a protein or RNA from a different organism. A transgene can be integrated into the genome of the target cell or can be present as part of an extrachromosomal expression construct.

[0056] As used herein, "operably linked" refers to a first molecule linked to a second molecule, the molecules being positioned so that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single, continuous molecule, and may or may not be adjacent. For example, if a NARE regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, the NARE is operably linked to the transcribable polynucleotide molecule. In addition, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without any intervening nucleotides.

[0057] NAREs particularly suitable for driving expression in the liver are provided herein. As used herein, NAREs beginning with "L" are particularly useful for expressing genes in the liver. Liver NAREs may be liver-specific, meaning that they exhibit significantly increased or preferential expression of an operably linked transgene in the liver compared to other tissues. Tables 5 and 7 provide NAREs particularly suitable for driving expression in the liver. Table 4 also provides NAREs suitable for driving expression in various tissues, including the liver.

[0058] In some embodiments, polynucleotide sequences are provided that comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE provided in Table 5 or Table 7. In some embodiments, polynucleotides are provided that comprise one or more NAREs provided in Table 5 or Table 7.

[0059] Provided herein are polynucleotide sequences comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of L2-L7, L9, L10, L10_v2, L11-L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16-L62, L64, L66, L69-L78, L78b, and L79-L140. Provided herein are polynucleotide sequences comprising a NARE selected from the group consisting of L2 to L7, L9, L10, L10_v2, L11 to L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16 to L62, L64, L66, L69 to L78, L78b, and L79 to L140.

[0060] As used herein, the term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides.Identity can be determined by comparing the position of each sequence that can be aligned for comparison purposes.For example, if the position of the nucleotide sequence being compared is occupied by the same base, the molecules are identical at that position.The degree of identity between nucleic acid sequences or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990), and the ALIGN program (version 2.0). The well-known Smith-Waterman algorithm may also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894, BLAST 2.0 at ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions, and other modifications when comparing sequences.

[0061] Provided herein are polynucleotides comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89. Provided herein are polynucleotides comprising a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89.

[0062] Provided herein is a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE selected from the group consisting of L45, L123, L132, and L133. Provided herein is a polynucleotide comprising a NARE selected from the group consisting of L45, L123, L132, and L133.

[0063] Provided herein are methods for expressing a transgene in the liver, wherein the transgene is operably linked to a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a NARE provided in Table 5 or Table 7. Provided herein are methods for expressing a transgene in the liver, wherein the transgene is operably linked to a NARE provided in Table 5 or Table 7.

[0064] Provided herein are methods for expressing a transgene in the liver, 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 L2-L7, L9, L10, L10_v2, L11-L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16-L62, L64, L66, L69-L78, L78b, and L79-L140. Provided herein is a method for expressing a transgene in the liver, wherein the transgene is operably linked to a NARE selected from the group consisting of L2-L7, L9, L10, L10_v2, L11-L13, L13_TISU, L13_v2, L14, L15, L14_L21, L15_L21_TISU, L16-L62, L64, L66, L69-L78, L78b, and L79-L140.

[0065] In embodiments, the transgene is an ATP7B or ATB7B minigene.

[0066] In some embodiments, the NARE comprises an intron or a portion of an intron, such as the ACTA1 intron, ACTA1e intron, ACTC1 intron, ACTC1.3 intron, ACTC1e intron, ACTC1e-ACTC1p intron, ALDOA intron, APOC intron, APOC1 intron, ATF5 intron, CAMK2A intron, CBA intron, chimeric intron, CMV-rabbit beta globulin intron, CRYAB intron, DES.4 intron, EEF1A1 intron, EEF1B2 intron, EF1a intron, FHL1 intron, FLOT 1 intron, FXYD1 intron, GFAP intron, HBB intron, hCPE intron, hEf1a2-intron, HPD intron, IFI27L2 intron, mouse IgG chimeric intron, MVM intron, MVMi-AATp intron, MVMi-SynE-mTTRp intron, Rabit beta globin intron, RBP4 intron, RPL26 intron, RPL27 intron, S100A6 intron, sEEF1A1 intron, SV40 intron, TMSB10 intron, and UCHL1 intron.

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

[0068] In embodiments, the NARE comprises a UTR or a portion of a UTR. The UTR or portion of a UTR may be selected from CTNNB1 UTR, hEf1a2-utr1, hEf1a2-utr2, hNSE utr1, hNSE utr2, hSyn1_utr1, hSyn1_utr2, HTLV 5'UTR, HTLV 5'UTR, L21 UTR, TMSB10_utr1, and TMSB10_utr2.

[0069] NAREs used as references or controls are also provided herein. Some NAREs are provided for constitutive expression of an operably linked transgene, i.e., for maintaining transgene expression at a constant level. Constitutive NAREs can drive expression of an operably linked transgene in various cell types and tissues. As used herein, promoters beginning with "C" are constitutive NAREs. The constitutive NAREs disclosed herein may be useful for expressing genes in the liver, muscle, and / or central nervous system. The sequences of control NAREs are provided in Table 4.

[0070] In some embodiments, the NARE comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3-8, 11-69, 71-73, 76-149, and 256.

[0071] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NOs: 3-8, 11-69, 71-73, 76-149, and 256.

[0072] In some embodiments, the NARE comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 15, 45-64, 71, and 98.

[0073] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 45-64 and 98.

[0074] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 151 or SEQ ID NO: 155.

[0075] In some embodiments, the NARE comprises (i) SEQ ID NO: 150, and (ii) SEQ ID NO: 151 or SEQ ID NO: 155.

[0076] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 151.

[0077] In some embodiments, the NARE comprises (i) SEQ ID NO: 150, and (ii) SEQ ID NO: 151.

[0078] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0079] In some embodiments, the NARE comprises (i) SEQ ID NO: 150, and (ii) SEQ ID NO: 155.

[0080] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; 5% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical, and (iii) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 169, 172, or 173.

[0081] In some embodiments, the NARE comprises (i) any one of SEQ ID NOs: 154, 157, or 158; (ii) any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, any one of SEQ ID NOs: 169, 172, or 173.

[0082] In some embodiments, the NARE comprises: (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 169, 172, or 173.

[0083] In some embodiments, the NARE comprises (i) SEQ ID NO: 157, (ii) any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174, and (iii) optionally, any one of SEQ ID NOs: 169, 172, or 173.

[0084] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 156, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0085] In some embodiments, the NARE comprises (i) SEQ ID NO: 156, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0086] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 156, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0087] In some embodiments, the NARE comprises (i) SEQ ID NO: 156, and (ii) SEQ ID NO: 157.

[0088] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 161, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0089] In some embodiments, the NARE comprises (i) SEQ ID NO: 161, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0090] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 161, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0091] In some embodiments, the NARE comprises (i) SEQ ID NO: 161, and (ii) SEQ ID NO: 157.

[0092] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0093] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0094] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0095] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, and (ii) SEQ ID NO: 157.

[0096] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 163, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0097] In some embodiments, the NARE comprises (i) SEQ ID NO: 163, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0098] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 163, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0099] In some embodiments, the NARE comprises (i) SEQ ID NO: 163, and (ii) SEQ ID NO: 157.

[0100] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 166, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0101] In some embodiments, the NARE comprises (i) SEQ ID NO: 166, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0102] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 166, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0103] In some embodiments, the NARE comprises (i) SEQ ID NO: 166, and (ii) SEQ ID NO: 157.

[0104] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 167, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0105] In some embodiments, the NARE comprises (i) SEQ ID NO: 167, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0106] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 167, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0107] In some embodiments, the NARE comprises (i) SEQ ID NO: 167, and (ii) SEQ ID NO: 157.

[0108] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0109] In some embodiments, the NARE comprises any one of (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, and (iii) SEQ ID NO: 154, 157, or 158.

[0110] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0111] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, and (iii) SEQ ID NO: 157.

[0112] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0113] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) SEQ ID NO: 157.

[0114] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 172.

[0115] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.

[0116] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 157, or 158, and (iii) SEQ ID NO: 172 or SEQ ID NO: 173.

[0117] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 157, and (iii) SEQ ID NO: 172.

[0118] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.

[0119] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 157, or 158, and (iii) SEQ ID NO: 172 or SEQ ID NO: 173.

[0120] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 173.

[0121] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 157, and (iii) SEQ ID NO: 173.

[0122] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0123] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0124] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0125] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) SEQ ID NO: 157.

[0126] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 174, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0127] In some embodiments, the NARE comprises (i) SEQ ID NO: 174, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0128] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 174, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 157.

[0129] In some embodiments, the NARE comprises (i) SEQ ID NO: 174, and (ii) SEQ ID NO: 157.

[0130] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 171; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, 158, or 176.

[0131] In some embodiments, the NARE comprises any one of (i) SEQ ID NO: 153, (ii) SEQ ID NO: 171, and (iii) SEQ ID NO: 154, 157, 158, or 176.

[0132] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 171, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 154 or SEQ ID NO: 176.

[0133] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 171, and (iii) SEQ ID NO: 154 or SEQ ID NO: 176.

[0134] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 171.

[0135] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 157, or 158, and (iii) SEQ ID NO: 171.

[0136] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 154, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 171.

[0137] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 154, and (iii) SEQ ID NO: 171.

[0138] In some embodiments, the NARE comprises: (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250; and (iii) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 171.

[0139] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250, and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171.

[0140] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 176, 188, or 250, and (iii) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 171.

[0141] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 176, 188, or 250, and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171.

[0142] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 183, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0143] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 188, and (iii) SEQ ID NO: 155.

[0144] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0145] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0146] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 154.

[0147] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) SEQ ID NO: 154.

[0148] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, and 158; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0149] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) any one of SEQ ID NOs: 154, 157, and 158, and (iii) SEQ ID NO: 155.

[0150] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 154, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0151] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 154, and (iii) SEQ ID NO: 155.

[0152] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 188.

[0153] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 188.

[0154] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) SEQ ID NO: 188.

[0155] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 250.

[0156] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) SEQ ID NO: 250.

[0157] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176.

[0158] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, and (ii) SEQ ID NO: 176.

[0159] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 171.

[0160] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 171.

[0161] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 153, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0162] In some embodiments, the NARE comprises (i) SEQ ID NO: 153, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0163] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 175 or 177-187.

[0164] In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) SEQ ID NO: 176, and (iii) any one of SEQ ID NOs: 175 or 177-187.

[0165] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 175; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0166] In some embodiments, the NARE comprises (i) SEQ ID NO: 175, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0167] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 177, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0168] In some embodiments, the NARE comprises (i) SEQ ID NO: 177, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0169] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 178, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0170] In some embodiments, the NARE comprises (i) SEQ ID NO: 178, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0171] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 179, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0172] In some embodiments, the NARE comprises (i) SEQ ID NO: 179, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0173] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 180, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0174] In some embodiments, the NARE comprises (i) SEQ ID NO: 180, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0175] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 181; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0176] In some embodiments, the NARE comprises (i) SEQ ID NO: 181, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0177] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 182, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0178] In some embodiments, the NARE comprises (i) SEQ ID NO: 182, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0179] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 183, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0180] In some embodiments, the NARE comprises (i) SEQ ID NO: 183, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0181] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 184, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0182] In some embodiments, the NARE comprises (i) SEQ ID NO: 184, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0183] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 185; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0184] In some embodiments, the NARE comprises (i) SEQ ID NO: 185, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0185] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 186, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0186] In some embodiments, the NARE comprises (i) SEQ ID NO: 186, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0187] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 187, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 176, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0188] In some embodiments, the NARE comprises (i) SEQ ID NO: 187, (ii) SEQ ID NO: 176, and (iii) SEQ ID NO: 155.

[0189] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 193, 194, or 195. a sequence, (iii) optionally a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (iv) optionally a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; and (v) optionally, SEQ ID NO: 169.

[0190] In some embodiments, the NARE comprises (i) SEQ ID NO: 190 or SEQ ID NO: 191, (ii) any one of SEQ ID NOs: 193, 194, or 195, (iii) optionally, SEQ ID NO: 162, (iv) optionally, SEQ ID NO: 168, and (v) optionally, SEQ ID NO: 169.

[0191] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 195; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 195. 162, (iv) optionally a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168, and (v) optionally a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168, and (v) optionally SEQ ID NO: 169.

[0192] In some embodiments, the NARE comprises (i) SEQ ID NO: 190, (ii) SEQ ID NO: 193 or SEQ ID NO: 195, (iii) optionally, SEQ ID NO: 168, (iv) optionally, SEQ ID NO: 168, and (v) optionally, SEQ ID NO: 169.

[0193] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0194] In some embodiments, the NARE comprises (i) SEQ ID NO: 190 or SEQ ID NO: 191, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0195] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0196] In some embodiments, the NARE comprises (i) SEQ ID NO: 190, and (ii) SEQ ID NO: 193.

[0197] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, at least 99% identical to SEQ ID NO: 169; (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; and (v) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0198] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, (ii) SEQ ID NO: 168, (iii) SEQ ID NO: 169, (iv) SEQ ID NO: 190 or SEQ ID NO: 191, and (v) SEQ ID NO: 193 or SEQ ID NO: 194.

[0199] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, at least 99% identical to SEQ ID NO: 169; (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; and (v) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0200] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, (ii) SEQ ID NO: 168, (iii) SEQ ID NO: 169, (iv) SEQ ID NO: 190 or SEQ ID NO: 191, and (v) SEQ ID NO: 193 or SEQ ID NO: 194.

[0201] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190; and (v) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0202] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, (ii) SEQ ID NO: 168, (iii) SEQ ID NO: 169, (iv) SEQ ID NO: 190, and (v) SEQ ID NO: 193.

[0203] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 195.

[0204] In some embodiments, the NARE comprises (i) SEQ ID NO: 190 or SEQ ID NO: 191, and (ii) SEQ ID NO: 195.

[0205] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 195.

[0206] In some embodiments, the NARE comprises (i) SEQ ID NO: 190, and (ii) SEQ ID NO: 195.

[0207] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 197, 202, or 203; (iv) optionally, a sequence selected from the group consisting of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206; (v) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 166, 193, or 194.

[0208] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) any one of SEQ ID NOs: 197, 202, or 203, (iv) optionally, any one of SEQ ID NOs: 190, 191, 192, 196, 203, 204, 205, or 206, (v) optionally, SEQ ID NO: 155 or SEQ ID NO: 162, and (vi) optionally, any one of SEQ ID NOs: 166, 193, or 194.

[0209] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197 or SEQ ID NO: 202; (iv) a sequence that is optionally a sequence of SEQ ID NO: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 6, 203, 204, or 206; (v) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 166 or SEQ ID NO: 193.

[0210] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197 or SEQ ID NO: 202, (iv) optionally, any one of SEQ ID NOs: 191, 192, 196, 203, 204, or 206, (v) optionally, SEQ ID NO: 155 or SEQ ID NO: 162, and (vi) optionally, SEQ ID NO: 166 or SEQ ID NO: 193.

[0211] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169. , (iii) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197, and (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191.

[0212] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, and (iv) SEQ ID NO: 190 or SEQ ID NO: 191.

[0213] In some embodiments, the NARE comprises: (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197; and (iv) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 191.

[0214] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, and (iv) SEQ ID NO: 191.

[0215] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 166; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168. (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (v) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197; and (vi) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 196.

[0216] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, (ii) SEQ ID NO: 166, (iii) SEQ ID NO: 168, (iv) SEQ ID NO: 169, (v) SEQ ID NO: 197, and (vi) SEQ ID NO: 196.

[0217] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, at least 99% identical to SEQ ID NO: 169; (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:202 or SEQ ID NO:203; and (vi) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:193 or SEQ ID NO:194.

[0218] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, (ii) SEQ ID NO: 168, (iii) SEQ ID NO: 169, (iv) SEQ ID NO: 202 or SEQ ID NO: 203, and (vi) SEQ ID NO: 193 or SEQ ID NO: 194.

[0219] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202; and (vi) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0220] In some embodiments, the NARE comprises (i) SEQ ID NO: 162, (ii) SEQ ID NO: 168, (iii) SEQ ID NO: 169, (iv) SEQ ID NO: 202, and (vi) SEQ ID NO: 193.

[0221] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169. , (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197, and (iv) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203.

[0222] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, and (iv) SEQ ID NO: 202 or SEQ ID NO: 203.

[0223] In some embodiments, the NARE comprises: (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197; and (iv) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 203.

[0224] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, and (iv) SEQ ID NO: 203.

[0225] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197; % identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:205 or SEQ ID NO:206, (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:205 or SEQ ID NO:206, and (v) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:155.

[0226] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, (iv) SEQ ID NO: 205 or SEQ ID NO: 206, and (v) SEQ ID NO: 155.

[0227] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least (iv) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 206; and (v) sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0228] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, (iv) SEQ ID NO: 206, and (v) SEQ ID NO: 155.

[0229] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 192; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 201-203.

[0230] In some embodiments, the NARE comprises (i) SEQ ID NO: 192, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194, and (iii) any one of SEQ ID NOs: 201-203.

[0231] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 192, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193, and (iii) SEQ ID NO: 201 or SEQ ID NO: 202.

[0232] In some embodiments, the NARE comprises (i) SEQ ID NO: 192, (ii) SEQ ID NO: 193, and (iii) SEQ ID NO: 201 or SEQ ID NO: 202.

[0233] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 201, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0234] In some embodiments, the NARE comprises (i) SEQ ID NO: 201, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0235] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 201, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0236] In some embodiments, the NARE comprises (i) SEQ ID NO: 201, and (ii) SEQ ID NO: 193.

[0237] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0238] In some embodiments, the NARE comprises (i) SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0239] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0240] In some embodiments, the NARE comprises (i) SEQ ID NO: 202, and (ii) SEQ ID NO: 193.

[0241] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238; (iii) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (iv) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197.

[0242] In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) any one of SEQ ID NOs: 204, 205, 206, or 236-238, (iii) optionally, SEQ ID NO: 168, (iv) optionally, SEQ ID NO: 169, and (v) optionally, SEQ ID NO: 197.

[0243] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 204, 205, or 236-238; (iii) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168. (iv) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 197.

[0244] In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) any one of SEQ ID NOs: 204, 205, or 236-238, (iii) optionally, SEQ ID NO: 168, (iv) optionally, SEQ ID NO: 169, and (v) optionally, SEQ ID NO: 197.

[0245] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 204, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0246] In some embodiments, the NARE comprises (i) SEQ ID NO: 204, and (ii) SEQ ID NO: 155.

[0247] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0248] In some embodiments, the NARE comprises (i) SEQ ID NO: 205 or SEQ ID NO: 206, and (ii) SEQ ID NO: 155.

[0249] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0250] In some embodiments, the NARE comprises (i) SEQ ID NO: 205, and (ii) SEQ ID NO: 155.

[0251] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 238, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0252] In some embodiments, the NARE comprises (i) SEQ ID NO: 238, and (ii) SEQ ID NO: 155.

[0253] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 236 or SEQ ID NO: 237, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0254] In some embodiments, the NARE comprises (i) SEQ ID NO: 236 or SEQ ID NO: 237, and (ii) SEQ ID NO: 155.

[0255] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 237, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0256] In some embodiments, the NARE comprises (i) SEQ ID NO: 237, and (ii) SEQ ID NO: 155.

[0257] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 236 or SEQ ID NO: 237, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0258] In some embodiments, the NARE comprises (i) SEQ ID NO: 236 or SEQ ID NO: 237, and (ii) SEQ ID NO: 155.

[0259] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 236, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0260] In some embodiments, the NARE comprises (i) SEQ ID NO: 236, and (ii) SEQ ID NO: 155.

[0261] In some embodiments, the NARE is (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 90% identical, at least 99% identical to SEQ ID NO: 197; (iv) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 204; (v) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0262] In some embodiments, the NARE comprises (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, (iv) SEQ ID NO: 204, and (v) SEQ ID NO: 155.

[0263] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 239-247.

[0264] In some embodiments, the NARE comprises (i) SEQ ID NO: 248, and (ii) any one of SEQ ID NOs: 239-247.

[0265] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 239, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0266] In some embodiments, the NARE comprises (i) SEQ ID NO: 239, and (ii) SEQ ID NO: 248.

[0267] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 240, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0268] In some embodiments, the NARE comprises (i) SEQ ID NO: 240, and (ii) SEQ ID NO: 248.

[0269] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 241, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0270] In some embodiments, the NARE comprises (i) SEQ ID NO: 241, and (ii) SEQ ID NO: 248.

[0271] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 242, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0272] In some embodiments, the NARE comprises (i) SEQ ID NO: 242, and (ii) SEQ ID NO: 248.

[0273] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 243, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0274] In some embodiments, the NARE comprises (i) SEQ ID NO: 243, and (ii) SEQ ID NO: 248.

[0275] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 244, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0276] In some embodiments, the NARE comprises (i) SEQ ID NO: 244, and (ii) SEQ ID NO: 248.

[0277] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 245, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0278] In some embodiments, the NARE comprises (i) SEQ ID NO: 245, and (ii) SEQ ID NO: 248.

[0279] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 246, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0280] In some embodiments, the NARE comprises (i) SEQ ID NO: 246, and (ii) SEQ ID NO: 248.

[0281] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 247, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 248.

[0282] In some embodiments, the NARE comprises (i) SEQ ID NO: 247, and (ii) SEQ ID NO: 248.

[0283] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 209-214.

[0284] In some embodiments, the NARE comprises (i) SEQ ID NO: 208, and (ii) any one of SEQ ID NOs: 209-214.

[0285] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0286] In some embodiments, the NARE comprises (i) SEQ ID NO: 207, and (ii) SEQ ID NO: 208.

[0287] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 209, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0288] In some embodiments, the NARE comprises (i) SEQ ID NO: 209, and (ii) SEQ ID NO: 208.

[0289] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 210, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0290] In some embodiments, the NARE comprises (i) SEQ ID NO: 210, and (ii) SEQ ID NO: 208.

[0291] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 211, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0292] In some embodiments, the NARE comprises (i) SEQ ID NO: 211, and (ii) SEQ ID NO: 208.

[0293] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 212, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0294] In some embodiments, the NARE comprises (i) SEQ ID NO: 212, and (ii) SEQ ID NO: 208.

[0295] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 213, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0296] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, and (ii) SEQ ID NO: 208.

[0297] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 214, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 208.

[0298] In some embodiments, the NARE comprises (i) SEQ ID NO: 214, and (ii) SEQ ID NO: 208.

[0299] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249.

[0300] In some embodiments, the NARE comprises (i) SEQ ID NO: 74, and (ii) any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249.

[0301] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 194, 207, 209-214, 216-232, 233-235, or 249.

[0302] In some embodiments, the NARE comprises (i) SEQ ID NO: 74, and (ii) any one of SEQ ID NOs: 194, 207, 209-214, 216-232, 233-235, or 249.

[0303] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0304] In some embodiments, the NARE comprises (i) SEQ ID NO: 207, and (ii) SEQ ID NO: 74.

[0305] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 209, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0306] In some embodiments, the NARE comprises (i) SEQ ID NO: 209, and (ii) SEQ ID NO: 74.

[0307] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 210, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0308] In some embodiments, the NARE comprises (i) SEQ ID NO: 210, and (ii) SEQ ID NO: 74.

[0309] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 211, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0310] In some embodiments, the NARE comprises (i) SEQ ID NO: 211, and (ii) SEQ ID NO: 74.

[0311] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 212, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0312] In some embodiments, the NARE comprises (i) SEQ ID NO: 212, and (ii) SEQ ID NO: 74.

[0313] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 213, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0314] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, and (ii) SEQ ID NO: 74.

[0315] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 214, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0316] In some embodiments, the NARE comprises (i) SEQ ID NO: 214, and (ii) SEQ ID NO: 74.

[0317] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 207 or 209-215.

[0318] In some embodiments, the NARE comprises any one of (i) SEQ ID NO: 193 or SEQ ID NO: 194, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 207 or 209-215.

[0319] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 207 or 209-215.

[0320] In some embodiments, the NARE comprises (i) SEQ ID NO: 193, (ii) SEQ ID NO: 215, and (iii) any one of SEQ ID NOs: 207 or 209-215.

[0321] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0322] In some embodiments, the NARE comprises (i) SEQ ID NO: 207, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0323] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0324] In some embodiments, the NARE comprises (i) SEQ ID NO: 207, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193.

[0325] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 209, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0326] In some embodiments, the NARE comprises (i) SEQ ID NO: 209, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0327] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 209, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0328] In some embodiments, the NARE comprises (i) SEQ ID NO: 209, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193.

[0329] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 210, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0330] In some embodiments, the NARE comprises (i) SEQ ID NO: 210, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0331] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 210, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0332] In some embodiments, the NARE comprises (i) SEQ ID NO: 210, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193.

[0333] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0334] In some embodiments, the NARE comprises (i) SEQ ID NO:211, (ii) SEQ ID NO:215, and (iii) SEQ ID NO:193 or SEQ ID NO:194.

[0335] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0336] In some embodiments, the NARE comprises (i) SEQ ID NO: 211, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193.

[0337] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 212, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0338] In some embodiments, the NARE comprises (i) SEQ ID NO: 212, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0339] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 212, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0340] In some embodiments, the NARE comprises (i) SEQ ID NO: 212, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193.

[0341] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 213, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0342] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0343] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 213, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0344] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, (ii) SEQ ID NO: 215, and (iii) SEQ ID NO: 193.

[0345] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 214, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0346] In some embodiments, the NARE comprises (i) SEQ ID NO:213, (ii) SEQ ID NO:214, and (iii) SEQ ID NO:193 or SEQ ID NO:194.

[0347] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 214, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 215, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0348] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, (ii) SEQ ID NO: 214, and (iii) SEQ ID NO: 193.

[0349] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 207 or 209-215.

[0350] In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) any one of SEQ ID NOs: 207 or 209-215.

[0351] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 207 or 209-215.

[0352] In some embodiments, the NARE comprises (i) SEQ ID NO: 155, (ii) SEQ ID NO: 205, and (iii) any one of SEQ ID NOs: 207 or 209-215.

[0353] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0354] In some embodiments, the NARE comprises (i) SEQ ID NO: 207, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) SEQ ID NO: 155.

[0355] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0356] In some embodiments, the NARE comprises (i) SEQ ID NO: 207, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0357] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 209, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0358] In some embodiments, the NARE comprises (i) SEQ ID NO: 209, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) SEQ ID NO: 155.

[0359] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 209, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0360] In some embodiments, the NARE comprises (i) SEQ ID NO: 209, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0361] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 210, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0362] In some embodiments, the NARE comprises (i) SEQ ID NO: 210, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) SEQ ID NO: 155.

[0363] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 210, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0364] In some embodiments, the NARE comprises (i) SEQ ID NO: 210, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0365] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0366] In some embodiments, the NARE comprises (i) SEQ ID NO:211, (ii) SEQ ID NO:205 or SEQ ID NO:206, and (iii) SEQ ID NO:155.

[0367] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0368] In some embodiments, the NARE comprises (i) SEQ ID NO: 211, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0369] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 212, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0370] In some embodiments, the NARE comprises (i) SEQ ID NO: 212, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) SEQ ID NO: 155.

[0371] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 212, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0372] In some embodiments, the NARE comprises (i) SEQ ID NO: 212, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0373] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 213, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0374] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) SEQ ID NO: 155.

[0375] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 213, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0376] In some embodiments, the NARE comprises (i) SEQ ID NO: 213, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0377] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 214, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0378] In some embodiments, the NARE comprises (i) SEQ ID NO: 214, (ii) SEQ ID NO: 205 or SEQ ID NO: 206, and (iii) SEQ ID NO: 155.

[0379] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 214, (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205, and (iii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 155.

[0380] In some embodiments, the NARE comprises (i) SEQ ID NO: 214, (ii) SEQ ID NO: 205, and (iii) SEQ ID NO: 155.

[0381] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 216, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0382] In some embodiments, the NARE comprises (i) SEQ ID NO: 216, and (ii) SEQ ID NO: 74.

[0383] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 217, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0384] In some embodiments, the NARE comprises (i) SEQ ID NO: 217, and (ii) SEQ ID NO: 74.

[0385] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 218, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0386] In some embodiments, the NARE comprises (i) SEQ ID NO: 218, and (ii) SEQ ID NO: 74.

[0387] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 219, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0388] In some embodiments, the NARE comprises (i) SEQ ID NO: 219, and (ii) SEQ ID NO: 74.

[0389] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 220, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0390] In some embodiments, the NARE comprises (i) SEQ ID NO: 220, and (ii) SEQ ID NO: 74.

[0391] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 221, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0392] In some embodiments, the NARE comprises (i) SEQ ID NO: 221, and (ii) SEQ ID NO: 74.

[0393] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 222, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0394] In some embodiments, the NARE comprises (i) SEQ ID NO: 222, and (ii) SEQ ID NO: 74.

[0395] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 223, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0396] In some embodiments, the NARE comprises (i) SEQ ID NO: 223, and (ii) SEQ ID NO: 74.

[0397] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 224, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0398] In some embodiments, the NARE comprises (i) SEQ ID NO: 224, and (ii) SEQ ID NO: 74.

[0399] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 225, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0400] In some embodiments, the NARE comprises (i) SEQ ID NO: 225, and (ii) SEQ ID NO: 74.

[0401] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 226, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0402] In some embodiments, the NARE comprises (i) SEQ ID NO: 226, and (ii) SEQ ID NO: 74.

[0403] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 227, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0404] In some embodiments, the NARE comprises (i) SEQ ID NO: 227, and (ii) SEQ ID NO: 74.

[0405] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 228, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0406] In some embodiments, the NARE comprises (i) SEQ ID NO: 228, and (ii) SEQ ID NO: 74.

[0407] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 229, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0408] In some embodiments, the NARE comprises (i) SEQ ID NO: 229, and (ii) SEQ ID NO: 74.

[0409] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 230, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0410] In some embodiments, the NARE comprises (i) SEQ ID NO: 230, and (ii) SEQ ID NO: 74.

[0411] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 231, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0412] In some embodiments, the NARE comprises (i) SEQ ID NO: 231, and (ii) SEQ ID NO: 74.

[0413] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 232, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0414] In some embodiments, the NARE comprises (i) SEQ ID NO: 232, and (ii) SEQ ID NO: 74.

[0415] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0416] In some embodiments, the NARE comprises (i) SEQ ID NO: 193 or SEQ ID NO: 194, and (ii) SEQ ID NO: 74.

[0417] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 194, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0418] In some embodiments, the NARE comprises (i) SEQ ID NO: 194, and (ii) SEQ ID NO: 74.

[0419] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 233, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0420] In some embodiments, the NARE comprises (i) SEQ ID NO: 233, and (ii) SEQ ID NO: 74.

[0421] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 234, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0422] In some embodiments, the NARE comprises (i) SEQ ID NO: 234, and (ii) SEQ ID NO: 74.

[0423] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 235, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0424] In some embodiments, the NARE comprises (i) SEQ ID NO: 235, and (ii) SEQ ID NO: 74.

[0425] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 249, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 74.

[0426] In some embodiments, the NARE comprises (i) SEQ ID NO: 249, and (ii) SEQ ID NO: 74.

[0427] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 165, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0428] In some embodiments, the NARE comprises (i) SEQ ID NO: 165, and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0429] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 165, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 158.

[0430] In some embodiments, the NARE comprises (i) SEQ ID NO: 165, and (ii) SEQ ID NO: 158.

[0431] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 199, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0432] In some embodiments, the NARE comprises (i) SEQ ID NO: 199, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0433] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 199, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0434] In some embodiments, the NARE comprises (i) SEQ ID NO: 199, and (ii) SEQ ID NO: 193.

[0435] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 164, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 200.

[0436] In some embodiments, the NARE comprises (i) SEQ ID NO: 164, and (ii) SEQ ID NO: 200.

[0437] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 189, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0438] In some embodiments, the NARE comprises (i) SEQ ID NO: 189, and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0439] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 189, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 193.

[0440] In some embodiments, the NARE comprises (i) SEQ ID NO: 189, and (ii) SEQ ID NO: 193.

[0441] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 195.

[0442] In some embodiments, the NARE comprises (i) SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) SEQ ID NO: 195.

[0443] In some embodiments, the NARE comprises (i) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202, and (ii) a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 195.

[0444] In some embodiments, the NARE comprises (i) SEQ ID NO: 202, and (ii) SEQ ID NO: 195.

[0445] Provided herein are methods for expressing a transgene in hepatocytes (e.g., in the liver), wherein the transgene is operably linked to a NARE disclosed herein and is particularly suitable for driving expression in hepatocytes (e.g., in the liver).

[0446] NARE Qualification Those skilled in the art will understand that certain modifications can be made to the NAREs disclosed herein without eliminating the ability of the NARE to drive gene expression in the desired tissue / cell type. Various in vitro and in vivo methods for confirming that a modified NARE is still able to drive gene expression are known in the art, including, but not limited to, the methods used herein. For example, the strength of a NARE can be assessed by operably linking a promoter to a transgene encoding a protein and measuring transgene expression, e.g., by detecting mRNA encoding the protein, or by measuring the presence or activity of the protein, e.g., by ELISA, Western blot, fluorescence, enzymatic activity of the protein, etc.

[0447] Provided herein are NAREs comprising one or more components. Provided herein are NAREs comprising one or more sequence components (or sequence variants thereof) listed in any one of Tables 1-7. Provided herein are NAREs comprising one or more sequence components (or sequence variants thereof) listed in Table 7. Provided herein are NAREs comprising one or more components (or sequence variants thereof) of a full-length NARE provided in Table 7. Simply by way of illustration and non-limiting example, provided are NAREs comprising one or more of the components (or sequence variants thereof) of NARE L4 (AATcre-mTTR): (1) AATcre, and (2) mTTRmd promoter. In embodiments, provided are NAREs comprising one or more components (or sequence variants thereof) of a full-length NARE provided in Table 7, whereby the components (or sequence variants thereof) are arranged 5' to 3' as shown in Table 7. In embodiments, NAREs are provided that comprise one or more components of a full-length NARE provided in Table 7 (or sequence variants thereof), whereby the components (or sequence variants thereof) are not arranged 5' to 3' as shown in Table 4, but are arranged in a different order.

[0448] In embodiments, in a NARE of Table 5 or Table 7, one or more of the components are replaced with a different sequence, comprising components (or sequence variants thereof) from different NAREs of Table 5 or Table 7. Additionally, NAREs comprising components (or sequence variants thereof) from two or more NAREs disclosed in Table 5 or Table 7 are also contemplated.

[0449] Furthermore, NARE variants are contemplated that contain sequences in addition to the elements of the NAREs disclosed herein (e.g., added to either end of the NARE sequence or inserted within the NARE sequence). Similarly, NARE variants are provided in which specific sequences have been removed from the NAREs disclosed herein (e.g., as terminal or internal deletions). In embodiments, NAREs comprising one or more components are provided without any additional nucleic acid sequence(s) linking the components of the NARE. In embodiments, NAREs comprising one or more components are provided, whereby individual components are connected by additional nucleic acid sequences. These additional nucleic acid sequences may or may not be related to the expression of an operably linked transgene. In embodiments, some of the components are directly linked, while other components are linked to other components via intervening sequences. Thus, variants of the NAREs disclosed in Table 5 or Table 7 are contemplated, in which additional sequences have been added between different components. Variants of the NAREs disclosed in Table 5 or Table 7 are further contemplated, which disclose the same components of a given NARE in Table 5 or Table 7, but differ in the sequence(s) connecting the individual components.

[0450] Nucleic Acid Constructs and Vectors In one aspect, nucleic acid constructs and vectors for introducing transgenes or expression constructs into cells, and their uses are provided. Nucleic acid constructs include expression constructs, including plasmids. The term "expression construct" refers to a recombinant polynucleotide construct that includes a nucleic acid encoding an RNA that can be transcribed in a cell. Methods for constructing expression constructs and plasmids via standard recombinant techniques are known in the art.

[0451] In some embodiments, the expression construct comprises a sequence encoding ATP7B, and the sequence encoding ATP7B is codon optimized.

[0452] In one aspect, a) a promoter; b) a sequence encoding ATP7B operably linked to a promoter; and c) a polyadenylation signal.

[0453] In some embodiments, a vector comprises a recombinant DNA construct containing additional DNA elements, including DNA segments that provide appropriate levels of DNA replication in host cells and expression of target genes in target cells. As used herein, "vector" refers to a vehicle containing a polynucleotide delivered to a host cell, either in vitro or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA minicircles, or viruses (containing viral-derived sequences). A vector may also refer to a virion containing a nucleic acid delivered to a host cell, either in vitro or in vivo. In some embodiments, a vector refers to a virion containing a recombinant viral genome, where the viral genome includes one or more ITRs and a transgene.

[0454] In one embodiment, the vector is a viral vector or a combination of viral vectors. In one aspect, a vector is provided comprising any of the nucleic acid constructs disclosed herein.

[0455] Provided herein are nucleic acid constructs and vectors comprising the nucleic acid regulatory elements disclosed herein operably linked to a transgene. In embodiments, the nucleic acid constructs or vectors disclosed herein comprise additional regulatory elements, including, but not limited to, a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer. In embodiments, the nucleic acid constructs or vectors disclosed herein comprise a polyadenylation sequence. In embodiments, the nucleic acid constructs or vectors disclosed herein comprise an internal ribosome entry site (IRES). IRES sequences can be used to produce multiple polypeptides from a single gene transcript. IRES (or other suitable sequences) can be used to produce proteins containing multiple polypeptide chains or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE, and ganglion cells. In one embodiment, the IRES is located 3' of the transgene.

[0456] viral vectors Viral vectors for expressing target genes in target cells, tissues, or organisms are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric, mosaic, or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles, or small molecules).

[0457] AAV vectors Adeno-associated viruses (AAVs) are small, single-stranded DNA viruses that require a helper virus to promote efficient replication. The 4.7-kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins primarily function in AAV replication and rescue, as well as in regulating AAV integration into host cell chromosomes. The Cap reading frame encodes three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. VP3 accounts for more than 80% of the total protein in AAV virions. Adjacent to the 5' and 3' ends of the Rep and Cap open reading frames are inverted terminal repeats (ITRs) approximately 145 bp long. The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with therapeutic or reporter transgenes.

[0458] Recombinant adeno-associated virus "rAAV" vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have one or more of the AAV wild-type genes, preferably the Rep and / or Cap genes, deleted in whole or in part, but retain functional flanking ITR sequences.

[0459] In some embodiments, the viral vector is a rAAV virion comprising a rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome comprises a nucleic acid construct disclosed herein.

[0460] In some embodiments, the viral vectors disclosed herein comprise a nucleic acid comprising AAV 5' and 3' ITRs located 5' and 3', respectively, of a sequence encoding a transgene. In embodiments, the transgene is UPF1, BDNF, or ATP7B. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5' and 3' ITR sequences arranged in tandem, e.g., 5'-3', or head-to-tail, or in another alternative arrangement. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs or to have the 5' ITR (or conversely, the 3' ITR) located both 5' and 3' to the transgene. The ITR sequences may be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences may be present. The ITRs need not be wild-type nucleotide sequences and can be modified (e.g., by nucleotide insertion, deletion, or substitution) so long as the sequences provide functional rescue, replication, and packaging. The ITRs can be selected from AAV2 or from other AAV serotypes, as described herein.

[0461] In some embodiments, a vector is provided comprising a nucleic acid sequence comprising (i) a nucleic acid construct disclosed herein and (ii) one or more inverted terminal repeats (ITRs). In one embodiment, the nucleic acid sequence comprises a 5'ITR and a 3'ITR. In one embodiment, the 5'ITR and the 3'ITR are derived from adeno-associated virus (AAV) serotype AAV2.

[0462] In some embodiments, the viral vector is an AAV vector, for example, AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., an AAV9 ITRs and AAV9 capsid protein), AAVrh.74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein).

[0463] In some embodiments, the viral vector is a pseudotyped AAV vector containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins).

[0464] In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV containing AAV2 ITRs and AAV 7m8 capsid proteins).

[0465] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, incorporated herein by reference).

[0466] In some embodiments, the AAV vector contains two or more capsid proteins selected from different serotypes. In some embodiments, the AAV vector contains rAAV2-retro and AAVrh.10 capsid proteins. In some embodiments, the AAV vector contains rAAV2-retro and AAVrh.10 capsid proteins at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively. In some embodiments, the AAV vector contains AAVrh.10 and rAAV2-retrocapsid proteins, respectively, in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0467] In some embodiments, a mixture of (1) an AAV vector comprising rAAV2-retro and (2) an AAV vector comprising AAVrh.10 is used. In some embodiments, a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively, of (1) an AAV vector comprising rAAV2-retro and (2) an AAV vector comprising AAVrh.10 is used. In some embodiments, a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively, of (1) an AAV vector comprising AAVrh.10 and (2) an AAV vector comprising rAAV2-retro is used.

[0468] Other viral vectors Other viral vectors include adenovirus (AV) vectors, such as those based on human adenovirus type 2 and human adenovirus type 5, which have been rendered replication-deficient by deletion of the E1 and E3 regions. A transcription cassette can be inserted into the E1 region to obtain an E1 / E3-deleted recombinant AV vector. Adenovirus vectors also include helper-dependent, large-capacity adenovirus vectors (also known as large-capacity, "gutless" or "gutted" vectors) that do not contain viral coding sequences. These vectors contain cis-acting elements necessary for viral DNA replication and packaging, primarily inverted terminal repeats (ITRs) and packaging signals (CYs). These helper-dependent AV vector genomes have the potential to carry foreign DNA from several hundred base pairs to approximately 36 kb.

[0469] Alternatively, other systems, such as lentiviral vectors, can be used. Lentiviral-based systems can transduce not only non-dividing cells but also dividing cells, making them useful for targeting non-dividing cells in the CNS, for example. Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome, providing the potential for very long-term gene expression.

[0470] Polynucleotides, including plasmids, YACs, minichromosomes, and minicircles, carrying target genes containing expression cassettes can also be introduced into cells or organisms by non-viral vector systems, for example, using cationic lipids, polymers, or both as carriers. Conjugated poly-L-lysine (PLL) polymer and polyethyleneimine (PEI) polymer systems can also be used to deliver vectors into cells. Other methods for delivering vectors into cells include hydrodynamic injection and electroporation, as well as the use of ultrasound, for both cell cultures and organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012;1:27), which is incorporated herein by reference.

[0471] rAAV virion production The rAAV virions disclosed herein can be constructed and produced using materials and methods described herein and known to those of skill in the art. Such engineering methods used to construct any embodiment of the present disclosure are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, and International Patent Publication No. WO 95 / 13598. Additionally, suitable methods for producing rAAV cassettes within adenovirus capsids are described in U.S. Patent Nos. 5,856,152 and 5,871,982.

[0472] Briefly, to package the rAAV genome into rAAV virions, host cells containing sequences necessary for expressing AAV rep and AAV cap or functional fragments thereof, as well as helper genes essential for AAV production, are used. The AAV rep and cap sequences are obtained from AAV sources found herein. The AAV rep and cap sequences can be introduced into host cells by any method known to those skilled in the art, including, but not limited to, transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into host cells via one or more nucleic acid molecules and stably present in the cells as episomes. In another embodiment, the rep and cap sequences are stably integrated into the cell's genome. In another embodiment, the rep and cap sequences are transiently expressed in the host cell. For example, a nucleic acid molecule useful for such transfection comprises, from 5' to 3', a NARE, an optional spacer inserted between the NARE and the start of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.

[0473] The rep and cap sequences, along with their expression control sequences, may be provided on a single vector, or each sequence may be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences may be provided on a vector containing other DNA sequences that can be introduced into host cells. Preferably, the promoter used in this construct may be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins may be in any form that transfers these components into host cells. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as sequences of marker genes. This molecule does not contain AAV ITRs and generally does not contain AAV packaging sequences. To avoid the occurrence of homologous recombination, other viral sequences, particularly adenoviral sequences, are avoided in this plasmid. Desirably, this plasmid is constructed so that it can be stably transfected into cells.

[0474] Although the molecules providing rep and cap can be transiently transfected into host cells, it is preferred that the host cells be stably transformed with the sequences necessary to express functional rep / cap proteins in the host cell, e.g., as an episome or by integration into the host cell chromosome. Depending on the promoter controlling expression in such stably transfected host cells, the rep / cap proteins can be transiently expressed (e.g., through the use of an inducible promoter).

[0475] The methods used to construct embodiments of the present disclosure are conventional genetic or recombinant engineering techniques, as described in the references cited above. For example, rAAV can be produced using a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.), according to the manufacturer's instructions. See also Herzog et al., 1999, Nature Medic., 5(1):56-63, for the methods used in the following examples, which utilize a plasmid carrying the transgene, a helper plasmid containing AAV rep and cap, and a plasmid providing adenoviral helper functions of E2A, E4Orf6, and VA. While the present specification provides illustrative examples of specific constructs using the information provided herein, those skilled in the art will be able to select and design other suitable constructs using their selection of spacers, promoters, and other elements, including at least one translational start and stop signal, and the optional addition of a polyadenylation site.

[0476] rAAV virions can be produced by culturing host cells containing the rAAV viruses described herein, which contain the rAAV genome packaged into rAAV virions, AAV rep sequences, and AAV cap sequences, under the control of regulatory sequences that direct their expression. Suitable viral helper genes, such as adenovirus E2A, E4Orf6, and VA, among other possible helper genes, can be provided to the culture by various methods known in the art, preferably on separate plasmids. Recombinant AAV virions directing the expression of the transgene are then isolated from the cells or cell culture in the absence of contaminating helper virus or wild-type AAV.

[0477] The expression of the transgene can be measured by methods known in the art. For example, target cells can be infected in vitro, and the copy number of the transgene in the cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). The RNA expression level can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the protein expression level can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by specific methods detailed in the following examples.

[0478] cell In embodiments, the nucleic acid constructs and vectors disclosed herein are used to deliver a NARE operably linked to a transgene to a cell. Provided herein are cells comprising a NARE, nucleic acid construct, or vector disclosed herein. In embodiments, the cell is a liver cell. The cell may be a mammalian cell. The cell may be a human cell. The cell may be isolated.

[0479] Pharmaceutical Composition Provided herein is a pharmaceutical composition comprising a nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.

[0480] The nucleic acid constructs or vectors disclosed herein are preferably evaluated for contamination by conventional methods and then formulated into pharmaceutical compositions suitable for storage and / or administration to a patient.

[0481] Formulation of the nucleic acid constructs or vectors disclosed herein involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly buffered saline or other buffers suitable for injection, such as HEPES, to maintain pH at an appropriate physiological level. The nucleic acid constructs or vectors disclosed herein can be formulated into pharmaceutical compositions. These compositions may contain, in addition to the vector, pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives known to those skilled in the art. Such materials must be non-toxic and not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials can be determined by those skilled in the art depending on the route of administration. Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, incorporated herein by reference. Physiological saline, magnesium chloride, dextrose, or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001%, may be used. In some cases, Ringer's solution, lactated Ringer's solution, or Hartmann's solution may be used. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0482] Pharmaceutical compositions comprising the nucleic acid constructs or vectors disclosed herein may be formulated with one or more pharmaceutically acceptable excipients, which may be pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, magnesium talc, calcium or zinc stearate, or steric acid), solvents or encapsulating materials, bulking agents, salts, surfactants, and / or preservatives, that are involved in carrying or transporting the therapeutic compound for administration to a subject. Some examples of substances that can act as pharmaceutically acceptable excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffers; and other non-toxic compatible substances used in pharmaceutical formulations.

[0483] Bulking agents are compounds that add mass to a pharmaceutical formulation and contribute to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol, and sorbitol.

[0484] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce particle formation in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes particle formation after reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton®; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, or the like. Myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic®, PF68, etc.).

[0485] Preservatives may be used in the formulations of the present invention. Suitable preservatives for use in the formulations of the present invention include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl-dimethylammonium chlorides, where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical textbooks, such as "Remington's Pharmaceutical Sciences," The Science and Practice of Pharmacy, 19th Ed., Mack Publishing Company, Easton, Pa., (1995).

[0486] For delayed release, the nucleic acid constructs or vectors disclosed herein may be included in pharmaceutical compositions formulated for delayed release, such as microcapsules formed from biocompatible polymers or liposome carrier systems, according to methods known in the art.

[0487] For long-term storage of the vector, the vector may be frozen in the presence of glycerol.

[0488] method Provided herein are methods for inducing expression of a transgene in cells and / or tissues. Provided herein are methods of inducing expression of a transgene, comprising providing a cell comprising a nucleic acid construct comprising a NARE disclosed herein operably linked to the transgene, and culturing the cell under conditions that allow expression of the transgene. In some embodiments, the cell is a liver cell. Provided herein are methods for inducing expression of a transgene in vivo. Provided herein are methods for inducing expression of a transgene in vitro. Provided herein are methods for inducing expression of a transgene ex vivo.

[0489] Provided herein are methods for treating a disease or disorder in a subject in need thereof using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein. In some embodiments, the disease is Wilson's disease (WD). In some embodiments, the subject has a mutation in ATP7B.

[0490] Provided herein are methods for treating or preventing dystonia or bradykinesia in a subject suffering from Wilson's disease in need thereof, the methods comprising administering to the subject a vector or pharmaceutical composition disclosed herein. Provided herein are vectors or pharmaceutical compositions disclosed herein for use in treating or preventing dystonia or bradykinesia in a subject suffering from Wilson's disease in need thereof. Provided herein is use of a vector in the manufacture of a medicament for treating or preventing dystonia or bradykinesia in a subject suffering from Wilson's disease in need thereof.

[0491] Provided herein are methods for reducing the incidence of leukopenia or anemia in a subject suffering from Wilson's disease in need thereof, comprising administering to the subject a vector or pharmaceutical composition disclosed herein. Provided herein are vectors or pharmaceutical compositions disclosed herein for use in reducing the incidence of leukopenia or anemia in a subject suffering from Wilson's disease in need thereof. Provided herein is use of a vector in the manufacture of a medicament for reducing the incidence of leukopenia or anemia in a subject suffering from Wilson's disease in need thereof.

[0492] Provided herein is a method for reducing the incidence of cirrhosis in a subject suffering from Wilson's disease in need thereof, comprising administering to the subject a vector or pharmaceutical composition disclosed herein. Provided herein is a vector or pharmaceutical composition disclosed herein for use in reducing the incidence of cirrhosis in a subject suffering from Wilson's disease in need thereof. Provided herein is the use of a vector in the manufacture of a medicament for reducing the incidence of cirrhosis in a subject suffering from Wilson's disease in need thereof.

[0493] In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals include, but are not limited to, humans or non-human mammals, such as cows, horses, dogs, sheep, or cats. An "individual" or "patient" is also a subject herein.

[0494] As used herein, the terms "treat," "treated," "treating," or "treatment" refer to therapeutic treatment, the purpose of which is to slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the severity of the condition, disorder, or disease; stabilization (i.e., not worsening) of the pathological condition, disorder, or disease state; delay in the onset of the condition, disorder, or disease, or delay in the progression of the condition, disorder, or disease; improvement in one or more symptoms of the condition, disorder, or disease state; and remission (partial or complete), or improvement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment. The terms "prevent", "preventing", and the like refer to acting before the onset of an overt disease or disorder to prevent the onset of the disease or disorder, minimize the extent of the disease or disorder, or delay the course of its development.

[0495] In some embodiments, treatment refers to increased survival (e.g., survival time). For example, treatment can result in an increased life expectancy of a patient. In some embodiments, treatment can result in an increased life expectancy of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about

[0013] In some embodiments, treatment results in an increase in patient life expectancy of more than 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more. In some embodiments, treatment results in an increase in patient life expectancy of more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or more, compared to the life expectancy of one or more control individuals with the disease or disorder who are not being treated. In some embodiments, treatment results in long-term survival of the patient. As used herein, the term "long-term survival" refers to a survival time or life expectancy of greater than about 40, 45, 50, 55, 60 years or more.

[0496] Methods of administration include, but are not limited to, intracisternal, intraventricular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intrathecal, intravaginal, transdermal, rectal, by inhalation, or topical, particularly to the ear, nose, eye, or skin. The mode of administration is left to the discretion of the physician.

[0497] In some cases, the nucleic acid constructs or vectors described herein are administered locally. This can be achieved, for example, by local infusion during surgery, by topical application (e.g., cream or lotion), by injection, by catheter, by suppository or enema, or by implant, which is a porous, non-porous, or gel-like material, including membranes such as silastic membranes or fibers. In some situations, the nucleic acid constructs or vectors described herein are introduced into the central nervous system, circulatory system, or gastrointestinal tract by any suitable route, including intraventricular injection, intrathecal injection, paraspinal injection, epidural injection, enema, and injection adjacent to a peripheral nerve.

[0498] The compositions described herein can be administered as a single dose or multiple doses. Such compositions can be administered at regular intervals depending on the nature, severity, and extent of the subject's condition. In some embodiments, a therapeutically effective amount of a nucleic acid construct or vector is administered intrathecally at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, every two months, every other month, every month, every other week, or every week).

[0499] The amount of the nucleic acid construct or vector described herein that is effective in treating a disease can be determined using standard clinical techniques known to those skilled in the art. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose used will also depend on the route of administration, the condition, the severity of the condition being treated, and various physical factors related to the individual being treated, and can be determined according to the judgment of a medical professional.

[0500] An effective amount of rAAV carrying a nucleic acid sequence encoding a transgene (including, but not limited to, UPF1, ATP7B, and BDNF) under the control of a NARE is, for example, about 1×10 9 ~Approx. 1×10 14The rAAV may be present in a range of about 1 x 10 rAAV genome particles (vg) / kg body weight. A "genome particle" is defined herein as an AAV capsid containing a single-stranded DNA molecule that can be quantified by a sequence-specific method (such as qPCR or ddPCR). In some embodiments, the rAAV is present in a range of about 1 x 10 12 ~Approx. 1×10 13 In some embodiments, the rAAV is administered at about 5 x 10 11 ~Approx. 5×10 12 In some embodiments, the rAAV is administered in a total of about 7.5 x 10 vg / mL of cerebrospinal fluid (CSF) volume per patient. 13 ~7.5×10 14 It is administered in vg.

[0501] In some embodiments, the rAAV is about 1 x 10 11 ~Approx. 1×10 14 rAAV genome particles (vg) / kg body weight are administered to animals.

[0502] In some embodiments, the rAAV genome particles are provided in a volume of about 20 uL to about 50 mL. In some embodiments, the rAAV genome particles are provided in a volume of about 30 uL to about 30 mL. In some embodiments, the rAAV genome particles are provided in a volume of about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 uL. In some embodiments, the rAAV genome particles are provided in a volume of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 mL.

[0503] Still other doses within these ranges can be selected by the attending physician. It is understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the nucleic acid constructs or vectors, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

[0504] The nucleic acid constructs or vectors disclosed herein can also be advantageously provided to cells ex vivo, followed by administering the live cells to a subject. Methods for treating disease by transplanting cells modified to express recombinant proteins are also well known. See, for example, U.S. Patent No. 5,399,346, which discloses a method for introducing nucleic acids into primary human cells for introduction into humans. In some embodiments, the use of human cells for ex vivo therapy is preferred, although other cells, such as bacterial cells, may be transplanted into the subject's vasculature to continuously release therapeutic agents. See, for example, U.S. Patent Nos. 4,309,776 and 5,704,910.

[0505] This application is related to U.S. Provisional Application No. 63 / 379,109, entitled "UPF1 EXPRESSION CONSTRUCTS," U.S. Provisional Application No. 63 / 379,113, entitled "ATP7B GENE THERAPY," and U.S. Provisional Application No. 63 / 379,114, entitled "BDNF GENE THERAPY," all of which were filed by the same applicant on October 11, 2022, and each of which is incorporated by reference in its entirety herein.

[0506] It is understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention. Furthermore, it should be understood that the disclosure of the invention herein includes all possible combinations of such particular features. For example, if a particular feature is disclosed in connection with a particular aspect or embodiment of the invention, or with a particular claim, that feature can also be used in combination with and / or in connection with other specific aspects and embodiments of the invention, and in the invention generally, to the extent possible.

[0507] When reference is made herein to a method that includes two or more defined steps, the defined steps can be performed in any order or simultaneously (unless the context excludes this possibility), and the method can include one or more other steps that occur before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes these possibilities).

[0508] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, to the extent that the definition or use of a term in a reference incorporated herein by reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0509] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given, which should not be construed as limiting or defining the entire scope of the invention. [Example]

[0510] Example 1: Design of NARE Potential NARE candidates were also identified from genes highly expressed in humans within the target tissue (liver). Promoter regions were then defined based on chromatin marks, accessibility, conservation, and other genome-wide datasets, or cis-regulatory elements were identified.

[0511] Motif 10 element (MTE) / downstream promoter element (DPE) modifications were incorporated to increase transcription, as were various cellular, viral, or synthetic 5'UTR motifs to increase transcription / translation. Intron selection was varied to include, for example, short cellular introns that naturally have their own standalone transcription start sites, indicating the presence of a promoter within this particular intron. Additionally, introns were inserted at different positions in the sequence and / or introns were truncated while maintaining splicing elements. Additionally, individual or multiple point mutations were introduced to increase expression or prevent inactivation. Different transcriptional enhancers were also introduced upstream of the core promoter. Insertion, substitution, and / or shuffling of transcriptional motifs were utilized in certain cases, as was removal of CpG sequences to reduce immunogenicity while maintaining promoter activity.

[0512] Gene regulatory elements were also optimized by the following methods: (i) highly expressed transcription factor binding sites (TFBS) upstream of the core promoter were identified, shuffled, and cloned; (ii) iterative mutation and scoring of promoters using artificial intelligence (AI) algorithms.

[0513] Materials and Methods AI-driven NARE design Core prediction model: Construct potency predictions were based on predictions from the Xpresso model (xpresso.gs.washington.edu). The original Xpresso model was developed considering a 10.5 kb region surrounding the transcription start site (TSS, 7.5 kb upstream, and 3 kb downstream of the TSS). Due to size constraints (fragments longer than approximately 1.5 kb were not used), the algorithm was modified to include a "padding" step before calculating predicted activity. This padding step took the sequence and added ambiguous ("N") nucleotides upstream and downstream of the shorter input fragment according to the expected / annotated TSS. The final model took the sequence, scanned for uppercase base pairs (the convention used to label TSSs), padded the sequence to length, converted it to a one-hot encoded data frame, and then passed it through a convolutional neural network (CNN) with weights and structure from Xpresso.

[0514] Custom software was written to generate and automate the scoring of the following analyses (Figure 1).

[0515] ENCODE+JeT (C6): ENCODE fragment coordinates were downloaded and then used to analyze such elements from the reference human genome (GRCh38). All ENCODE fragments were individually placed upstream of the JeT promoter and then tested through the modified prediction model.

[0516] ENCODE+JeT spacing optimization: A custom script was used to insert 300-bp, 400-bp, 500-bp, or 600-bp spacing elements between the top 2,000 scoring ENCODE elements and the JeT promoter. The new "spacing" constructs were again tested with the modified prediction model.

[0517] Point mutation / saturation mutagenesis: A custom script was used to generate all possible point mutation versions of the input sequence. The resulting sequences were then scored using a modified prediction model. A custom script was also developed to iterate this process through a greedy search (introducing the best result at each round).

[0518] Through the CNN model, the effects of 708,157 enhancer fragments cloned in silico upstream of the C6 promoter sequence were predicted. The majority of enhancers were predicted to have a positive effect on C6 potency, with approximately 2,000 constructs resulting in an approximately 2-fold improvement. This subset was further analyzed to identify optimal spacing between the enhancer element and the promoter sequence. Introducing spaced sequences improved the potency of constructs, with the greatest improvement observed at 400 bp spacing. Selected constructs (C118–C127) were synthesized and tested in vitro.

[0519] Promoter syntax and rational design of NARE Potential NARE candidates were also identified from genes highly expressed in humans within the target tissue (liver). Promoter regions were then defined based on chromatin marks, accessibility, conservation, and other genome-wide datasets, or cis-regulatory elements were identified.

[0520] Motif 10 element (MTE) / downstream promoter element (DPE) modifications were incorporated to increase transcription, as were various cellular, viral, or synthetic 5'UTR motifs to increase transcription / translation. For CAG-based gene regulatory elements (but also others described herein), intron selection was varied to include, for example, short cellular introns that naturally have their own standalone transcription start sites, indicating the presence of a promoter within this particular intron. Additionally, introns were inserted at different positions in the sequence and / or introns were truncated while maintaining splicing elements. Additionally, individual or multiple point mutations were introduced to increase expression or prevent inactivation. Different transcriptional enhancers were also introduced upstream of the core promoter. Insertion, substitution, and / or shuffling of transcriptional motifs were utilized in certain cases, as was removal of CpG sequences to reduce immunogenicity while maintaining promoter activity.

[0521] Gene regulatory elements were also optimized by the following methods: (i) highly expressed transcription factor binding sites (TFBS) upstream of the core promoter were identified, shuffled, and cloned; (ii) iterative mutation and scoring of promoters using artificial intelligence (AI) algorithms.

[0522] Example 2: Dual reporter protein expression assay To test the ability of NARE candidates to drive gene expression, we utilized a dual-reporter assay using flow cytometry. A diagram of the dual-reporter expression construct and assay is provided in Figure 2A. Different NARE promoter sequences were cloned upstream of a transgene encoding mClover3 (green / yellow fluorescent protein), which was then positioned upstream of a posttranscriptional regulatory element from woodchuck hepatitis virus (WPRE250) and a synthetic polyadenylation (polyA) signal. mClover3 fluorescence was used as a measure of NARE intensity. The plasmid also contained a separate expression cassette (5' to 3': SV40 promoter-tdTomato (red fluorescent protein)-SV40 polyA), which was the same in all NARE test constructs (Figure 2A). tdTomato fluorescence was used as an internal normalization control, allowing us to account for variations in transfection efficiency.

[0523] Various cell lines were transiently transfected with the dual reporter plasmids using TurboFect® transfection reagent. After 48 hours, cells were trypsinized and resuspended for flow cytometry. Raw data were processed to select only live single cells for analysis. A gate for tdTomato signal was set using a sample containing untransfected cells. This gate was then applied to all live single cells to identify tdTomato-positive (and therefore transfected) cells (Figure 2B). mClover3 fluorescence was then determined for the tdTomato-positive population. Because transfections varied slightly between different experiments, relative NARE potency was calculated by the ratio of mClover3 fluorescence to tdTomato fluorescence. NARE candidates were tested alongside known constitutive and tissue-specific reference promoters, such as CAG, CMV, or AAT.

[0524] Example 3: Intensity of different NAREs in liver cells (in vitro) Materials and Methods cell culture Huh-7, AML-12, and HEK293 immortalized cell lines were maintained at 37°C in 5% CO2. Cells were transiently transfected with plasmids using TurboFect™. After 48 hours (72 hours for AML-12), cell lysates were collected for protein analysis. After 20 passages, a new aliquot of cells was thawed and passaged twice before use in subsequent experiments.

[0525] Primary hepatocyte culture Gibco-derived mouse and human hepatocyte cell lines were thawed and placed in suspension medium, then centrifuged at 100 x g for 10 minutes at 4°C. The supernatant was aspirated, and the cells were resuspended in maintenance medium (+10% FBS) and then plated at 400,000 cells per well onto a 24-well plate. Four hours after plating, the plate was gently tapped to loosen debris, and the medium was then aspirated and replaced with non-FBS containing maintenance medium. Cells were maintained at 37°C in 5% CO2. A complete medium change was performed daily to feed the cultures.

[0526] Transduction For immortalized and primary cell cultures, AAV8 particles were added to the culture medium at a multiplicity of infection (MOI) of 50,000 on days 1 and 3 in vitro, respectively. Doxorubicin-containing medium was added to the cells to accelerate transduction. The medium was replaced with non-doxorubicin-containing medium after 72 hours, according to standard protocols.

[0527] result We developed a library of NAREs suitable for gene expression in the liver. The promoters conferred stronger expression in immortalized hepatocytes (Huh7, HepG2, and AML-12) than commonly used reference promoters. The Huh7 cell line is a hepatocyte-derived carcinoma cell line. HepG2 is a hepatoblastoma cell line. AML-12 cells are an immortal epithelial cell line derived from mouse hepatocytes. The reference promoters included the CAG promoter, alpha-1-antitrypsin (AAT) promoter, human thyroxine-binding globulin (TBG) promoter, liver-specific promoter 1 (LP1), hybrid liver promoter (HLP), and liver combinatorial bundle promoter (HCB). ATT is a small liver-specific promoter currently used in gene therapy clinical trials. HCB is an effective NARE that is more potent than AAT. The promoters in the promoter library were as strong as or stronger than CAG, but also significantly weaker.

[0528] Additional NAREs were bioinformatically identified from human top liver-expressed genes and defined using various epigenetic and genomic features (histone acetylation, TF occupancy, sequence conservation, etc.). The potency of selected NAREs in liver cells is shown in Figures 3A and 3C. L45 was 22-fold stronger than the AAT promoter (Figures 3C and 3D). NAREs L10, L13, and L15 are specific to liver cells (Figure 3B).

[0529] Further optimization of NARE L45 resulted in even increased expression (Table 1, Figures 4A and 4B). The optimized NARE was up to approximately three times stronger than the 1.6 kb CAG promoter in liver cells. The efficacy of additional promoters in the human liver cell line Huh7 is shown in Figure 4C. The efficacy of derivatives of the AAT promoter (L1 and HCB promoter) in different cell lines is shown in Tables 6 and 7, respectively. [Table 1] [Table 2]

Table 3

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 6-1

Table 6-2

Table 6-3

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

Table 7-21

Table 7-22

Table 7-23

Table 7-24

Table 7-25

Table 7-26

Table 7-27

Table 7-28

Table 7-29

Table 7-30

Table 7-31

Table 7-32

Table 7-33

Table 7-34

Table 7-35

Table 7-36

Table 7-37

Table 7-38

Table 7-39

Table 7-40

Table 7-41

Table 7-42

Table 7-43

Table 7-44

Table 7-45

Table 7-46

Table 7-47

Table 7-48

Table 7-49

Table 7-50

Table 7-51

Table 7-52

Table 7-53

Table 7-54

Table 7-55

Table 7-56

Table 7-57

Table 7-58

Table 7-59

Table 7-60

Table 7-61

Table 7-62

Table 7-63

Table 7-64

Table 7-65

Table 7-66

Table 7-67

Table 7-68

Table 7-69

Table 7-70

Table 7-71

Table 7-72

Table 7-73

Table 7-74

Table 7-75

Table 7-76

Table 7-77

Table 7-78

Table 7-79

Table 7-80

Table 7-81

Table 7-82

Table 7-83

Table 7-84

Table 7-85

Table 7-86

Table 7-87

Table 7-88

Table 7-89

Table 7-90

Table 7-91

Table 7-92

Table 7-93

Table 7-94

Table 7-95

Table 7-96

Table 7-97

Table 7-98

Table 7-99

Table 7-100

Table 7-101

Table 7-102

Table 7-103

Table 7-104

Table 7-105

Table 7-106

Table 7-107

Table 7-108

Table 7-109

Table 7-110

Table 7-111

Table 7-112

Table 7-113

Table 7-114

Table 7-115

Table 7-116

Table 7-117

Table 7-118

Table 7-119

Table 7-120

Table 7-121

Table 7-122

Table 7-123

Table 7-124

Table 7-125

Table 7-126

Table 7-127

Table 7-128

Table 7-129

Table 7-130

Table 7-131

Table 7-132

Table 7-133

Table 7-134

Table 7-135

Table 7-136

Table 7-137

Table 7-138

Table 7-139

Table 7-140

Table 7-141

Table 7-142

Table 7-143

Table 7-144

Table 7-145

Table 7-146

Table 7-147

Table 7-148

Table 7-149

Table 7-150

Table 7-151

Table 7-152

Table 7-153

Table 7-154

Table 7-155

Table 7-156

Table 7-157

Table 7-158

Table 7-159

Table 7-160

Table 7-161

Table 7-162

Table 7-163

Claims

1. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO:74, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs:193, 194, 207, 209-214, 216-232, 233-235, or 249.

2. 2. The NARE of claim 1, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 74; and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249.

3. 2. The NARE of claim 1, comprising: (i) SEQ ID NO: 74; and (ii) any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249.

4. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 151 or SEQ ID NO:

155.

5. 5. The NARE of claim 4, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 150, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 151 or SEQ ID NO:

155.

6. 5. The NARE of claim 4, comprising (i) SEQ ID NO: 150, and (ii) SEQ ID NO: 151 or SEQ ID NO:

155.

7. A NARE comprising: (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, or 158; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 169, 172, or 173.

8. 8. The NARE of claim 7, comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, or 158; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 169, 172, or 173.

9. 8. The NARE of claim 7, comprising: (i) any one of SEQ ID NOs: 154, 157, or 158; (ii) any one of SEQ ID NOs: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, any one of SEQ ID NOs: 169, 172, or 173.

10. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 153; (ii) a sequence that is at least 90% identical to SEQ ID NO: 171; and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, 158, or 176.

11. 11. The NARE of claim 10, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 153; (ii) a sequence that is at least 95% identical to SEQ ID NO: 171; and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, 158, or 176.

12. 11. The NARE of claim 10, comprising: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 171; and (iii) any one of SEQ ID NOs: 154, 157, 158, or 176.

13. A NARE comprising: (i) a sequence at least 90% identical to SEQ ID NO: 153; (ii) a sequence at least 90% identical to any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO:

171.

14. 14. The NARE of claim 13, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 153; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO:

171.

15. 14. The NARE of claim 13, comprising: (i) SEQ ID NO: 153; (ii) any one of SEQ ID NOs: 154, 157, 158, 176, 188, or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO:

171.

16. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 155; (ii) a sequence that is at least 90% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 175 or 177-187.

17. 17. The NARE of claim 16, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 153; (ii) a sequence that is at least 95% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 175 or 177-187.

18. 17. The NARE of claim 16, comprising: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 176; and (iii) any one of SEQ ID NOs: 175 or 177-187.

19. A NARE comprising: (i) a sequence at least 90% identical to SEQ ID NO: 190 or SEQ ID NO: 191; (ii) a sequence at least 90% identical to any one of SEQ ID NOs: 193, 194, or 195; and (iii) optionally, a sequence at least 90% identical to SEQ ID NO: 162; (iv) optionally, a sequence at least 90% identical to SEQ ID NO: 168; and (v) optionally, SEQ ID NO:

169.

20. 20. The NARE of claim 19, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 190 or SEQ ID NO: 191; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 193, 194, or 195; and (iii) optionally, a sequence that is at least 95% identical to SEQ ID NO: 162; (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 168; and (v) optionally, SEQ ID NO:

169.

21. 20. The NARE of claim 19, comprising: (i) SEQ ID NO: 190 or SEQ ID NO: 191; (ii) any one of SEQ ID NOs: 193, 194, or 195; (iii) optionally, SEQ ID NO: 162; (iv) optionally, SEQ ID NO: 168; and (v) optionally, SEQ ID NO:

169.

22. (i) a sequence that is at least 90% identical to SEQ ID NO: 168; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 197, 202, or 203; (iv) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206; (v) optionally, a sequence that is at least 90% identical to SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, a sequence that is at least 90% identical to any one of SEQ ID NOs: 166, 193, or 194.

23. 23. The NARE of claim 22, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 168; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 197, 202, or 203; (iv) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206; (v) optionally, a sequence that is at least 95% identical to SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, a sequence that is at least 95% identical to any one of SEQ ID NOs: 166, 193, or 194.

24. 23. The NARE of claim 22, comprising: (i) SEQ ID NO: 168; (ii) any one of SEQ ID NOs: 197, 202, or 203; (iv) optionally, any one of SEQ ID NOs: 190, 191, 192, 196, 202, 203, 204, 205, or 206; (v) optionally, SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, any one of SEQ ID NOs: 166, 193, or 194.

25. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 192; (ii) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO: 194; and (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 201-203.

26. 26. The NARE of claim 25, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 192; (ii) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO: 194; and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 201-203.

27. 26. The NARE of claim 25, comprising: (i) SEQ ID NO: 192; (ii) SEQ ID NO: 193 or SEQ ID NO: 194; and (iii) any one of SEQ ID NOs: 201-203.

28. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 155; (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238; (iii) optionally, a sequence that is 90% identical to SEQ ID NO: 168; (iv) optionally, a sequence that is at least 90% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 90% identical to SEQ ID NO:

197.

29. 29. The NARE of claim 28, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 155; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238; (iii) optionally, a sequence that is 90% identical to SEQ ID NO: 168; (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 95% identical to SEQ ID NO:

197.

30. 29. The NARE of claim 28, comprising: (i) SEQ ID NO: 155; (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 204, 205, 206, or 236-238; (iii) optionally, a sequence that is 90% identical to SEQ ID NO: 168; (iv) optionally, a sequence that is at least 95% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 95% identical to SEQ ID NO:

197.

31. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 248, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 239-247.

32. 32. The NARE of claim 31, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 248; and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 239-247.

33. 32. The NARE of claim 31, comprising: (i) SEQ ID NO: 248; and (ii) any one of SEQ ID NOs: 239-247.

34. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 208, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 209-214.

35. 35. The NARE of claim 34, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 208, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 209-214.

36. 35. The NARE of claim 34, comprising: (i) SEQ ID NO: 208; and (ii) any one of SEQ ID NOs: 209-214.

37. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO: 194; (ii) a sequence that is at least 90% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 90% identical to SEQ ID NOs: 207 or any one of 209-215.

38. 38. The NARE of claim 37, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO: 194; (ii) a sequence that is at least 95% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 207 or 209-215.

39. 38. The NARE of claim 37, comprising: (i) SEQ ID NO: 193 or SEQ ID NO: 194; (ii) SEQ ID NO: 215; and (iii) any one of SEQ ID NOs: 207 or 209-215.

40. A NARE comprising: (i) a sequence that is at least 90% identical to SEQ ID NO: 155; (ii) a sequence that is at least 90% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 90% identical to SEQ ID NOs: 207 or any one of 209-215.

41. 41. The NARE of claim 40, comprising: (i) a sequence that is at least 95% identical to SEQ ID NO: 155; (ii) a sequence that is at least 95% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 95% identical to SEQ ID NOs: 207 or any one of 209-215.

42. 41. The NARE of claim 40, comprising: (i) SEQ ID NO: 155; (ii) SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) any one of SEQ ID NOs: 207 or 209-215.

43. a) (i) a sequence that is at least 90% identical to SEQ ID NO: 53; b) (i) a sequence that is at least 90% identical to SEQ ID NO: 195, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 202; c) (i) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 168, (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 169, (iii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 197, and (iv) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 203; d) (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 220; e) (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO: 229, or f) A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 90% identical to a sequence that is at least 90% identical to SEQ ID NO:

230.

44. a) (i) a sequence that is at least 95% identical to SEQ ID NO: 53; b) (i) a sequence that is at least 95% identical to SEQ ID NO: 195, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 202; c) (i) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 168, (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 169, (iii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 197, and (iv) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 203; d) (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 220; e) (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO: 229, or f) A NARE according to claim 43, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 74, and (ii) a sequence that is at least 95% identical to a sequence that is at least 95% identical to SEQ ID NO:

230.

45. a) (i) SEQ ID NO: 53; b) (i) SEQ ID NO: 195, and (ii) SEQ ID NO: 202; c) (i) SEQ ID NO: 168, (ii) SEQ ID NO: 169, (iii) SEQ ID NO: 197, and (iv) SEQ ID NO: 203; d) (i) SEQ ID NO: 74, and (ii) SEQ ID NO: 220; e) (i) SEQ ID NO: 74, and (ii) SEQ ID NO: 229, or f) The NARE of claim 43, comprising (i) SEQ ID NO: 74, and (ii) SEQ ID NO:

230.

46. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 166, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, or 158.

47. 47. The NARE of claim 46, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 166, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, or 158.

48. 47. The NARE of claim 46, comprising: (i) SEQ ID NO: 166; and (ii) any one of SEQ ID NOs: 154, 157, or 158.

49. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 165, and (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 154, 157, or 158.

50. 50. The NARE of claim 49, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 165, and (ii) a sequence that is at least 95% identical to any one of SEQ ID NOs: 154, 157, or 158.

51. 50. The NARE of claim 49, comprising: (i) SEQ ID NO: 165; and (ii) any one of SEQ ID NOs: 154, 157, or 158.

52. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 199, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO:

194.

53. 53. The NARE of claim 52, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 199, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO:

194.

54. 53. The NARE of claim 52, comprising: (i) SEQ ID NO: 199; and (ii) SEQ ID NO: 193 or SEQ ID NO:

194.

55. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 164, and (ii) a sequence that is at least 90% identical to SEQ ID NO:

200.

56. 56. The NARE of claim 55, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 164, and (ii) a sequence that is at least 95% identical to SEQ ID NO:

200.

57. 56. The NARE of claim 55, comprising: (i) SEQ ID NO: 164; and (ii) SEQ ID NO:

200.

58. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 189, and (ii) a sequence that is at least 90% identical to SEQ ID NO: 193 or SEQ ID NO:

194.

59. 59. The NARE of claim 58, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 189, and (ii) a sequence that is at least 95% identical to SEQ ID NO: 193 or SEQ ID NO:

194.

60. 59. The NARE of claim 58, comprising (i) SEQ ID NO: 189, and (ii) SEQ ID NO: 193 or SEQ ID NO:

194.

61. A NARE comprising (i) a sequence that is at least 90% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 90% identical to SEQ ID NO:

195.

62. 62. The NARE of claim 61, comprising (i) a sequence that is at least 95% identical to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a sequence that is at least 95% identical to SEQ ID NO:

195.

63. 62. The NARE of claim 61, comprising: (i) SEQ ID NO: 202 or SEQ ID NO: 203; and (ii) SEQ ID NO:

195.

64. A nucleic acid regulatory element (NARE) comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 3-8, 11-69, 71-73, or 76-149.

65. 65. The NARE of claim 64, comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 3-8, 11-69, 71-73, or 76-149.

66. 65. The NARE of claim 64, comprising any one of SEQ ID NOs: 3-8, 11-69, 71-73, or 76-149.

67. 65. The NARE of claim 64, comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 53, 80, 81, 132, 141, or 142.

68. 68. The NARE of claim 67, comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 53, 80, 81, 132, 141, or 142.

69. 68. The NARE of claim 67, comprising any one of SEQ ID NOs: 53, 80, 81, 132, 141, or 142.

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

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

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

73. The vector of claim 70, wherein the vector is a non-viral vector.

74. The vector of claim 70, wherein the vector is a viral vector.

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

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

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

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

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

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

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

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

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

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

85. 82. A method of treating Wilson's disease in a subject in need thereof, said method comprising administering to said subject an expression construct of claim 70, a vector of any one of claims 72-78, or a pharmaceutical composition of claim 81.

86. 86. The method of claim 85, wherein the subject has a mutation in the ATP7B gene.

87. 87. The method of claim 86, wherein the subject is a human.

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