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

Through artificial intelligence optimization convolutional neural network and computer simulated mutation technology, efficient liver-specific nucleic acid regulatory elements are built, solving the problem of time-consuming and costly NARE optimization in the existing technology, and achieving efficient and safe expression of gene therapy vectors.

CN120322447APending Publication Date: 2025-07-15MEIRAGTX GENE REGULATION LTD
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Patent Information

Application Number
CN202380084516.6
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Priority Date
2023-04-17
Filing Date
2023-10-11
Publication Date
2025-07-15

AI Technical Summary

Technical Problem

In the prior art, when optimizing nucleic acid regulatory elements (NAREs), there are expensive and time-consuming low-throughput and high-throughput methods, which are difficult to effectively improve their effectiveness, reduce their size and achieve tissue-specific expression.

Method used

The artificial intelligence model is used to optimize the effectiveness of the convolutional neural network (CNN) to predict promoter effectiveness, combine computer simulation of saturation mutations, build a NARE library, and test the combination of enhancer elements and promoter sequences in vitro to optimize the expression of liver-specific NARE.

Benefits of technology

It improves the expression efficacy of NARE, achieves liver-specific expression, reduces the use of gene therapy vectors, reduces immune response and safety risks, and allows packaging of larger genetically modified goods.

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Abstract

The present application relates to a nucleic acid regulatory element (NAR E) capable of enhancing the expression of a gene in the liver. The present application also relates to methods of employing the NARE and uses of the NARE. Expression cassettes and vectors containing the NARE are also disclosed. These are particularly useful for applications using gene therapy.
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Description

[0001] Cross - reference to related applications

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

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (SeqList - 162027 - 54276.xml; size: 359,832 bytes; and creation date: October 6, 2023) are hereby incorporated by reference in their entireties into this application. Technical field

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

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

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

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

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

[0010] Provided herein is a polynucleotide sequence comprising a sequence that is 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 is a polynucleotide sequence comprising a sequence that is at least 90% 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 polynucleotide sequence comprising 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.

[0011] The present disclosure provides a polynucleotide comprising a sequence that is at least 80% identical to a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89. The present disclosure provides a polynucleotide comprising a sequence that is at least 90% identical to a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89. The present disclosure provides a polynucleotide comprising a NARE selected from the group consisting of L10, L13, L15, L17, L45, L56-L61, L71-L79, and L89.

[0012] The present disclosure provides a polynucleotide comprising a sequence that is at least 80% identical to a NARE selected from the group consisting of L45, L123, L132, and L133. The present disclosure provides a polynucleotide comprising a sequence that is at least 90% identical to a NARE selected from the group consisting of L45, L123, L132, and L133. The present disclosure provides a polynucleotide comprising a NARE selected from the group consisting of L45, L123, L132, and L133.

[0013] In some embodiments, provided is a polynucleotide sequence comprising a sequence that is at least 80% identical to a NARE sequence provided in Table 7. In some embodiments, provided is a polynucleotide sequence comprising a sequence that is at least 90% identical to a NARE sequence provided in Table 7. In some embodiments, provided is a polynucleotide comprising a NARE sequence provided in Table 7.

[0014] In one aspect, provided is 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 NO: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 NO: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 NO:193, 194, 207, 209-214, 216-232, 233-235, or 249.

[0015] On the one hand, a NARE is provided, which 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.

[0016] On the one hand, a NARE is provided, which comprises: (i) a sequence that is at least 90% identical to any one of SEQ ID NO: 154, 157 or 158; (ii) a sequence that is at least 90% identical to any one of SEQ ID NO: 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 NO: 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 NO: 154, 157 or 158; (ii) a sequence that is at least 95% identical to any one of SEQ ID NO: 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 NO: 169, 172 or 173. In some embodiments, the NARE comprises: (i) any one of SEQ ID NO: 154, 157 or 158; (ii) any one of SEQ ID NO: 153, 156, 161, 162, 163, 166, 167, 168 or 174; and (iii) optionally, any one of SEQ ID NO: 169, 172 or 173.

[0017] On the one hand, a NARE is provided, which 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 NO: 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 NO: 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 NO: 154, 157, 158 or 176.

[0018] On the one hand, a NARE is provided, which 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 NO: 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 NO: 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 NO: 154, 157, 158, 176, 188 or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171.

[0019] On the one hand, a NARE is provided, which 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 either SEQ ID NO: 175 or any one of SEQ ID NOs: 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 either SEQ ID NO: 175 or any one of SEQ ID NOs: 177 - 187. In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 176; and (iii) either SEQ ID NO: 175 or any one of SEQ ID NOs: 177 - 187.

[0020] On the one hand, a NARE is provided, which 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.

[0021] On the one hand, a NARE is provided, the NARE comprising: (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 NO: 197, 202, or 203; (iv) optionally, a sequence that is at least 90% identical to any one of SEQ ID NO: 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 NO: 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 NO: 197, 202, or 203; (iv) optionally, a sequence that is at least 95% identical to any one of SEQ ID NO: 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 NO: 166, 193, or 194. In some embodiments, the NARE comprises: (i) SEQ ID NO: 168; (ii) any one of SEQ ID NO: 197, 202, or 203; (iv) optionally, any one of SEQ ID NO: 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 NO: 166, 193, or 194.

[0022] On the one hand, a NARE is provided, which 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.

[0023] On the one hand, a NARE is provided, which 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 at least 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 at least 95% 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 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 at least 95% 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.

[0024] On the one hand, a NARE is provided, the 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. 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. On the one hand, a NARE is provided, the 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.

[0025] 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. On the one hand, a NARE is provided, the 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 NO: 207 or any one of SEQ ID NOs: 209 - 215.

[0026] 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 SEQ ID NO: 207 or any one of SEQ ID NOs: 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) SEQ ID NO: 207 or any one of SEQ ID NOs: 209 - 215. In one aspect, provided is a NARE 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 SEQ ID NO: 207 or any one of SEQ ID NOs: 209 - 215.

[0027] 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 SEQ ID NOs: 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) SEQ ID NO: 207 or any one of SEQ ID NOs: 209 - 215.

[0028] In one aspect, provided is a NARE that comprises:

[0029] a) (i) A sequence that is at least 90% identical to SEQ ID NO: 53;

[0030] 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;

[0031] c) (i) A sequence that is at least 90% identical to SEQ ID NO: 168; (ii) A sequence that is at least 90% identical to SEQ ID NO: 169; (iii) A sequence that is at least 90% identical to SEQ ID NO: 197; and (iv) A sequence that is at least 90% identical to SEQ ID NO: 203;

[0032] 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 SEQ ID NO: 220;

[0033] 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 SEQ ID NO: 229; or

[0034] f) (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: 230.

[0035] In some embodiments, the NARE comprises:

[0036] a) (i) A sequence that is at least 95% identical to SEQ ID NO: 53;

[0037] 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;

[0038] c) (i) A sequence that is at least 95% identical to SEQ ID NO: 168; (ii) A sequence that is at least 95% identical to SEQ ID NO: 169; (iii) A sequence that is at least 95% identical to SEQ ID NO: 197; and (iv) A sequence that is at least 95% identical to SEQ ID NO: 203;

[0039] 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 SEQ ID NO: 220;

[0040] 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

[0041] 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.

[0042] In some embodiments, the NARE comprises:

[0043] a) (i) SEQ ID NO:53;

[0044] b) (i) SEQ ID NO:195; and (ii) SEQ ID NO:202;

[0045] c) (i) SEQ ID NO:168; (ii) SEQ ID NO:169; (iii) SEQ ID NO:197; and (iv) SEQ ID NO:203;

[0046] d) (i) SEQ ID NO:74; and (ii) SEQ ID NO:220;

[0047] e) (i) SEQ ID NO:74; and (ii) SEQ ID NO:229; or

[0048] f) (i) SEQ ID NO:74; and (ii) SEQ ID NO:230.

[0049] On the one hand, provided is a NARE, which 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 NO: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 NO:154, 157 or 158. In some embodiments, the NARE comprises: (i) SEQ ID NO:166; and (ii) any one of SEQ ID NO:154, 157 or 158.

[0050] On the one hand, a NARE is provided, which 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 NO: 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 NO: 154, 157 or 158. In some embodiments, the NARE comprises: (i) SEQ ID NO: 165; and (ii) any one of SEQ ID NO: 154, 157 or 158.

[0051] On the one hand, a NARE is provided, which 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.

[0052] On the one hand, a NARE is provided, which 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.

[0053] On the one hand, a NARE is provided, which 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.

[0054] On the one hand, a NARE is provided, which 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.

[0055] On the one hand, a NARE is provided, which 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.

[0056] On the one hand, an expression construct is provided, which comprises the NARE disclosed herein and an operably linked transgene. In some embodiments, the expression construct further comprises a polyadenylation sequence.

[0057] On the one hand, a vector is provided, which comprises the 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 which comprises (i) an expression construct comprising the NARE disclosed herein and an operably linked transgene, and (ii) one or more inverted terminal repeats (ITRs). In some embodiments, the vector comprises a nucleic acid sequence which comprises (i) the expression construct disclosed herein, and (ii) one or more inverted terminal repeats (ITRs). In some embodiments, the 5′ ITR and 3′ ITR are derived from adeno-associated virus (AAV) serotype AAV2.

[0058] On the one hand, a cell is provided, which comprises the expression construct disclosed herein or the vector disclosed herein. In one embodiment, the cell is a liver cell.

[0059] On the one hand, a pharmaceutical composition is provided, which comprises: (i) the expression construct disclosed herein or the vector disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0060] On the one hand, a method for expressing a transgene in a cell comprising the expression construct disclosed herein or the vector disclosed herein is provided. On the one hand, a method for regulating the expression of a transgene in a cell comprising the expression construct disclosed herein or the vector disclosed herein is provided. In one embodiment, the cell is a liver cell.

[0061] On the one hand, a method for treating a subject in need of treatment for Wilson's disease is provided, which method comprises administering to the subject the expression construct disclosed herein, the vector disclosed herein or the pharmaceutical composition disclosed herein. In one embodiment, the subject has a mutation in the ATP7B gene. In one embodiment, the subject is a human. Description of the Drawings

[0062] Figure 1Bioinformatics flowchart for using AI to design and optimize a given promoter sequence. The process is divided into two parallel methods: (i) Promoter enhancement, whereby all known / annotated enhancer fragments are collected from the ENCODE database and inserted upstream of the promoter sequence; and (ii) Saturation mutagenesis, where all possible point mutations (without insertions or deletions) are generated for each base pair of the original input promoter sequence by in silico simulation. Next, the best candidates from both methods are tested in vitro in a preferred cell line, and the activity is recorded by fluorescence. Then, in the final in vitro screening, the best in vitro performers from both strategies are combined by rational design.

[0063] Figure 2A and Figure 2B . Dual-reporter gene design and measurement. Figure 2A Illustrates the design of the dual-reporter gene construct. The candidate NARE is cloned upstream (5′) of the mClover3 coding sequence. Each construct contains a constant region that includes a tdTomato transgene used as a normalization control. Figure 2B Provides an example of flow cytometry data obtained with the dual-reporter gene system, where mClover3 expression in tdTomato+ cells reflects the level of promoter activity.

[0064] Figure 3A 、 Figure 3B 、 Figure 3C and Figure 3D . Potency of selected NAREs in vitro in liver cells. NAREs specifically suitable for expression in the liver were rationally designed by adopting established or putative liver-specific core NAREs and enhancers and incorporating other novel variations based on various bioinformatics methods. Expression was evaluated using a dual-reporter gene assay. Figure 3A : Potency of the indicated NAREs in Huh7 cells compared to the reference promoters CAG, CMV, and AAT. Figure 3B : Tissue specificity of the selected NAREs (relative expression in HepG2 liver cells versus relative expression in HEK293T cells). Figure 3C : Potency of the indicated NAREs in the human liver cell line Huh7 cells compared to the AAT promoter (L1, 305 bp). Figure 3D : Bright field and mClover3 fluorescence of NARE L45 compared to the AAT promoter (L1). There are similar numbers of cells in the bright field images, but stronger mClover3 fluorescence is observed in L45. After transfection in Huh7 cells for 48 h, mClover3 expression driven by NARE L45 is higher than that of AAT.

[0065] Figure 4A 、Figure 4B and Figure 4C . Potency of selected NAREs in vitro in liver cells. Figure 4A and Figure 4B : Optimization of NARE L45 generated new NAREs that were up to 9-fold more potent than the parental (L45). NARE modifications included enhancers, cis-regulatory elements, introns, or other sequences identified bioinformatically that were predicted to enhance activity. Figure 4A : Expression of L45 and modified NAREs relative to CAG in Huh7 cells, as determined using a dual-reporter assay. Figure 4B : Expression levels of 12 liver-specific NAREs were up to 4-fold stronger than the CAG reference promoter and stronger than currently clinically used promoters (AAT, LP1, TBG). Dual-reporter plasmids were transiently transfected into the human liver cell line (Huh7), and expression of the mClover3 transgene was measured by flow cytometry. Figure 4C : Potency of the indicated NAREs in Huh7 cells. Detailed Description

[0066] Nucleic acid regulatory elements (NAREs), including promoters, are essential components of gene therapy, controlling the expression level and persistence of therapeutic genes. NAREs can drive cell-specific expression of transgenes independent of, for example, capsid selection. Incorporating stronger NAREs can increase potency and efficacy at lower viral vector doses, potentially reducing safety risks, immune responses, and vector production costs. Additionally, reducing the NARE size while maintaining strength and specificity allows for efficient packaging of larger transgenes or expression cassettes into AAVs. This application provides a library of NAREs to improve the safety, efficacy, and persistence of therapeutic transgene expression.

[0067] Although multiple NAREs and especially promoter sequences have been characterized / optimized by conventional low-throughput assays (rational design) or newer high-throughput methods such as MPRA, these methods still require expensive and time-consuming in vitro optimization. Here, using advanced artificial intelligence models, a convolutional neural network (CNN) was repurposed and optimized to predict promoter efficacy. A library of NAREs was computationally constructed by cloning all known enhancer elements reported in the ENCODE database upstream of active, compact constitutive promoters. Subsequently, the correct spacing between enhancer elements and promoter sequences that performed particularly well was optimized. At the same time, in silico saturation mutagenesis was performed, thereby introducing all possible point mutations within the promoter sequence and selecting those that had the greatest impact on promoter efficacy. Elements that performed particularly well were generated and tested in vitro. Although most NAREs exhibited better or the same performance as the original promoter, the best enhancer elements and point mutations were selected and combined in a second round of optimization. After this new set was synthesized and tested in vitro, an enhanced NARE that exhibited increased efficacy was obtained.

[0068] NARE

[0069] The present disclosure provides NAREs that may include a promoter and / or an enhancer as part of their sequence. Traditionally, a promoter has been defined as the DNA region that initiates transcription. A promoter includes specific DNA motifs to which transcription factors (TFs) and their complexes can access. On the other hand, an enhancer has been defined as the DNA region that amplifies transcription initiation by directly interacting with the target promoter. Similarly, enhancer sequences that are distant from the target promoter contain DNA motifs that serve as binding sites for TFs and cofactors. Sometimes, the term promoter can be used as a shorthand to refer to a nucleic acid sequence that contains multiple regulatory elements. For example, the control promoter CAG contains the CMV enhancer, the β-actin promoter region, and an intron, but can also be referred to as a promoter. Specifically, the CAG promoter consists of: (1) the cytomegalovirus (CMV) early enhancer element, (2) the promoter, the first exon, and the first intron of the chicken β-actin gene, and (3) the splice acceptor of the rabbit β-globin gene. As used herein, the term "nucleic acid regulatory element" can refer to a promoter as defined in the traditional sense, as well as an element combination that contains a promoter and / or other nucleic acid regulatory elements that regulate the expression of a gene operably linked to the element. A nucleic acid regulatory element can be or include, for example, one or more of a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0070] As used herein, the term "NARE" can refer to a promoter as traditionally defined, as well as a combination of nucleic acid regulatory elements that includes a promoter and / or other nucleic acid regulatory elements that regulate the expression of a gene operably linked to the element. The nucleic acid regulatory element can be or include, for example, one or more of a promoter, an enhancer, a translation initiation signal, an intron, and / or a splicing enhancer.

[0071] As used herein, a "transgene" refers to, for example, a gene (particularly the coding sequence of the gene) transferred into one or more cells of an organism using a vector as described herein. The transgene can encode a protein or RNA that is normally expressed in the cells of the target organism, or can encode a protein or RNA from a different organism. The transgene can be integrated into the genome of the target cell, or can exist as part of an extrachromosomal expression construct.

[0072] As used herein, "operatively linked" means that a first molecule is joined to a second molecule, where the arrangement of the molecules is such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. By way of example, if a NARE regulates the transcription in a cell of a transcribable polynucleotide molecule of interest, the NARE is operatively linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are joined such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion, they are operatively linked to each other. Two transcriptional regulatory elements can be operatively linked to each other by a linker nucleic acid (e.g., intervening non-coding nucleic acid), or can be operatively linked to each other in the absence of intervening nucleotides.

[0073] The present disclosure provides NAREs that are particularly suitable for driving expression in the liver. As used herein, a NARE that begins with "L" is particularly useful for the expression of a gene in the liver. A liver NARE can be liver-specific, meaning that they show a significant increase or preferential expression of an operably linked transgene in the liver compared to other tissues. Tables 5 and 7 provide NAREs that are particularly suitable for driving expression in the liver. Table 4 also provides NAREs that are suitable for driving expression in multiple tissues, including the liver.

[0074] In some embodiments, a polynucleotide sequence is provided that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the NARE provided in Table 5 or Table 7. In some embodiments, a polynucleotide is provided that comprises one or more of the NAREs provided in Table 5 or Table 7.

[0075] A polynucleotide sequence is provided herein that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the 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. A polynucleotide sequence is provided herein that comprises 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.

[0076] As used herein, the term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each of the sequences being aligned for purposes of comparison. For example, when a position in the compared nucleotide sequences is occupied by the same base, then the molecules are identical at that position. The degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at the shared positions. Methods and computer programs for determining sequence identity and similarity are publicly available, including but not limited to the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990)) and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm can also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0, ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions and other modifications when comparing sequences.

[0077] This disclosure provides a polynucleotide that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a NARE selected from the group consisting of: L10, L13, L15, L17, L45, L56-L61, L71-L79 and L89. This disclosure provides a polynucleotide that comprises a NARE selected from the group consisting of: L10, L13, L15, L17, L45, L56-L61, L71-L79 and L89.

[0078] This disclosure provides a polynucleotide that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a NARE selected from the group consisting of: L45, L123, L132 and L133. This disclosure provides a polynucleotide that comprises a NARE selected from the group consisting of: L45, L123, L132 and L133.

[0079] The present disclosure provides a method 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 the NARE provided in Table 5 or Table 7. The present disclosure provides a method for expressing a transgene in the liver, wherein the transgene is operably linked to the NARE provided in Table 5 or Table 7.

[0080] The present disclosure provides a method 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 the 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. The present disclosure provides a method for expressing a transgene in the liver, wherein the transgene is operably linked to the 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.

[0081] In an embodiment, the transgene is an ATP7B or an ATB7B minigene.

[0082] In an embodiment, the NARE comprises an intron or a part of an intron. The intron or the part of the intron may be selected from: ACTA1 intron, ACTA1e intron, ACTC1 intron, ACTC1.3 intron, ACTC1e intron, ACTC1e-ACTC1p intron, ALDOA intron, APOC intron, APOC1 intron, ATF5 intron, CAMK2A intron, CBA intron, chimeric intron, CMV-rabbit β-globin intron, CRYAB intron, DES.4 intron, EEF1A1 intron, EEF1B2 intron, EF1a intron, FHL1 intron, FLOT1 intron, FXYD1 intron, GFAP intron, HBB intron, hCPE intron, hEf1a2-intron, HPD intron, IFI27L2 intron, murine IgG chimeric intron, MVM intron, MVMi-AATp intron, MVMi-SynE-mTTRp intron, rabbit β-globin intron, RBP4 intron, RPL26 intron, RPL27 intron, S100A6 intron, sEEF1A1 intron, SV40 intron, TMSB10 intron, and UCHL1 intron.

[0083] In an embodiment, the NARE comprises an enhancer or a part of an enhancer. The enhancer or the part of the enhancer may be selected from: ACTA1 enhancer, ACTC1 enhancer, CKM enhancer, CMV enhancer, CMV enhancer, MCK enhancer, mDES enhancer, mDES.1 enhancer, minCKM enhancer, minCKM2 enhancer, minDes enhancer, NRGN enhancer 1.1, NRGN enhancer 1.2, NRGN enhancer 2.1, NRGN enhancer 2.2, SV40 enhancer, and SV40 enhancer (SV40e).

[0084] In an embodiment, the NARE comprises a UTR or a part of a UTR. The UTR or the part of the 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.

[0085] The present disclosure also provides NAREs for use as reference or control. Some NAREs provide for constitutive expression of an operably linked transgene, i.e., the expression of the transgene is maintained at a constant level. Constitutive NAREs can drive the expression of an operably linked transgene in a variety of cell types and tissues. As used herein, a promoter starting with "C" is a constitutive NARE. The constitutive NAREs disclosed herein can be used for gene expression in the liver, muscle, and / or central nervous system. Table 4 provides the sequences of the control NAREs.

[0086] In some embodiments, the NARE comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3-8, 11-69, 71-73, 76-149, and 256.

[0087] 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.

[0088] In some embodiments, the NARE comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 15, 45-64, 71, and 98.

[0089] In some embodiments, the NARE comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 45-64, and 98.

[0090] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 150; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 151 or SEQ ID NO: 155.

[0091] In some embodiments, the NARE comprises: (i) SEQ ID NO: 150; and (ii) SEQ ID NO: 151 or SEQ ID NO: 155.

[0092] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 150; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 151.

[0093] In some embodiments, the NARE comprises: (i) SEQ ID NO: 150; and (ii) SEQ ID NO: 151.

[0094] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 150; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0095] In some embodiments, the NARE comprises: (i) SEQ ID NO: 150; and (ii) SEQ ID NO: 155.

[0096] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 169, 172, or 173.

[0097] In some embodiments, the NARE comprises: (i) any one of SEQ ID NO:154, 157, or 158; (ii) any one of SEQ ID NO:153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, any one of SEQ ID NO:169, 172, or 173.

[0098] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:157; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:169, 172, or 173.

[0099] In some embodiments, the NARE comprises: (i) SEQ ID NO:157; (ii) any one of SEQ ID NO:153, 156, 161, 162, 163, 166, 167, 168, or 174; and (iii) optionally, any one of SEQ ID NO:169, 172, or 173.

[0100] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:156; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:154, 157, or 158.

[0101] In some embodiments, the NARE comprises: (i) SEQ ID NO:156; and (ii) any one of SEQ ID NO:154, 157, or 158.

[0102] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 156; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0103] In some embodiments, the NARE comprises: (i) SEQ ID NO: 156; and (ii) SEQ ID NO: 157.

[0104] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 161; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158.

[0105] In some embodiments, the NARE comprises: (i) SEQ ID NO: 161; and (ii) any one of SEQ ID NO: 154, 157, or 158.

[0106] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 161; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0107] In some embodiments, the NARE comprises: (i) SEQ ID NO: 161; and (ii) SEQ ID NO: 157.

[0108] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 162; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 154, 157 or 158.

[0109] In some embodiments, the NARE comprises: (i) SEQ ID NO: 162; and (ii) any one of SEQ ID NO: 154, 157 or 158.

[0110] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 162; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 157.

[0111] In some embodiments, the NARE comprises: (i) SEQ ID NO: 162; and (ii) SEQ ID NO: 157.

[0112] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 163; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 154, 157 or 158.

[0113] In some embodiments, the NARE comprises: (i) SEQ ID NO: 163; and (ii) any one of SEQ ID NO: 154, 157 or 158.

[0114] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 163; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0115] In some embodiments, the NARE comprises: (i) SEQ ID NO: 163; and (ii) SEQ ID NO: 157.

[0116] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 166; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158.

[0117] In some embodiments, the NARE comprises: (i) SEQ ID NO: 166; and (ii) any one of SEQ ID NO: 154, 157, or 158.

[0118] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 166; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0119] In some embodiments, the NARE comprises: (i) SEQ ID NO: 166; and (ii) SEQ ID NO: 157.

[0120] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 167; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158.

[0121] In some embodiments, the NARE comprises: (i) SEQ ID NO: 167; and (ii) any one of SEQ ID NO: 154, 157, or 158.

[0122] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 167; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0123] In some embodiments, the NARE comprises: (i) SEQ ID NO: 167; and (ii) SEQ ID NO: 157.

[0124] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158.

[0125] In some embodiments, the NARE comprises: (i) SEQ ID NO:168; (ii) SEQ ID NO:169; and (iii) any one of SEQ ID NO:154, 157 or 158.

[0126] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:169; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:157.

[0127] In some embodiments, the NARE comprises: (i) SEQ ID NO:168; (ii) SEQ ID NO:169; and (iii) SEQ ID NO:157.

[0128] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:157.

[0129] In some embodiments, the NARE comprises (i) SEQ ID NO:153; and (ii) SEQ ID NO:157.

[0130] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 157; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172.

[0131] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 154, 157 or 158; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.

[0132] In some embodiments, the NARE comprises (i) SEQ ID NO: 153; (ii) any one of SEQ ID NO: 154, 157 or 158; and (iii) SEQ ID NO: 172 or SEQ ID NO: 173.

[0133] In some embodiments, the NARE comprises (i) SEQ ID NO: 153; (ii) SEQ ID NO: 157; and (iii) SEQ ID NO: 172.

[0134] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 172 or SEQ ID NO: 173.

[0135] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) any one of SEQ ID NO: 154, 157, or 158; and (iii) SEQ ID NO: 172 or SEQ ID NO: 173.

[0136] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 173.

[0137] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 157; and (iii) SEQ ID NO: 173.

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

[0139] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0140] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0141] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; and (ii) SEQ ID NO: 157.

[0142] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 174; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0143] In some embodiments, the NARE comprises: (i) SEQ ID NO: 174; and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0144] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 174; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157.

[0145] In some embodiments, the NARE comprises: (i) SEQ ID NO: 174; and (ii) SEQ ID NO: 157.

[0146] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 171; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, 158, or 176.

[0147] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 171; and (iii) any one of SEQ ID NO: 154, 157, 158, or 176.

[0148] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 171; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154 or SEQ ID NO: 176.

[0149] 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.

[0150] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 171.

[0151] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) any one of SEQ ID NO: 154, 157, or 158; and (iii) SEQ ID NO: 171.

[0152] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 171.

[0153] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 154; and (iii) SEQ ID NO: 171.

[0154] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, 158, 176, 188, or 250; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 171.

[0155] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) any one of SEQ ID NO: 154, 157, 158, 176, 188, or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171.

[0156] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 153; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 154, 176, 188 or 250; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 171.

[0157] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) any one of SEQ ID NO: 154, 176, 188 or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO: 171.

[0158] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 183; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0159] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 188; and (iii) SEQ ID NO: 155.

[0160] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, or 158.

[0161] In some embodiments, the NARE comprises (i) SEQ ID NO: 153; and (ii) any one of SEQ ID NO: 154, 157, or 158.

[0162] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154.

[0163] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; and (ii) SEQ ID NO: 154.

[0164] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 154, 157, and 158; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0165] 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.

[0166] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 154; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0167] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 154; and (iii) SEQ ID NO: 155.

[0168] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 188.

[0169] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 188.

[0170] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; and (ii) SEQ ID NO: 188.

[0171] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 250.

[0172] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; and (ii) SEQ ID NO: 250.

[0173] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176.

[0174] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; and (ii) SEQ ID NO: 176.

[0175] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 171.

[0176] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 171.

[0177] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 153; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0178] In some embodiments, the NARE comprises: (i) SEQ ID NO: 153; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to either SEQ ID NO: 175 or any one of SEQ ID NOs: 177 - 187.

[0180] In some embodiments, the NARE comprises: (i) SEQ ID NO: 155; (ii) SEQ ID NO: 176; and (iii) either SEQ ID NO: 175 or any one of SEQ ID NOs: 177 - 187.

[0181] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 175; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0182] In some embodiments, the NARE comprises: (i) SEQ ID NO: 175; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 177; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0184] In some embodiments, the NARE comprises: (i) SEQ ID NO: 177; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 178; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0186] In some embodiments, the NARE comprises: (i) SEQ ID NO: 178; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 179; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0188] In some embodiments, the NARE comprises: (i) SEQ ID NO: 179; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0189] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 180; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0190] In some embodiments, the NARE comprises: (i) SEQ ID NO: 180; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0191] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 181; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0192] In some embodiments, the NARE comprises: (i) SEQ ID NO: 181; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0193] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 182; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0194] In some embodiments, the NARE comprises: (i) SEQ ID NO: 182; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0195] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 183; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0196] In some embodiments, the NARE comprises: (i) SEQ ID NO: 183; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0197] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 184; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0198] In some embodiments, the NARE comprises: (i) SEQ ID NO: 184; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0199] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 185; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0200] In some embodiments, the NARE comprises: (i) SEQ ID NO: 185; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0201] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 186; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0202] In some embodiments, the NARE comprises: (i) SEQ ID NO: 186; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0203] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 187; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 176; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0204] In some embodiments, the NARE comprises: (i) SEQ ID NO: 187; (ii) SEQ ID NO: 176; and (iii) SEQ ID NO: 155.

[0205] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 193, 194, or 195; (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; and (v) optionally, SEQ ID NO: 169.

[0206] In some embodiments, the NARE comprises: (i) SEQ ID NO: 190 or SEQ ID NO: 191; (ii) any one of SEQ ID NO: 193, 194, or 195; (iii) optionally, SEQ ID NO: 162; (iv) optionally, SEQ ID NO: 168; and (v) optionally, SEQ ID NO: 169.

[0207] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 190; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 195; and (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 162; (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 168; and (v) optionally, SEQ ID NO: 169.

[0208] 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.

[0209] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0210] 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.

[0211] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0212] In some embodiments, the NARE comprises: (i) SEQ ID NO: 190; and (ii) SEQ ID NO: 193.

[0213] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; and (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0214] 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.

[0215] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; and (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0216] 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.

[0217] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190; and (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[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: 190; and (v) SEQ ID NO: 193.

[0219] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 195.

[0220] In some embodiments, the NARE comprises (i) SEQ ID NO: 190 or SEQ ID NO: 191; and (ii) SEQ ID NO: 195.

[0221] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 190; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 195.

[0222] In some embodiments, the NARE comprises: (i) SEQ ID NO: 190; and (ii) SEQ ID NO: 195.

[0223] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 197, 202 or 203; (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 190, 191, 192, 196, 202, 203, 204, 205 or 206; (v) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155 or SEQ ID NO: 162; and (vi) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 166, 193 or 194.

[0224] In some embodiments, the NARE comprises: (i) SEQ ID NO:168; (ii) SEQ ID NO:169; (iii) any one of SEQ ID NO:197, 202 or 203; (iv) optionally, any one of SEQ ID NO: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 NO:166, 193 or 194.

[0225] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:197 or SEQ ID NO:202; (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO:191, 192, 196, 203, 204 or 206; (v) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:155 or SEQ ID NO:162; and (vi) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:166 or SEQ ID NO:193.

[0226] 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 NO: 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.

[0227] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 197; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 190 or SEQ ID NO: 191.

[0228] 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.

[0229] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 191.

[0230] 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.

[0231] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 166; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (vi) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 196.

[0232] 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.

[0233] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203; and (vi) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0234] 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.

[0235] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 162; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 202; and (vi) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0236] 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.

[0237] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203.

[0238] 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.

[0239] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; and (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 203.

[0240] 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.

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

[0242] 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.

[0243] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 206; and (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0244] 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.

[0245] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 192; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 201 - 203.

[0246] 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.

[0247] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 192; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193; and (iii) SEQ ID NO: 201 or SEQ ID NO: 202.

[0248] 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.

[0249] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 201; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0250] In some embodiments, the NARE comprises: (i) SEQ ID NO: 201; and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0251] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 201; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0252] In some embodiments, the NARE comprises: (i) SEQ ID NO: 201; and (ii) SEQ ID NO: 193.

[0253] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0254] 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.

[0255] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 202 or SEQ ID NO: 203; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0256] In some embodiments, the NARE comprises: (i) SEQ ID NO: 202; and (ii) SEQ ID NO: 193.

[0257] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 204, 205, 206, or 236 - 238; (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197.

[0258] In some embodiments, the NARE comprises: (i) SEQ ID NO: 155; (ii) any one of SEQ ID NO: 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.

[0259] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 204, 205, or 236 - 238; (iii) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (iv) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; and (v) optionally, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197.

[0260] In some embodiments, the NARE comprises: (i) SEQ ID NO: 155; (ii) any one of SEQ ID NO: 204, 205, or 236 - 238; (iii) optionally, SEQ ID NO: 168; (iv) optionally, SEQ ID NO: 169; and (v) optionally, SEQ ID NO: 197.

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

[0262] In some embodiments, the NARE comprises: (i) SEQ ID NO: 204; and (ii) SEQ ID NO: 155.

[0263] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0264] In some embodiments, the NARE comprises: (i) SEQ ID NO: 205 or SEQ ID NO: 206; and (ii) SEQ ID NO: 155.

[0265] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0266] In some embodiments, the NARE comprises: (i) SEQ ID NO: 205; and (ii) SEQ ID NO: 155.

[0267] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 238; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0268] In some embodiments, the NARE comprises (i) SEQ ID NO: 238; and (ii) SEQ ID NO: 155.

[0269] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 236 or SEQ ID NO: 237; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0270] In some embodiments, the NARE comprises: (i) SEQ ID NO: 236 or SEQ ID NO: 237; and (ii) SEQ ID NO: 155.

[0271] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 237; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0272] In some embodiments, the NARE comprises: (i) SEQ ID NO: 237; and (ii) SEQ ID NO: 155.

[0273] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 236 or SEQ ID NO: 237; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0274] In some embodiments, the NARE comprises: (i) SEQ ID NO: 236 or SEQ ID NO: 237; and (ii) SEQ ID NO: 155.

[0275] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 236; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0276] In some embodiments, the NARE comprises: (i) SEQ ID NO: 236; and (ii) SEQ ID NO: 155.

[0277] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169; (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 197; (iv) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 204; (v) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0278] 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.

[0279] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 239-247.

[0280] In some embodiments, the NARE comprises: (i) SEQ ID NO: 248; and (ii) any one of SEQ ID NOs: 239-247.

[0281] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 239; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0282] In some embodiments, the NARE comprises: (i) SEQ ID NO: 239; and (ii) SEQ ID NO: 248.

[0283] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 240; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0284] In some embodiments, the NARE comprises: (i) SEQ ID NO: 240; and (ii) SEQ ID NO: 248.

[0285] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 241; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0286] In some embodiments, the NARE comprises: (i) SEQ ID NO: 241; and (ii) SEQ ID NO: 248.

[0287] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 242; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0288] In some embodiments, the NARE comprises: (i) SEQ ID NO: 242; and (ii) SEQ ID NO: 248.

[0289] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 243; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0290] In some embodiments, the NARE comprises: (i) SEQ ID NO: 243; and (ii) SEQ ID NO: 248.

[0291] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 244; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0292] In some embodiments, the NARE comprises: (i) SEQ ID NO: 244; and (ii) SEQ ID NO: 248.

[0293] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 245; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0294] In some embodiments, the NARE comprises: (i) SEQ ID NO: 245; and (ii) SEQ ID NO: 248.

[0295] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 246; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0296] In some embodiments, the NARE comprises: (i) SEQ ID NO: 246; and (ii) SEQ ID NO: 248.

[0297] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 247; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 248.

[0298] In some embodiments, the NARE comprises: (i) SEQ ID NO: 247; and (ii) SEQ ID NO: 248.

[0299] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 209 - 214.

[0300] In some embodiments, the NARE comprises: (i) SEQ ID NO: 208; and (ii) any one of SEQ ID NOs: 209 - 214.

[0301] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0302] In some embodiments, the NARE comprises: (i) SEQ ID NO: 207; and (ii) SEQ ID NO: 208.

[0303] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 209; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0304] In some embodiments, the NARE comprises: (i) SEQ ID NO: 209; and (ii) SEQ ID NO: 208.

[0305] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0306] In some embodiments, the NARE comprises: (i) SEQ ID NO: 210; and (ii) SEQ ID NO: 208.

[0307] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0308] In some embodiments, the NARE comprises: (i) SEQ ID NO: 211; and (ii) SEQ ID NO: 208.

[0309] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 212; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0310] In some embodiments, the NARE comprises: (i) SEQ ID NO: 212; and (ii) SEQ ID NO: 208.

[0311] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 213; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0312] In some embodiments, the NARE comprises: (i) SEQ ID NO: 213; and (ii) SEQ ID NO: 208.

[0313] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 214; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 208.

[0314] In some embodiments, the NARE comprises: (i) SEQ ID NO: 214; and (ii) SEQ ID NO: 208.

[0315] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 193, 194, 207, 209-214, 216-232, 233-235, or 249.

[0316] 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.

[0317] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 194, 207, 209-214, 216-232, 233-235, or 249.

[0318] 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.

[0319] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:207; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74.

[0320] In some embodiments, the NARE comprises: (i) SEQ ID NO:207; and (ii) SEQ ID NO:74.

[0321] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:209; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:74.

[0322] In some embodiments, the NARE comprises: (i) SEQ ID NO:209; and (ii) SEQ ID NO:74.

[0323] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:210; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:74.

[0324] In some embodiments, the NARE comprises: (i) SEQ ID NO:210; and (ii) SEQ ID NO:74.

[0325] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:211; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:74.

[0326] In some embodiments, the NARE comprises: (i) SEQ ID NO:211; and (ii) SEQ ID NO:74.

[0327] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 212; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0328] In some embodiments, the NARE comprises: (i) SEQ ID NO: 212; and (ii) SEQ ID NO: 74.

[0329] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 213; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0330] In some embodiments, the NARE comprises: (i) SEQ ID NO: 213; and (ii) SEQ ID NO: 74.

[0331] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 214; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0332] In some embodiments, the NARE comprises: (i) SEQ ID NO: 214; and (ii) SEQ ID NO: 74.

[0333] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0334] In some embodiments, the NARE comprises: (i) SEQ ID NO: 193 or SEQ ID NO: 194; (ii) SEQ ID NO: 215; and (iii) SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0335] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0336] In some embodiments, the NARE comprises: (i) SEQ ID NO: 193; (ii) SEQ ID NO: 215; and (iii) SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0337] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0338] 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.

[0339] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0340] In some embodiments, the NARE comprises: (i) SEQ ID NO: 207; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 209; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0342] 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.

[0343] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 209; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0344] In some embodiments, the NARE comprises: (i) SEQ ID NO: 209; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0346] 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.

[0347] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0348] In some embodiments, the NARE comprises: (i) SEQ ID NO: 210; (ii) SEQ ID NO: 215; and (iii) SEQ ID NO: 193.

[0349] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0350] 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.

[0351] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0352] In some embodiments, the NARE comprises: (i) SEQ ID NO: 211; (ii) SEQ ID NO: 215; and (iii) SEQ ID NO: 193.

[0353] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 212; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0354] 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.

[0355] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 212; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0356] In some embodiments, the NARE comprises: (i) SEQ ID NO: 212; (ii) SEQ ID NO: 215; and (iii) SEQ ID NO: 193.

[0357] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 213; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0358] 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.

[0359] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 213; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 193.

[0360] In some embodiments, the NARE comprises: (i) SEQ ID NO: 213; (ii) SEQ ID NO: 215; and (iii) SEQ ID NO: 193.

[0361] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 214; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0362] 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.

[0363] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 214; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 215; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0364] In some embodiments, the NARE comprises: (i) SEQ ID NO: 213; (ii) SEQ ID NO: 214; and (iii) SEQ ID NO: 193.

[0365] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0366] In some embodiments, the NARE comprises: (i) SEQ ID NO: 155; (ii) SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0367] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0368] In some embodiments, the NARE comprises: (i) SEQ ID NO: 155; (ii) SEQ ID NO: 205; and (iii) SEQ ID NO: 207 or any one of SEQ ID NOs: 209-215.

[0369] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0370] 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.

[0371] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 207; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0372] In some embodiments, the NARE comprises: (i) SEQ ID NO: 207; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 209; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0374] 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.

[0375] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 209; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0376] In some embodiments, the NARE comprises: (i) SEQ ID NO: 209; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0378] 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.

[0379] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 210; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0380] In some embodiments, the NARE comprises: (i) SEQ ID NO: 210; (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%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0382] 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.

[0383] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0384] In some embodiments, the NARE comprises: (i) SEQ ID NO: 211; (ii) SEQ ID NO: 205; and (iii) SEQ ID NO: 155.

[0385] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 212; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0386] 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.

[0387] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 212; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0388] In some embodiments, the NARE comprises: (i) SEQ ID NO: 212; (ii) SEQ ID NO: 205; and (iii) SEQ ID NO: 155.

[0389] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 213; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0390] 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.

[0391] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 213; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 155.

[0392] In some embodiments, the NARE comprises: (i) SEQ ID NO: 213; (ii) SEQ ID NO: 205; and (iii) SEQ ID NO: 155.

[0393] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 214; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205 or SEQ ID NO: 206; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0394] 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.

[0395] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 214; (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 205; and (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 155.

[0396] In some embodiments, the NARE comprises: (i) SEQ ID NO: 214; (ii) SEQ ID NO: 205; and (iii) SEQ ID NO: 155.

[0397] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 216; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0398] In some embodiments, the NARE comprises: (i) SEQ ID NO: 216; and (ii) SEQ ID NO: 74.

[0399] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 217; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0400] In some embodiments, the NARE comprises: (i) SEQ ID NO: 217; and (ii) SEQ ID NO: 74.

[0401] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 218; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0402] In some embodiments, the NARE comprises: (i) SEQ ID NO: 218; and (ii) SEQ ID NO: 74.

[0403] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 219; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0404] In some embodiments, the NARE comprises: (i) SEQ ID NO: 219; and (ii) SEQ ID NO: 74.

[0405] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 220; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0406] In some embodiments, the NARE comprises: (i) SEQ ID NO: 220; and (ii) SEQ ID NO: 74.

[0407] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 221; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0408] In some embodiments, the NARE comprises: (i) SEQ ID NO: 221; and (ii) SEQ ID NO: 74.

[0409] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 222; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 74.

[0410] In some embodiments, the NARE comprises: (i) SEQ ID NO: 222; and (ii) SEQ ID NO: 74.

[0411] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 223; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 74.

[0412] In some embodiments, the NARE comprises: (i) SEQ ID NO: 223; and (ii) SEQ ID NO: 74.

[0413] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 224; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 74.

[0414] In some embodiments, the NARE comprises: (i) SEQ ID NO: 224; and (ii) SEQ ID NO: 74.

[0415] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 225; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0416] In some embodiments, the NARE comprises: (i) SEQ ID NO: 225; and (ii) SEQ ID NO: 74.

[0417] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 226; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0418] In some embodiments, the NARE comprises: (i) SEQ ID NO: 226; and (ii) SEQ ID NO: 74.

[0419] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 227; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0420] In some embodiments, the NARE comprises: (i) SEQ ID NO: 227; and (ii) SEQ ID NO: 74.

[0421] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 228; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0422] In some embodiments, the NARE comprises: (i) SEQ ID NO: 228; and (ii) SEQ ID NO: 74.

[0423] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 229; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0424] In some embodiments, the NARE comprises: (i) SEQ ID NO: 229; and (ii) SEQ ID NO: 74.

[0425] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 230; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0426] In some embodiments, the NARE comprises: (i) SEQ ID NO: 230; and (ii) SEQ ID NO: 74.

[0427] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 231; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0428] In some embodiments, the NARE comprises: (i) SEQ ID NO: 231; and (ii) SEQ ID NO: 74.

[0429] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 232; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0430] In some embodiments, the NARE comprises: (i) SEQ ID NO: 232; and (ii) SEQ ID NO: 74.

[0431] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0432] In some embodiments, the NARE comprises: (i) SEQ ID NO: 193 or SEQ ID NO: 194; and (ii) SEQ ID NO: 74.

[0433] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 194; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0434] In some embodiments, the NARE comprises: (i) SEQ ID NO: 194; and (ii) SEQ ID NO: 74.

[0435] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 233; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0436] In some embodiments, the NARE comprises: (i) SEQ ID NO: 233; and (ii) SEQ ID NO: 74.

[0437] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 234; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0438] In some embodiments, the NARE comprises: (i) SEQ ID NO: 234; and (ii) SEQ ID NO: 74.

[0439] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 235; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0440] In some embodiments, the NARE comprises: (i) SEQ ID NO: 235; and (ii) SEQ ID NO: 74.

[0441] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 249; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74.

[0442] In some embodiments, the NARE comprises: (i) SEQ ID NO: 249; and (ii) SEQ ID NO: 74.

[0443] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 165; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 154, 157, or 158.

[0444] In some embodiments, the NARE comprises (i) SEQ ID NO: 165; and (ii) any one of SEQ ID NOs: 154, 157, or 158.

[0445] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 165; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 158.

[0446] In some embodiments, the NARE comprises (i) SEQ ID NO: 165; and (ii) SEQ ID NO: 158.

[0447] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0448] In some embodiments, the NARE comprises (i) SEQ ID NO: 199; and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0449] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 199; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0450] In some embodiments, the NARE comprises (i) SEQ ID NO: 199; and (ii) SEQ ID NO: 193.

[0451] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 200.

[0452] In some embodiments, the NARE comprises: (i) SEQ ID NO: 164; and (ii) SEQ ID NO: 200.

[0453] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 189; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193 or SEQ ID NO: 194.

[0454] In some embodiments, the NARE comprises: (i) SEQ ID NO: 189; and (ii) SEQ ID NO: 193 or SEQ ID NO: 194.

[0455] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 189; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 193.

[0456] In some embodiments, the NARE comprises: (i) SEQ ID NO: 189; and (ii) SEQ ID NO: 193.

[0457] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:202 or SEQ ID NO:203; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:195.

[0458] In some embodiments, the NARE comprises: (i) SEQ ID NO:202 or SEQ ID NO:203; and (ii) SEQ ID NO:195.

[0459] In some embodiments, the NARE comprises: (i) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:202; and (ii) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:195.

[0460] In some embodiments, the NARE comprises: (i) SEQ ID NO:202; and (ii) SEQ ID NO:195.

[0461] Provided herein is a method for expressing a transgene in hepatocytes (e.g., in the liver), wherein the transgene is operably linked to a NARE that is particularly suitable for driving expression in hepatocytes (e.g., in the liver) as disclosed herein.

[0462] NARE modification

[0463] 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 are known in the art for confirming that modified NAREs are still capable of driving gene expression, including but not limited to the methods used herein. By way of example, the strength of the promoter can be assessed by operably linking the NARE to a transgene encoding a protein and measuring transgene expression, such as by detecting the mRNA encoding the protein, or by measuring the presence or activity of the protein, such as by ELISA, Western Blot, fluorescence, enzymatic activity of the protein, etc.

[0464] The present disclosure provides a NARE comprising one or more components. The present disclosure provides a NARE comprising one or more sequence components (or sequence variants thereof) listed in any of Tables 1-7. The present disclosure provides a NARE comprising one or more sequence components (or sequence variants thereof) listed in Table 7. The present disclosure provides a NARE comprising one or more components (or sequence variants thereof) of the full-length NARE provided in Table 7. By way of non-limiting example only, a NARE is provided that comprises one or more components (or sequence variants thereof) of NAREL4 (AATcre-mTTR): (1) AATcre; and (2) the mTTRmd promoter. In an embodiment, a NARE is provided that comprises one or more components (or sequence variants thereof) of the 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 an embodiment, a NARE is provided that comprises one or more components (or sequence variants thereof) of the full-length NARE provided in Table 7, whereby the components (or sequence variants thereof) are arranged in a different order 5' to 3' than shown in Table 4.

[0465] In an embodiment, in the NAREs of Table 5 or Table 7, one or more components have been replaced with different sequences, including components (or sequence variants thereof) from different NAREs of Table 5 or Table 7. In addition, NAREs are contemplated that comprise components (or sequence variants thereof) derived from two or more NAREs disclosed in Table 5 or Table 7.

[0466] In addition, NARE variants are contemplated that include sequences other than the elements of the NAREs disclosed herein (e.g., added to either end or inserted within the NARE sequence). Also provided are NARE variants in which certain sequences have been removed from the NAREs disclosed herein (e.g., as terminal or internal deletions). In an embodiment, a NARE is provided that comprises one or more components without any additional nucleic acid sequence joining the components of the NARE. In an embodiment, a NARE is provided that comprises one or more components, whereby the individual components are joined by additional nucleic acid sequences. These additional nucleic acid sequences may or may not be related to the expression of an operably linked transgene. In an embodiment, some components are directly joined, while other components are joined to other components by intervening sequences. Thus, variants of the NAREs disclosed in Table 5 or Table 7 are contemplated in which additional sequences are added between different components. Further contemplated are variants of the NAREs disclosed in Table 5 or Table 7 that disclose the same components of a given NARE in Table 5 or Table 7, but differ in the sequences joining the individual components.

[0467] Nucleic acid constructs and vectors

[0468] On the one hand, nucleic acid constructs and vectors are provided, as well as their use for introducing a transgene or an expression construct into a cell. The nucleic acid construct includes an expression construct, including a plasmid. The term "expression construct" refers to a recombinant polynucleotide construct that includes a nucleic acid encoding an RNA capable of being transcribed in a cell. Methods for constructing expression constructs and plasmids by standard recombinant techniques are known in the art.

[0469] In some embodiments, the expression construct contains a sequence encoding ATP7B, wherein the ATP7B coding sequence is codon-optimized.

[0470] On the one hand, an expression construct is provided, the expression construct comprising:

[0471] a) a promoter;

[0472] b) a sequence encoding ATP7B, operably linked to the promoter; and

[0473] c) a polyadenylation signal.

[0474] In some embodiments, the vector contains a recombinant DNA construct, the recombinant DNA construct including additional DNA elements, including DNA segments providing for DNA replication in a host cell and expression of a target gene at an appropriate level in a target cell. As used herein, "vector" means an agent containing a polynucleotide to be delivered to a host cell in vitro or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA microcircles, or viruses (including virus-derived sequences). A vector can also refer to a viral particle containing a nucleic acid to be delivered to a host cell in vitro or in vivo. In some embodiments, a vector refers to a viral particle containing a recombinant viral genome, wherein the viral genome contains one or more ITRs and a transgene.

[0475] In one embodiment, the vector is a viral vector or a combination of multiple viral vectors. On the one hand, a vector is provided, the vector containing any nucleic acid construct disclosed herein.

[0476] The present disclosure provides nucleic acid constructs and vectors that comprise nucleic acid regulatory elements disclosed herein operably linked to a transgene. In embodiments, a nucleic acid construct or vector disclosed herein comprises additional regulatory elements, including but not limited to promoters, enhancers, translation initiation signals, introns, and / or splicing enhancers. In embodiments, a nucleic acid construct or vector disclosed herein comprises a polyadenylation sequence. In embodiments, a nucleic acid construct or vector disclosed herein comprises an internal ribosome entry site (IRES). IRES sequences can be used to produce more than one polypeptide from a single gene transcript. The IRES (or other suitable sequence) is used to produce a protein containing more than one polypeptide chain, or to express two different proteins in or from the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE, and ganglion cells. In one embodiment, the IRES is located 3′ to the transgene.

[0477] Viral vector

[0478] Viral vectors for expressing a target gene in a target cell, tissue, or organism are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxviral vectors, baculoviral vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric viruses, mosaic viruses, or pseudotyped viruses and / or viruses containing exogenous proteins, synthetic polymers, nanoparticles, or small molecules).

[0479] AAV vector

[0480] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires a helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames that encode the Rep and Cap proteins, respectively. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. The functions of these proteins are mainly to regulate AAV replication and to rescue AAV and integrate it into the host cell chromosome. The Cap reading frame encodes three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the viral particle capsid. More than 80% of the total protein in the AAV viral particle comprises VP3. Flanking the rep and cap open reading frames at the 5′ and 3′ termini are inverted terminal repeats (ITRs) that are approximately 145 bp in length. The two ITRs are the only cis-elements required for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter gene transgene.

[0481] The recombinant adeno-associated virus "rAAV" vector includes any vector derived from any adeno-associated virus serotype. The rAAV vector may have one or more of the AAV wild-type genes that are completely or partially deleted, preferably the Rep and / or Cap genes, but retains the functional flanking ITR sequences.

[0482] In some embodiments, the viral vector is an rAAV virion that contains an rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome contains the nucleic acid construct disclosed herein.

[0483] In some embodiments, the viral vector disclosed herein contains a nucleic acid that includes AAV 5′ ITR and 3′ ITR located 5′ and 3′, respectively, of a sequence encoding a transgene. In an embodiment, the transgene is UPF1, BDNF, or ATP7B. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5′ ITR and 3′ ITR sequences arranged in tandem, such as 5′ to 3′ or head-to-tail or in another alternative configuration. In other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITR, or to have 5′ ITRs (or conversely, 3′ ITRs) located both 5′ and 3′ of the transgene. The ITR sequences may be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences may be present. The ITRs need not be wild-type nucleotide sequences and may be altered (e.g., by nucleotide insertion, deletion, or substitution), provided that the sequences provide functional rescue, replication, and packaging. The ITRs may be selected from AAV2 or from other AAV serotypes as described herein.

[0484] In some embodiments, a vector containing a nucleic acid sequence is provided, the nucleic acid sequence comprising (i) the 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 the adeno-associated virus (AAV) serotype AAV2.

[0485] In some embodiments, the viral vector is an AAV vector, such as AAV1 (i.e., AAV containing AAV1 ITR and AAV1 capsid protein), AAV2 (i.e., AAV containing AAV2 ITR and AAV2 capsid protein), AAV3 (i.e., AAV containing AAV3 ITR and AAV3 capsid protein), AAV4 (i.e., AAV containing AAV4 ITR and AAV4 capsid protein), AAV5 (i.e., AAV containing AAV5 ITR and AAV5 capsid protein), AAV6 (i.e., AAV containing AAV6 ITR and AAV6 capsid protein), AAV7 (i.e., AAV containing AAV7 ITR and AAV7 capsid protein), AAV8 (i.e., AAV containing AAV8 ITR and AAV8 capsid protein), AAV9 (i.e., AAV containing AAV9 ITR and AAV9 capsid protein), AAVrh74 (i.e., AAV containing AAVrh74 ITR and AAVrh74 capsid protein), AAVrh.8 (i.e., AAV containing AAVrh.8 ITR and AAVrh.8 capsid protein), or AAVrh.10 (i.e., AAV containing AAVrh.10 ITR and AAVrh.10 capsid protein).

[0486] In some embodiments, the viral vector is a pseudotyped AAV vector that contains ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., AAV containing AAV2 ITR and AAV9 capsid protein). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., AAV containing AAV2 ITR and AAV10 capsid protein).

[0487] In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., AAV containing AAV2 ITR and AAV7m8 capsid protein).

[0488] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein of a chimeric protein containing one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In an embodiment, the capsid is a variant AAV capsid, such as AAV2 variant rAAV2-retro (SEQ ID NO:44, from WO 2017 / 218842, incorporated herein by reference).

[0489] 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-retro 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.

[0490] In some embodiments, a mixture of (1) an AAV vector comprising rAAV2-retro and (2) an AAV vector comprising AAVrh.10 is used. In some embodiments, the ratio of (1) the AAV vector comprising rAAV2-retro to (2) the AAV vector comprising AAVrh.10 is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively. In some embodiments, the ratio of (1) the AAV vector comprising AAVrh.10 to (2) the AAV vector comprising rAAV2-retro is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, respectively.

[0491] Other viral vectors

[0492] Other viral vectors include adenovirus (AV) vectors, such as those based on human adenovirus type 2 and human adenovirus type 5, which have been rendered replication-deficient by deletions in the E1 and E3 regions. A transcription cassette can be inserted into the E1 region, thereby generating a recombinant E1 / E3-deleted AV vector. Adenovirus vectors also include helper-dependent high-capacity adenovirus vectors (also referred to as high-capacity "gutless" or "viremic" vectors), which contain no viral coding sequences. These vectors contain the cis-acting elements required for viral DNA replication and packaging, mainly the inverted terminal repeats (ITRs) and the packaging signal (CY). The genomes of these helper-dependent AV vectors have the potential to carry exogenous DNA ranging from a few hundred base pairs to approximately 36 kb.

[0493] Alternatively, other systems such as lentiviral vectors can be used. Lentivirus-based systems can transduce both non-dividing cells and dividing cells, making them useful for applications targeting non-dividing cells such as those in the CNS. Lentiviral vectors are derived from the human immunodeficiency virus and, like the virus, integrate into the host genome, thereby providing the potential for very long-term gene expression.

[0494] Cationic lipids, polymers, or both can also be used as vectors to introduce polynucleotides (including plasmids, YACs, minichromosomes, and microcircles) carrying a target gene containing an expression cassette into cells or organisms by non-viral vector systems. Conjugated poly-L-lysine (PLL) polymers and polyethyleneimine (PEI) polymer systems can also be used to deliver the vectors to cells. Other methods for delivering vectors to cells include hydrodynamic injection and electroporation, as well as the use of ultrasound, both for cell culture and for organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012;1:27), which is incorporated herein by reference.

[0495] rAAV virion production

[0496] The rAAV viral particles disclosed herein can be constructed and produced using the materials and methods described herein and those known to those of skill in the art. Such engineering methods for constructing any embodiment of the present disclosure are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989); and International Patent Publication No. WO 95 / 13598. In addition, methods suitable for generating rAAV cassettes in an adenovirus capsid have been described in U.S. Patent Nos. 5,856,152 and 5,871,982.

[0497] Briefly, to package the rAAV genome into rAAV viral particles, a host cell containing sequences necessary for expressing AAV rep and AAV cap or functional fragments thereof and helper genes necessary for AAV production is used. The AAV rep and cap sequences are obtained from AAV sources as identified herein. The AAV rep and cap sequences can be introduced into the host cell in any manner known to those of skill in the art, including but not limited to transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into the host cell by one or more nucleic acid molecules and stably exist in the cell as episomes. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has rep and cap sequences that are transiently expressed in the host cell. For example, a nucleic acid molecule useful for such transfection contains, from 5' to 3', a NARE, an optional spacer inserted between the NARE and the start site of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.

[0498] The rep and cap sequences, along with their expression control sequences, can be provided on a single vector, or each sequence can be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences can be provided on a vector containing other DNA sequences to be introduced into the host cell. Preferably, the promoter used in such constructs can be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins can be in any form that transfers these components into the host cell. Ideally, such a molecule is in the form of a plasmid, which can contain other non-viral sequences, such as those of marker genes. Such a molecule does not contain the AAV ITR, and generally does not contain the AAV packaging sequence. To avoid the occurrence of homologous recombination, other viral sequences, especially those of adenovirus, are avoided in such plasmids. The plasmid is ideally constructed such that it can be stably transfected into cells.

[0499] Although the molecule providing the rep and cap can be transiently transfected into the host cell, it is preferred to stably transform the host cell with the sequences necessary for expressing functional rep / cap proteins in the host cell, for example, as an episome or by integration into the chromosome of the host cell. Depending on the promoter controlling the expression of such stably transfected host cells, the rep / cap proteins can be transiently expressed (e.g., by using an inducible promoter).

[0500] The methods for constructing the embodiments of the present disclosure are conventional genetic engineering or recombinant engineering techniques, such as those described in the above references. For example, rAAV can be produced using the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.) with a triple transfection method according to the manufacturer's instructions. See also Herzog et al., 1999, Nature Medic., 5(1):56 - 63, for the method used in the following examples, using a plasmid with a transgene, a helper plasmid containing AAV rep and cap, and a plasmid providing the adenovirus helper functions of E2A, E4 Orf6, and VA. Although this specification provides illustrative examples of specific constructs, using the information provided herein, those skilled in the art can select and design other suitable constructs using the selection of spacers, promoters, and other elements (including at least one translation start and stop signal) and optionally adding polyadenylation sites.

[0501] rAAV viral particles can be produced by culturing host cells containing an rAAV virus as described herein, the host cells containing an rAAV genome to be packaged into the rAAV viral particles, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression. Suitable viral helper genes (such as adenovirus E2A, E4 Orf6, and VA, and other possible helper genes) can be provided to the culture in a variety of ways known in the art, preferably on a separate plasmid. Thereafter, recombinant AAV viral particles directing the expression of the transgene are isolated from the cells or cell culture in the absence of contaminating helper virus or wild-type AAV.

[0502] Expression of the transgene can be measured in a manner known in the art. For example, target cells can be infected in vitro, and the copy number of the transgene in the cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression levels can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and protein expression levels can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by specific methods detailed in the Examples below.

[0503] Cell

[0504] In embodiments, the nucleic acid constructs and vectors disclosed herein are used to deliver a NARE operably linked to a transgene to a cell. Cells containing a NARE, nucleic acid construct, or vector disclosed herein are provided. In embodiments, the cell is a liver cell. The cell can be a mammalian cell. The cell can be a human cell. The cell may be isolated.

[0505] Pharmaceutical composition

[0506] Pharmaceutical compositions are provided, the pharmaceutical compositions comprising a nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.

[0507] Preferably, contamination of the nucleic acid constructs or vectors disclosed herein is assayed by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0508] The formulation of the nucleic acid constructs or vectors disclosed herein involves the use of pharmaceutically and / or physiologically acceptable agents or carriers, particularly agents or carriers suitable for injection, such as buffered saline or other buffers (e.g., HEPES), to maintain the pH at an appropriate physiological level. The nucleic acid constructs or vectors disclosed herein can be formulated into pharmaceutical compositions. In addition to the carrier, these compositions can also contain pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials can be determined by the skilled person according to the route of administration. Pharmaceutical compositions are generally in liquid form. Liquid pharmaceutical compositions generally include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, which is incorporated herein by reference. It can include saline solutions, magnesium chloride, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, surfactants such as Pluronic acid (PF68) 0.001% can be used. In some cases, Ringer's injection solution, lactated Ringer's injection solution, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.

[0509] Pharmaceutical compositions containing the nucleic acid constructs or vectors disclosed herein can be formulated with one or more pharmaceutically acceptable excipients, which can be pharmaceutically acceptable materials, compositions, or agents such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate, or zinc stearate or stearic acid), solvents, or encapsulating materials, which are involved in carrying or transporting a therapeutic compound for administration to a subject, swelling agents, salts, surfactants, and / or preservatives. Some examples of materials that can be used as pharmaceutically acceptable excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations.

[0510] An expander is a compound that increases the mass of a pharmaceutical preparation and contributes to the physical structure of the preparation in lyophilized form. Suitable expanders according to the present invention include mannitol, glycine, polyethylene glycol, and sorbitol.

[0511] The use of a surfactant can reduce the aggregation of the reconstituted protein and / or reduce the formation of microparticles in the reconstituted preparation. The amount of surfactant added should be such that it reduces the aggregation of the reconstituted protein and minimizes the formation of microparticles after reconstitution. Suitable surfactants according to the present invention include polysorbates (such as polysorbate 20 or 80); poloxamers (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-betaine (such as lauramidopropyl); myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or sodium methyl oleoyl taurate; and copolymers of polyethylene glycol, polypropylene glycol, and ethylene glycol and propylene glycol (such as Pluronics, PF68, etc.).

[0512] Preservatives can be used in the preparations of the present invention. Suitable preservatives for the preparations of the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl-dimethyl ammonium chloride, where the alkyl is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols (such as phenol, butanol, and benzyl alcohol), alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, such as "Remington’s Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Edition, Mack Publishing Company, Easton, Pa., (1995).

[0513] For delayed release, the nucleic acid constructs or vectors disclosed herein can be included in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from a biocompatible polymer or in a liposomal carrier system according to methods known in the art.

[0514] If the carrier is intended for long-term storage, it can be frozen in the presence of glycerol.

[0515] Method

[0516] The present disclosure provides methods for inducing the expression of a transgene in a cell and / or tissue. The present disclosure provides a method for inducing the expression of a transgene, the method comprising providing a cell comprising a nucleic acid construct comprising a NARE disclosed herein operably linked to a transgene, and culturing the cell under conditions that permit the expression of the transgene. In some embodiments, the cell is a hepatocyte. The present disclosure provides a method for inducing the expression of a transgene in vivo. The present disclosure provides a method for inducing the expression of a transgene in vitro. The present disclosure provides a method for inducing the expression of a transgene ex vivo.

[0517] The present disclosure provides methods for treating a disease or disorder in a subject in need thereof using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein. In some embodiments, the disease is Wilson's disease (WD). In some embodiments, the subject has a mutation in ATP7B.

[0518] The present disclosure provides a method for treating or preventing dystonia or bradykinesia in a subject suffering from Wilson's disease, the method comprising administering to the subject a vector or pharmaceutical composition disclosed herein. The present disclosure provides a vector or pharmaceutical composition disclosed herein for treating or preventing dystonia or bradykinesia in a subject suffering from Wilson's disease. The present disclosure provides the 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.

[0519] The present disclosure provides a method for reducing the incidence of leukopenia or anemia in a subject suffering from Wilson's disease, the method comprising administering to the subject a vector or pharmaceutical composition disclosed herein. The present disclosure provides a vector or pharmaceutical composition disclosed herein for reducing the incidence of leukopenia or anemia in a subject suffering from Wilson's disease. The present disclosure provides the 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.

[0520] The present disclosure provides a method for reducing the incidence of liver cirrhosis in a subject suffering from Wilson's disease, the method comprising administering to the subject a vector or pharmaceutical composition disclosed herein. The present disclosure provides a vector or pharmaceutical composition disclosed herein for reducing the incidence of liver cirrhosis in a subject suffering from Wilson's disease. The present disclosure provides the use of a vector in the manufacture of a medicament for reducing the incidence of liver cirrhosis in a subject suffering from Wilson's disease.

[0521] In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but not be limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals include, but are not limited to, human or non-human mammals such as cows, equines, canines, sheep, or felines, among others. An individual and a patient are also subjects herein.

[0522] As used herein, the terms "treat / treated / treating / treatment" refer to therapeutic treatment, where the goal is to slow down (alleviate) an undesired physiological disorder, condition, or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the degree of a disorder, condition, or disease; stabilization (i.e., not worsening) of the state of a disorder, condition, or disease; delay in the onset or slowing of the progression of a disorder, condition, or disease; improvement in one or more symptoms of a disorder, condition, or disease state; and remission (whether partial or complete) or enhancement or improvement of a disorder, condition, or disease. Treatment includes eliciting a clinically significant response without excessive side effects. Treatment also includes prolonging the survival period as compared to the expected survival period in the absence of treatment. The terms "prevent / prevention", etc. refer to acting before the onset of an apparent disease or condition to prevent the development of the disease or condition or to minimize the degree of the disease or condition, or to slow down its development process.

[0523] In some embodiments, treatment refers to an increase in survival (e.g., survival time). For example, treatment can result in an increase in the patient's life expectancy. In some embodiments, treatment results in an increase in the patient's life expectancy of more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more compared to the average life expectancy of one or more untreated control individuals with the disease or disorder. In some embodiments, treatment results in an increase in the patient's life expectancy of more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or longer compared to the average life expectancy of one or more untreated control individuals with the disease or disorder. In some embodiments, treatment results in long-term survival of the patient. As used herein, the term "long-term survival" refers to a survival time or life expectancy of more than about 40 years, 45 years, 50 years, 55 years, 60 years or longer.

[0524] Administration methods include, but are not limited to, intracisternal, intraventricular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intrathecal, intravaginal, transdermal, rectal, by inhalation or topically (especially to the ear, nose, eye or skin). The mode of administration is at the discretion of the practitioner.

[0525] In some cases, the nucleic acid constructs or vectors described herein are administered locally. By way of example, this can be achieved by local infusion during surgery, surface application (e.g., in a cream or lotion), by injection, via a catheter, via a suppository or enema or via an implant, which is a porous, non-porous or gel-like material, including membranes, such as silicone rubber membranes or fibers. In some cases, the nucleic acid constructs or vectors described herein are introduced into the central nervous system, the circulatory system or the gastrointestinal tract by any suitable route, including intraventricular injection, intrathecal injection, paravertebral injection, epidural injection, enema and injection adjacent to a peripheral nerve.

[0526] The compositions described herein can be administered as a single dose or multiple doses. Such compositions can be administered at regular time intervals depending on the nature, severity, and extent of the disorder in the subject. In some embodiments, a therapeutically effective amount of the nucleic acid construct or vector is administered intrathecally at regular time intervals (e.g., once a year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (monthly / once every month), biweekly (once every two weeks), or weekly).

[0527] The amount of the nucleic acid construct or vector described herein effective to treat a disease can be determined using standard clinical techniques known to those of skill in the art. Additionally, in vitro or in vivo assays can optionally be used to assist in identifying the optimal dosage range. The exact dosage to be employed will also depend on the route of administration, the disorder, the severity of the disorder being treated, and various physical factors associated with the individual being treated, and can be decided according to the judgment of the healthcare practitioner.

[0528] An effective amount of rAAV carrying a nucleic acid sequence encoding a transgene (including but not limited to UPF1, ATP7B, and BDNF) under the control of a NARE can be, for example, in the range of about 1×10 9 to about 1x10 14 rAAV genome particles (vg) / kg body weight. "Genome particles" are defined herein as AAV capsids containing single-stranded DNA molecules, which can be quantified by sequence-specific methods such as qPCR or ddPCR. In some embodiments, rAAV is administered at about 1×10 12 to about 1x 10 13 rAAV vg / kg body weight. In some embodiments, rAAV is administered at about 5x 10 11 to about 5x 10 12 vg / mL cerebrospinal fluid (CSF) volume. In some embodiments, rAAV is administered at a total of about 7.5x 10 13 to 7.5x 10 14 vg per patient.

[0529] In some embodiments, rAAV is administered to an animal at about 1×10 11 to about 1x 10 14 rAAV genome particles (vg) / kg body weight.

[0530] In some embodiments, the rAAV genomic particles are provided in a volume between about 20 μL and about 50 mL. In some embodiments, the rAAV genomic particles are provided in a volume between about 30 μL and about 30 mL. In some embodiments, the rAAV genomic particles are provided in a volume of about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 μL. In some embodiments, the rAAV genomic 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.

[0531] The attending physician may also select other doses within these ranges. It should be understood that for any particular subject, the specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the nucleic acid construct or vector, and the dosage ranges described herein are only exemplary and are not intended to limit the scope or practice of the claimed invention.

[0532] The nucleic acid constructs or vectors disclosed herein may also be advantageously provided ex vivo to cells, which are then administered to a subject. Methods of treating diseases by implanting cells that have been modified to express a recombinant protein are also well known. See, for example, U.S. Patent No. 5,399,346, which discloses methods for introducing nucleic acids into primary human cells for introduction into the human body. Although human cells are preferably used for in vitro therapy in some embodiments, other cells (such as bacterial cells) may also be implanted into the vasculature of a subject to provide a sustained release of a therapeutic agent. See, for example, U.S. Patent Nos. 4,309,776 and 5,704,910.

[0533] 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 U.S. provisional applications were filed on October 11, 2022 by the same applicant, and each of which is incorporated herein by reference in its entirety.

[0534] It should be understood that the present invention is not limited to the specific molecules, compositions, methods or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention. It should also be understood that the disclosure of the present invention in this specification includes all possible combinations of such specific features. For example, in the case where a specific feature is disclosed in the context of a particular aspect or embodiment or a particular claim of the present invention, the feature can also be used in combination with and / or in the context of other particular aspects and embodiments of the present invention to the extent possible, and is generally used in the present invention.

[0535] In the case where a method involving two or more defined steps is mentioned herein, the defined steps can be carried out in any order or simultaneously (unless the context excludes such possibilities), and the method can include one or more other steps carried out before any defined step, between two defined steps, or after all defined steps (unless the context excludes those possibilities).

[0536] All other cited patents and applications are incorporated herein by reference in their entirety. In addition, when the definition or use of a term in a reference incorporated herein by reference is inconsistent or contradictory to the definition of the term provided herein, the definition of the term provided herein shall prevail and the definition of the term in the reference shall not apply.

[0537] For better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be construed as limiting or defining the entire scope of the present invention.

[0538] Examples

[0539] Example 1: NARE Design

[0540] Potential NARE candidates were also identified from genes highly expressed in human target tissue (liver). Subsequently, promoter regions were defined or cis-regulatory elements were identified based on chromatin marks, accessibility, conservation, and other genome-wide datasets.

[0541] Incorporated ten - motif element (MTE) / downstream promoter element (DPE) modifications to increase transcription, and incorporated various cellular, viral, or synthetic 5′UTR motifs to increase transcription / translation. The choice of introns was changed, for example to include short cellular introns that naturally have unique independent transcription start sites, indicating the presence of a promoter within this particular intron. In addition, introns were inserted at different positions of the insertion sequence, and / or the introns were truncated while maintaining splicing elements. In addition, single or multiple point mutations were introduced to increase expression or prevent inactivation. Additionally, different transcriptional enhancers were introduced upstream of the core promoter. In some cases, transcriptional motif insertions, replacements, and / or shuffling were employed, and CpG sequences were removed to reduce immunogenicity while maintaining promoter activity.

[0542] Gene regulatory elements were also optimized by the following methods: (i) identifying, shuffling, and cloning highly expressed transcription factor binding sites (TFBS) upstream of the core promoter; (ii) using an artificial intelligence (AI) algorithm to iteratively mutate and score the promoter.

[0543] Materials and Methods

[0544] AI-driven NARE design

[0545] Core prediction model: The prediction of construct potency was based on the prediction of the Xpresso model (xpresso.gs.washington.edu). The original Xpresso model was developed by considering a 10.5 kb region around the transcription start site (TSS, 7.5 kb upstream and 3 kb downstream of the TSS). Due to size limitations (not using fragments longer than about 1.5 kb), the algorithm was modified to include a "padding" step before calculating the predicted activity. This padding step takes a sequence and adds ambiguous ("N") nucleotides upstream and downstream of the shorter input fragment according to the expected / annotated TSS. The final model takes a sequence, scans for uppercase base pairs (the convention for marking the TSS), pads the sequence length, converts it to a one - hot encoded data frame, and then passes it through a convolutional neural network (CNN) with the weights and architecture of Xpresso.

[0546] Custom software was written to create scores for and automate the following analyses ( Figure 1 )

[0547] ENCODE+JeT (C6): ENCODE fragment coordinates were downloaded and subsequently used to parse such elements from the reference human genome (GRCh38). All ENCODE fragments were individually positioned upstream of the JeT promoter and then tested by the modified prediction model.

[0548] ENCODE + JeT Spacing Optimization: A custom script was used to insert spacer elements of 300 bp, 400 bp, 500 bp, or 600 bp between the top ~2000 ENCODE elements with the highest scores and the JeT promoter. The new "spaced" constructs were then retested with the modified prediction model.

[0549] Point Mutation / Saturation Mutagenesis: A custom script was used to generate all possible point-mutated forms of the input sequence. The resulting sequences were then scored with the modified prediction model. A custom script was also developed to iterate this process through greedy search (introducing the best results in each round).

[0550] Using a CNN model, the effects of in silico cloning of 708,157 enhancer fragments upstream of the C6 promoter sequence were predicted. Most enhancers were predicted to have a positive effect on C6 potency, with approximately 2,000 constructs showing approximately a 2-fold improvement. This subset was further analyzed to identify the optimal spacing between enhancer elements and the promoter sequence. Introducing spacer sequences improved the potency of the constructs, with the largest improvement observed at a 400-bp spacing. The selected constructs were synthesized and tested in vitro (C118 to C127).

[0551] Promoter syntax and rational design of NARE

[0552] Potential NARE candidates were also identified from genes highly expressed in human target tissue (liver). Subsequently, promoter regions were defined or cis-regulatory elements were identified based on chromatin marks, accessibility, conservation, and other genome-wide datasets.

[0553] Incorporating decamer motif element (MTE) / downstream promoter element (DPE) modifications to increase transcription, and incorporating various cellular, viral, or synthetic 5′UTR motifs to increase transcription / translation. In CAG-based gene regulatory elements (and in other elements described herein), the choice of introns was varied, for example, to include short cellular introns that naturally have unique independent transcription start sites, indicating the presence of a promoter within this specific intron. Additionally, introns were inserted at different positions within the sequence, and / or introns were truncated while maintaining splicing elements. Furthermore, single or multiple point mutations were introduced to increase expression or prevent inactivation. Additionally, different transcriptional enhancers were introduced upstream of the core promoter. In some cases, transcriptional motif insertions, replacements, and / or shuffles were employed, and CpG sequences were removed to reduce immunogenicity while maintaining promoter activity.

[0554] Gene regulatory elements were also optimized by the following methods: (i) identifying, shuffling, and cloning highly expressed transcription factor binding sites (TFBS) upstream of the core promoter; (ii) using artificial intelligence (AI) algorithms to iteratively mutate and score promoters.

[0555] Example 2: Dual reporter protein expression assay

[0556] To test the ability of NARE candidates to drive gene expression, a dual reporter assay using flow cytometry was utilized. Figure 2A Diagrams of the dual reporter gene expression constructs and assays were provided. Different NARE promoter sequences were cloned upstream of a transgene encoding mClover3, a green / yellow fluorescent protein, which was in turn upstream of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE250) and a synthetic polyadenylation (polyA) signal. mClover3 fluorescence served as a measure of NARE strength. The plasmid also contained a separate expression cassette (5' to 3': SV40 promoter - tdTomato, a red fluorescent protein - SV40 polyA), which was the same in all NARE test constructs ( Figure 2A ). tdTomato fluorescence served as an internal normalization control, which could account for variations in transfection efficiency.

[0557] Using a transfection reagent, various cell lines were transiently transfected with the dual reporter plasmid. After 48 hours, the cells were trypsinized and resuspended for flow cytometry. The raw data was processed to select only live single cells for analysis. A sample containing untransfected cells was used to set the gating for the tdTomato signal. Subsequently, this gating was applied to all live single cells to identify tdTomato-positive (and thus transfected) cells ( Figure 2B ). Subsequently, mClover3 fluorescence was determined for the tdTomato-positive population. Since transfection varied slightly between different experiments, the relative NARE potency was calculated by the ratio of mClover3 fluorescence to tdTomato fluorescence. NARE candidates were tested together with known constitutive and tissue-specific reference promoters such as CAG, CMV, or AAT.

[0558] Example 3: Strengths of different NAREs in liver cells (in vitro)

[0559] Materials and methods

[0560] Cell culture

[0561] The Huh-7, AML-12, and HEK 293 immortalized cell lines were maintained at 37 °C in 5% CO2. Cells were transiently transfected with plasmids using TurboFect TM After 48 h (72 h for AML-12), cell lysates were collected for protein analysis. After 20 passages, new cell aliquots were thawed and passaged twice, and then used for subsequent experiments.

[0562] Primary hepatocyte culture

[0563] Mouse and human hepatocyte cell lines from Gibco were thawed and placed in suspension medium, then centrifuged at 100 x g for 10 min at 4 °C. The supernatant was aspirated, and the cells were resuspended in maintenance medium (+10% FBS), then seeded at 400,000 cells per well on a 24-well plate. Four hours after seeding, the plate was gently tapped to loosen debris, then the medium was aspirated and replaced with maintenance medium without FBS. The cells were maintained at 37 °C in 5% CO2. Complete medium changes were performed daily to feed the cultures.

[0564] Transduction

[0565] For immortalized cells and primary cell cultures, AAV8 particles were added to the medium at a multiplicity of infection (MOI) of 50,000 on day 1 and day 3 in vitro, respectively. Medium containing doxorubicin was added to the cells to accelerate transduction. After 72 h, the medium was changed to medium without doxorubicin according to the standard protocol.

[0566] Results

[0567] A NARE library suitable for gene expression in the liver was developed. The promoter provided stronger expression in immortalized hepatocytes (Huh7, HepG2, AML-12) compared to commonly used reference promoters. The Huh7 cell line is a hepatocyte-derived cancer cell line. HepG2 is a hepatoblastoma cell line. AML-12 cells are an immortalized epithelial cell line derived from mouse liver cells. Reference promoters include: CAG promoter, α-1-antitrypsin (AAT) promoter, human thyroxine-binding globulin (TBG) promoter, liver-specific promoter 1 (LP1), hybrid liver promoter (HLP), and hepatic combinatorial bundle promoter (HCB). ATT is a compact liver-specific promoter currently used in gene therapy clinical trials. HCB is an effective NARE that is stronger than AAT. The promoters in the promoter library have equal or greater strength compared to CAG, while also being significantly smaller.

[0568] Additional NAREs were identified from genes highly expressed in the human liver by bioinformatics and defined using various epigenetic and genomic features (histone acetylation, TF occupancy, sequence conservation, etc.). Figure 3A and Figure 3C show the potency of the selected NAREs in liver cells. L45 is 22-fold stronger than the AAT promoter ( Figure 3C and Figure 3D ). NAREs L10, L13, and L15 are liver cell-specific ( Figure 3B ).

[0569] NARE L45 was further optimized, resulting in an additional increase in expression (Table 1, Figure 4A and Figure 4B ). The optimized NARE is up to about 3-fold stronger than the 1.6 kb CAG promoter in liver cells. Figure 4C show the potency of the additional promoters in the human liver cell line Huh7. Tables 6 and 7 show the potencies of derivatives of the AAT promoter (L1) and the HCB promoter in different cell lines, respectively.

[0570] Table 1. Potency of L45-derived NAREs. Plasmid transfection.

[0571]

[0572]

[0573] Table 2: Potency of AAT promoter-derived NAREs. Plasmid transfection.

[0574]

[0575] Table 3. Optimization of the HCB promoter. Plasmid transfection.

[0576] Promoter Size (bp) Human liver (Huh7) C19:CAG 1761 0.8 L1:AAT 317 0.1 L75:HCB1 255 1.0 L76:HCB2 283 1.5 L77:HCB3 332 1.1 L78:HCB4 455 1.3 L79:HCB5 593 1.4 L89:HCB6 621 2.2

[0577] Table 4. Control or reference NAREs.

[0578]

[0579]

[0580]

[0581]

[0582]

[0583] Table 5. NAREs.

[0584]

[0585]

[0586]

[0587]

[0588] Table 6. NARE components.

[0589]

[0590]

[0591] Table 7. NARE and NARE components. SN = SEQ ID NO.

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

Claims

1. A NARE, the 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 NO:193, 194, 207, 209 - 214, 216 - 232, 233 - 235, or 249.

2. The NARE according to claim 1, the NARE 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 NO:193, 194, 207, 209 - 214, 216 - 232, 233 - 235, or 249.

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

4. A NARE, the 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. The NARE according to claim 4, the NARE 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. The NARE according to claim 4, the NARE comprising: (i) SEQ ID NO:150; and (ii) SEQ ID NO:151 or SEQ ID NO:

155.

7. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to any one of SEQ ID NO:154, 157, or 158; (ii) a sequence that is at least 90% identical to any one of SEQ ID NO: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 NO:169, 172, or 173.

8. The NARE according to claim 7, the NARE comprising: (i) a sequence that is at least 95% identical to any one of SEQ ID NO:154, 157, or 158; (ii) a sequence that is at least 95% identical to any one of SEQ ID NO: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 NO:169, 172, or 173.

9. The NARE according to claim 7, wherein the NARE comprises: (i) any one of SEQ ID NO: 154, 157 or 158; (ii) any one of SEQ ID NO: 153, 156, 161, 162, 163, 166, 167, 168 or 174; and (iii) optionally, any one of SEQ ID NO: 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 NO: 154, 157, 158 or 176.

11. The NARE according to claim 10, wherein 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 NO: 154, 157, 158 or 176.

12. The NARE according to claim 10, wherein the NARE comprises: (i) SEQ ID NO: 153; (ii) SEQ ID NO: 171; and (iii) any one of SEQ ID NO: 154, 157, 158 or 176.

13. 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 any one of SEQ ID NO: 154, 157, 158, 176, 188 or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO:

171.

14. The NARE according to claim 13, wherein 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 NO: 154, 157, 158, 176, 188 or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO:

171.

15. The NARE according to claim 13, wherein the NARE comprises: (i) SEQ ID NO: 153; (ii) any one of SEQ ID NO: 154, 157, 158, 176, 188 or 250; and (iii) optionally, SEQ ID NO: 155 or SEQ ID NO:

171.

16. A NARE, the 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 either SEQ ID NO: 175 or any one of SEQ ID NOs: 177 - 187.

17. The NARE according to claim 16, the NARE 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 either SEQ ID NO: 175 or any one of SEQ ID NOs: 177 - 187.

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

19. A NARE, the NARE comprising: (i) a sequence that is at least 90% identical to either 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.

20. The NARE according to claim 19, the NARE comprising: (i) a sequence that is at least 95% identical to either 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. The NARE according to claim 19, the NARE comprising: (i) either 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. A NARE, wherein the NARE 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 NO: 197, 202 or 203; (iv) optionally, a sequence that is at least 90% identical to any one of SEQ ID NO: 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 NO: 166, 193 or 194.

23. The NARE according to claim 22, wherein 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 NO: 197, 202 or 203; (iv) optionally, a sequence that is at least 95% identical to any one of SEQ ID NO: 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 NO: 166, 193 or 194.

24. The NARE according to claim 22, wherein the NARE comprises: (i) SEQ ID NO: 168; (ii) any one of SEQ ID NO: 197, 202 or 203; (iv) optionally, any one of SEQ ID NO: 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 NO: 166, 193 or 194.

25. A NARE, wherein the NARE 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 NO: 201 - 203.

26. The NARE according to claim 25, wherein 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 NO: 201 - 203.

27. The NARE according to claim 25, wherein 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.

28. A NARE, wherein the NARE 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 at least 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. The NARE according to claim 28, wherein 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 at least 95% 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. The NARE according to claim 28, wherein the NARE comprises: (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 at least 95% 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, wherein the NARE 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.

32. The NARE according to claim 31, wherein 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.

33. The NARE according to claim 31, wherein the NARE comprises: (i) SEQ ID NO: 248; and (ii) any one of SEQ ID NOs: 239 - 247.

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

37. A NARE, the 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 NO: 207 or any one of SEQ ID NOs: 209 - 215.

38. The NARE according to claim 37, the NARE 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 SEQ ID NO: 207 or any one of SEQ ID NOs: 209 - 215.

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

40. A NARE, the 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 NO: 207 or any one of SEQ ID NOs: 209 - 215.

41. The NARE according to claim 40, the NARE 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 NO: 207 or any one of SEQ ID NOs: 209 - 215.

42. The NARE as recited in claim 40, wherein 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.

43. A NARE, comprising: 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) (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. The NARE as recited in claim 43, wherein the NARE comprises: 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.

45. The NARE according to claim 43, wherein the NARE comprises: 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) (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 NO:154, SEQ ID NO:157, or SEQ ID NO:

158.

47. The NARE according to claim 46, wherein 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 NO:154, SEQ ID NO:157, or SEQ ID NO:

158.

48. The NARE according to claim 46, wherein the NARE comprises: (i) SEQ ID NO:166; and (ii) Any one of SEQ ID NO:154, SEQ ID NO:157, or SEQ ID NO:

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 NO:154, SEQ ID NO:157, or SEQ ID NO:

158.

50. The NARE according to claim 49, wherein 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 NO:154, SEQ ID NO:157, or SEQ ID NO:

158.

51. The NARE according to claim 49, wherein the NARE comprises: (i) SEQ ID NO:165; and (ii) Any one of SEQ ID NO:154, SEQ ID NO:157, or SEQ ID NO:

158.

52. A NARE, wherein the NARE 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.

53. The NARE according to claim 52, wherein 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.

54. The NARE according to claim 52, wherein the NARE comprises: (i) SEQ ID NO: 199; and (ii) SEQ ID NO: 193 or SEQ ID NO:

194.

55. A NARE, wherein the NARE 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.

56. The NARE according to claim 55, wherein 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.

57. The NARE according to claim 55, wherein the NARE comprises: (i) SEQ ID NO: 164; and (ii) SEQ ID NO:

200.

58. A NARE, wherein the NARE 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.

59. The NARE according to claim 58, wherein 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.

60. The NARE according to claim 58, wherein the NARE comprises: (i) SEQ ID NO: 189; and (ii) SEQ ID NO: 193 or SEQ ID NO:

194.

61. A NARE, wherein the NARE 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.

62. The NARE according to claim 61, wherein 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.

63. The NARE as claimed in claim 61, wherein the NARE comprises: (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. The NARE as claimed in claim 64, wherein 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.

66. The NARE as claimed in claim 64, wherein the NARE comprises any one of SEQ ID NOs: 3 - 8, 11 - 69, 71 - 73 or 76 - 149.

67. The NARE as claimed in claim 64, wherein 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.

68. The NARE as claimed in claim 67, wherein 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.

69. The NARE as claimed in claim 67, wherein the NARE comprises any one of SEQ ID NOs: 53, 80, 81, 132, 141 or 142.

70. An expression construct comprising the NARE as described in any one of the preceding claims and an operably linked transgene.

71. The expression construct as claimed in claim 70, wherein the expression construct further comprises a polyadenylation sequence.

72. A vector comprising the expression construct as claimed in claim 70.

73. The vector as claimed in claim 70, wherein the vector is a non - viral vector.

74. The vector as claimed in claim 70, wherein the vector is a viral vector.

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

76. A vector comprising a nucleic acid sequence comprising (i) the expression construct as claimed in claim 70, and (ii) one or more inverted terminal repeats (ITRs).

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

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

79. A cell comprising the expression construct as claimed in claim 70 or the vector as claimed in any one of claims 72 - 78.

80. The cell as claimed in claim 79, wherein the cell is a hepatocyte.

81. A pharmaceutical composition, the pharmaceutical composition comprising: (i) an expression construct as claimed in claim 70 or a vector as claimed in any one of claims 72 - 78, and (ii) a pharmaceutically acceptable excipient.

82. A method for expressing a transgene in a cell, the cell comprising an expression construct as claimed in claim 70 or a vector as claimed in any one of claims 72 - 78.

83. A method for regulating transgene expression in a cell, the cell comprising an expression construct as claimed in claim 70 or a vector as claimed in any one of claims 72 - 78.

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

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

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

87. The method according to claim 86, wherein the subject is human.

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