Libraries for novel protein design

Abstract

Conventional methods for developing therapeutics involve developing a range of candidate molecules, screening the candidates, identifying candidates that are therapeutically valuable, and later identifying targets that bind the therapeutic candidate. In contrast, the present disclosure provides a top-down approach, wherein a target is chosen first, and therapeutic agents are identified based on their ability to bind said target. Specifically, the present disclosure provides nucleic acid library production methods that enable efficient identification of therapeutic candidates. Moreover, the present disclosure provides screening methods that enable assaying of candidates based on their ability to bind therapeutically relevant targets. Further aspects of the disclosure provide methods for producing recombinant adeno-associated viral particles, such as, those comprising engineered capsid proteins that bind target molecules on the surfaces of cells. Thus, the present disclosure provides for the development of novel gene therapy delivery systems.

Description

[0001]UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 LIBRARIES FOR NOVEL PROTEIN DESIGN RELATED APPLICATIONS The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63 / 502,623 filed May 16, 2023, which is incorporated by reference in its entirety. BACKGROUND Adeno-associated viruses are a viral platform which can be engineered to function as a gene therapy delivery system. Discovery of new AAV capsid proteins can expand tissue- targeting capabilities for gene therapy delivery. SUMMARY Conventional methods for developing therapeutics involve developing a range of candidate molecules, screening the candidates, identifying candidates that are therapeutically valuable, and later identifying targets that bind the therapeutic candidate. In contrast, the present disclosure provides methods for fast development of viral vector-based therapies that are intentionally engineered to bind chosen target proteins. More specifically, the present disclosure provides for production and / or screening of candidate libraries featuring reduced coding sequence complexity. This enables faster identification of candidates, reduction of the number of candidates that are likely to recognize the target of interest, more focused assaying of candidate- target binding activities, and broadly applicable library design strategies that may be readily customized for specific therapeutic purposes. Further aspects of the disclosure provide methods for producing recombinant adeno-associated viral particles, such as, those comprising engineered AAV capsid proteins that bind target molecules on the surfaces of cells. Accordingly, in some aspects, the present disclosure provides a method comprising: identifying variant amino acid positions of interest relative to a parent sequence; designing engineered nucleic acid sequences encoding variants of the parent protein sequence (e.g., encoding one or more regions of a protein in which combinations of amino acid substitutions are being introduced, and / or encoding entire protein variants containing different combinations of amino acid substitutions relative to the parent sequence), wherein the engineered nucleic acid sequence comprises either a wild-type codon or a heterologous codon substituted for the wild- type codon, relative to the parent sequence, corresponding to each variant amino acid position of - 1 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 interest identified in step; and assembling a nucleic acid library comprising one or more engineered nucleic acids, wherein assembly comprises linking trimer nucleotide fragments as building blocks to produce one or more engineered nucleic acids comprising the engineered nucleic acid sequences that were designed. In some embodiments, the library is cloned into a plurality of vectors, thereby producing a vector library. In some embodiments, the vector library is sequenced. In some embodiments, the vector library is replicated in a cell population. In some embodiments, a vector includes one or more promoter, terminator, or other regulatory sequences. In some embodiments, a vector encodes portions of the protein of interest (e.g., an N-terminal portion and / or a C-terminal portion) that are not being varied and the library that is cloned into the vector encodes a region that is being varied (e.g., a portion comprising one or more variable regions). In some embodiments, the vector library is transfected (e.g., in the presence of a helper nucleic acid) into host cells, thereby producing a viral particle library. In some embodiments, the viral particles comprise adeno-associated virus (AAV) particles. In some embodiments, the parent sequence comprises or encodes viral surface proteins. In some embodiments, the parent sequence comprises or encodes adeno-associated virus (AAV) capsid proteins. In some embodiments, the AAV capsid proteins comprise at least one variable region (VR) of AAV capsid proteins. In some embodiments, one or more of the variant amino acid positions of interest are in a variable region (VR) of an AAV capsid protein. In some embodiments, the AAV capsid proteins are of a mammalian serotype. In some embodiments, the serotype of the parent sequence is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, or AAVrh.74; and the variant amino acid position is found in a different serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, or AAVrh.74. In some embodiments, the present disclosure provides for a nucleic acid encoding a protein variant of the library or a recombinantly expressed protein variant of the library. In some embodiments, the protein variant is an engineered viral capsid protein. In some embodiments, a nucleic acid encodes said engineered viral capsid protein. In some embodiments, the engineered viral capsid protein is an engineered AAV capsid protein. In some embodiments, a nucleic acid encodes said engineered AAV capsid protein. In some embodiments, an engineered AAV capsid protein comprises one or more amino acid substitutions that were identified using methods described in this application. In some embodiments, an engineered AAV capsid protein comprises one or more amino acid substitutions illustrated in FIG. 13 or FIG. 14. In some embodiments, an engineered AAV capsid protein comprises an amino acid sequence of one of - 2 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 the clones of FIG. 14 (e.g., 1-3, 2-4, 6-4, or 11-3). Sequences shown in FIG. 13 and FIG. 14 represent a portion of an AAV capsid protein (e.g., positions 261-520 of a variant AAV-rh74 VP1 sequence in FIG. 13, or positions 201-600 of a variant AAV-rh74 VP1 sequence in FIG. 14). Recombinant full length VP1 sequences can comprise these variant regions in the context of an N-terminal portion (e.g., N-terminal amino acids 1-260 or 1-200 of AAV-rh74 VP1) and / or C-terminal portion (e.g., amino acids C-terminal to position 520 or position 600 of AAV-rh74 VP1) of an AAV VP1 protein. In some embodiments, the N-terminal and / or C-terminal portions are from AAV-rh74. However, in some embodiments the N-terminal and / or C-terminal portions can be corresponding portions from one or more other AAV serotypes. In some embodiments, one or more of the amino acid substitutions identified according to the methods described in this application can be introduced into variable regions of other AAV serotypes (e.g., at positions corresponding to the positions shown in FIGs. 13 and 14). In some embodiments, an engineered nucleic acid comprises a nucleotide sequence encoding a variant AAV capsid amino acid sequence (e.g., comprising one or more of the amino acid substitutions shown in FIGs. 13 and 14). In some embodiments, the present disclosure provides for a library produced by methods described herein. In some embodiments, the present disclosure provides for a cell, or cell population thereof, comprising the protein variant, or nucleic acid encoding said protein variant. In some embodiments, the present disclosure provides for a cell population comprising the library produced by methods described herein. In some embodiments, the cell or cell population are mammalian cells. In some embodiments, the mammalian cells are human cells. In other aspects, the present disclosure provides a method of screening a viral particle library comprising: providing a viral particle library comprising different proteins of interest; contacting the viral particle library with a first host cell population prepared from a first host cell lineage, wherein the first host cell population comprises a target protein on the surface of the cells to which the proteins of interest putatively bind, optionally wherein contacting further comprises contacting the first host cell population with a helper nucleic acid; obtaining viral particles that transduce the first host cell population; contacting the viral particles that were obtained with a second host cell population prepared from the first host cell lineage, wherein the second host cell population does not comprise the target protein; and obtaining viral particles that do not bind to second host cell population. In some embodiments, said method steps are repeated one or more times with first and second populations prepared from a host cell lineage different from the first host cell lineage, wherein each repetition of the method steps does not use host cells of the same lineage more than once. In some embodiments, the viral particle library of said - 3 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 method is produced by library production methods described herein. In some embodiments, at least one of the proteins of interest comprise a protein variant described herein or produced by a method described herein. In some embodiments, the host cells are mammalian. In some embodiments, the host cells are human. In some embodiments, the present disclosure provides for a viral particle isolated by the methods described herein. In some embodiments, the viral particle is an AAV particle. In some embodiments, a cell, or cell population thereof, are transduced with the viral particle. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In other aspects, the present disclosure provides an engineered AAV capsid protein comprising any one of the clones (e.g., 1-3, 2-4, 6-4, or 11-3) described herein, or a nucleic acid encoding any one of said clones. In other aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle comprising a heterologous nucleic acid flanked by AAV inverted terminal repeats (ITRs) and at least one AAV capsid protein comprising an engineered AAV capsid protein that was produced and / or screened by methods described herein. In other aspects, the present disclosure provides an rAAV particle comprising a heterologous nucleic acid flanked by AAV ITRs and at least one AAV capsid protein, wherein the at least one AAV capsid protein comprises an engineered AAV capsid protein described herein. In some embodiments, the heterologous nucleic acid encodes a therapeutic RNA or a therapeutic protein. In some embodiments, the rAAV particle is administered to a subject. In some embodiments, the subject has, is suspected of having, or is at risk of developing, a disease or disorder. In some embodiments, the therapeutic RNA or therapeutic protein is therapeutic for the disease or disorder. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 illustrates a non-limiting embodiment of a process 100 for designing and producing a library. FIG. 2 illustrates a non-limiting embodiment of a process 200 for screening viral particles of interest from a viral particle library. FIG. 3 shows an example of an alignment for generation of mutational diversity in virtual family shuffling approach. The brackets above the sequences represent regions comprising amino acid positions of variation between the VP1 amino acid sequences from the indicated AAV serotypes. - 4 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 FIG. 4 shows usage of IUPAC coding approach for assembly of an AAV library. FIG. 5 shows a comparison of library complexity generated by trimer, IUPAC, or ambiguous (degenerate) synthesis. FIG. 6 shows an example of a designed mutational profile for AAV-rh74 A and B sub- libraries synthesized by the trimer approach. FIG. 7 shows an example of Sanger sequencing analysis of pooled PCR products for sub-library A. FIG. 8 shows an example of an alignment of 66 randomly selected capsid variants from AAV-rh74-AB library vs. parental AAV-rh74. FIG. 9 shows a dendrogram representation of clustering of sequenced AAV-rh74-AB library capsids vs parental AAV-rh74, based on point accepted mutation (PAM). FIG. 10 shows Weblogo sequence diversity based on 58 capsid variants from assembled AAV-rh74-AB library. FIGs. 11A-11B show a non-limiting embodiment of a strategy for AAV rh74-based library generation to minimize potential intra-liver distribution of AAVs. FIG. 11A shows an overview of the strategy. FIG. 11B shows an alignment of VR1 between AAV rh74 in AAV- rh74-AB library (top) and AAV1 in AAV-rh74-AB1-library (bottom). FIG. 12 illustrates a non-limiting embodiment of a flow chart for isolating viral particles that bind to target proteins. FIG. 13 shows examples of selected clones displaying significant motifs’ homologies in Variable Regions VI, VII, and VIII, but divergent in VR-IV. Note that the sequences of the variants shown on the lines labeled “1” and “2” comprise the same sequence at the indicated positions (positions 261-520) and share the same sequence identifier (SEQ ID NO: 97). FIG. 14 shows an example of an alignment of partial amino-acid capsid sequences from selected isolated clones. Apart from the indicated variant amino acid positions, the clones comprise the wild-type VP1 sequence of serotype AAVrh.74. Highlighted sequences incorporate homologous motifs. Note that the sequences of the variants shown on the lines labeled “1-3” and “2-4” comprise the same sequence at the indicated positions (positions 201-600) and share the same sequence identifier (SEQ ID NO: 109). FIG. 15 shows an alignment of the wild-type VP1 amino acid sequences from serotypes AAVHSC16, AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAVrh10, and AAV44.9. Amino acid positions 346, 501, 505, and 706 in VP1 from AAVHSC16 and examples of corresponding positions in VP1 from AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAVrh10, and AAV44.9 are shown above the alignment. - 5 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 DETAILED DESCRIPTION Aspects of the application relate to methods and compositions for generating libraries of biological variants. In some embodiments, methods and compositions for generating a library of nucleic acids encoding protein variants (e.g., viral capsid variants, such as AAV capsid variants) are provided. In some embodiments, methods and compositions for generating a library of viral variants (e.g., AAV variants), for example, comprising different capsid protein variants, are provided. Aspects of the application also relate to methods and compositions for processing libraries of biological variants to identify and / or isolate one or more variants having properties of interest. In some embodiments, methods and compositions for identifying and / or isolating viral variants (e.g., AAV variants) that selectively bind to one or more proteins on the surface of cells are provided. FIG. 1 illustrates a non-limiting embodiment of a process 100 for designing and producing a library. In act 110, protein sequences are analyzed to identify amino acid variants of interest relative to a parent sequence. In act 120, codons for the amino acid variants of interest are identified. In act 130, a library of nucleic acids encoding the protein variants of interest is produced. In some embodiments, this library is constructed via nucleic acid synthesis using trinucleotide building blocks corresponding to the codons identified in act 120. In act 140, the library of nucleic acid variants from act 130 is cloned into a vector to produce a library of vectors comprising different coding sequences for the protein variants that were identified in act 110. In some embodiments, this library of vectors can be used to produce a library of cells and / or virus particles. In act 150, host cells are transfected with the vector library from act 140 to produce a library of viral particles (e.g., AAV particles) each comprising a capsid containing a different capsid protein variant. In some embodiments, the method of FIG. 1 is used to produce a library of AAV variants. However, this method can be used to produce other libraries of protein coding sequences. In some embodiments, one or more of the acts illustrated in FIG. 1 can be omitted and / or combined with other acts. In some embodiments, in act 110, a plurality of sequences comprising a “parent sequence” and one or more “foster sequences” are aligned to identify amino acid variations of interest relative to the parent sequence. Alignments may be done on DNA, RNA, or protein forms of said sequences as long as encoded amino acid differences are ultimately identified. Accordingly, in some embodiments, parent and foster sequences comprise coding sequences (e.g., coding sequences in a DNA molecule). In some embodiments, parent and foster sequences - 6 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 comprise amino acid sequences. In some embodiments, the one or more foster sequences comprises at least 2 foster sequences, wherein each foster sequence differs from each other by one or more amino acids and differ from the parent sequence by one or more amino acids. In some embodiments, the one or more foster sequences comprises, 3, 4, 5, 6, 7, 8, 910, or more than 10 sequences, wherein each foster sequences differs from each other by one or more amino acids and differ from the parent sequence by one or more amino acids. Non-limiting examples of amino acid variations of interest relative to the parent sequence include amino acid substitutions, amino acid deletions, and amino acid insertions. In some embodiments, the variations comprise conservative amino acid substitutions. In some embodiments, the variations comprise non- conservative mutations. In some embodiments, the variations comprise both non-conservative and conservative amino acid substitutions. In some embodiments, alignment is used to identify all amino acid variations within a region of interest in the parent sequence relative to one or more foster sequences. In some embodiments, the region of interest is one or more variable regions (VRs) of an AAV capsid protein. As such, in some embodiments, the entire gene sequence of the parent and foster sequences need not be aligned. Alignment may be performed with any suitable alignment tool known in the art. Non-limiting examples of alignment tools include BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), COBALT, OPAL, Multlin, Clustal Omega, Clustal W2.0, and Clustal X2.0. As used herein, a “parent sequence” refers to an amino acid sequence or a nucleotide sequence that can be engineered to comprise a substitution, an insertion, or a deletion at one or more positions. In some embodiments, the one or more positions that can be varied in the parent sequence is determined after aligning the parent sequence with one or more foster sequences and identifying positions in the parent sequence that vary relative to the one or more foster sequences. Accordingly, as used herein, a “foster sequence” refers to an amino acid sequence or a nucleotide sequence that differs at one or more positions relative to a parent sequence. In some embodiments, in act 110, the parent sequence and the one or more foster sequences are genes or proteins that are related in sequence, genes that encode proteins related by function, or both. Non-limiting examples of nucleic acids or proteins which are related in sequence and / or genes encoding proteins related by function include, but are not limited to, genes within same family, genes within the same superfamily, separate genes arising from a duplication event, genes arising from a relocation event, divergent genes, homologs, paralogs, and orthologs. In some embodiments, in act 110, known structural and functional information about the proteins encoded by the parent sequence and the one or more foster sequences informs what regions of the corresponding genes will be aligned. Such information may include, but is - 7 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 not limited to, domains shared by the proteins, motifs shared by the proteins, supersecondary structures shared by the proteins, and folds shared by the proteins. In some embodiments, 3D modeling of parent and foster sequences is used to identify the plurality of sequences used in the alignment. Non-limiting examples of tools which are useful for prediction of protein structure and may be used to model peptides, polypeptides, and / or proteins in the methods described herein include AlphaFold, Rossetta, I-TASSER, Robetta, Phyre2, RaptorX, and SWISS- MODEL. In some embodiments, in act 110, it may be desirable to choose a parent amino acid sequence and / or one or more foster amino acid sequences based on the respective binding properties of said proteins. For example, in some embodiments, it may be desirable to engineer a protein variant of the parent sequence to take on binding properties similar to a protein encoded by a foster sequence by identifying foster sequences responsible for interaction with a target. In some embodiments, in act 110, the parent and one or more foster sequences comprise a viral surface protein. In some embodiments, the viral surface protein is an AAV capsid protein. In some embodiments, the AAV capsid protein is an AAV of a mammalian serotype, or derivative thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC16, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8- C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, AAV44.9, or AAVr3.45. In some embodiments, in act 110, the parent and the one or more foster sequences are AAV VP1 nucleic acid sequences or amino acid sequences from different serotypes (see, e.g., FIGs 3 and 15, and SEQ ID NOs: 1, 112-125). In some embodiments, the AAV serotype(s) corresponding to the one or more foster sequences bind to a target, and the wild-type AAV counterpart corresponding to the parent sequence either binds to the target with lower affinity or has no binding affinity towards the target. Accordingly, in some embodiments, the methods described herein engineer AAV variant capsid proteins that bind to: different targets than their naturally occurring counterparts, targets with higher affinity than their naturally occurring counterparts, and / or targets with lower affinity than their naturally occurring counterparts. - 8 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 In some embodiments, the parent and one or more foster sequences comprise a variable region (VR) of an AAV capsid protein. In some embodiments, the parent and one or more foster sequences are found in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, or VR- IX of an AAV VP1, wherein the parent sequence and the one or more foster sequences are of different serotypes. In some embodiments, the serotypes of the parent and one or more foster sequences are selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC16, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8- C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, AAV44.9, and AAVr3.45. In some embodiments, the parent and one or more foster sequences are found in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, or VR-IX of an AAV VP1, wherein the parent sequence and the one or more foster sequences are of different serotypes and are selected from: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAVrh.74, and AAV44.9. In some embodiments, in act 110, the protein sequences analyzed (e.g., the parent sequence and one or more foster sequences) are AAV VP1 amino acid sequences. Examples of AAV VP1 amino acid sequences are set forth below and in SEQ ID NOs: 1, 112-125. The first six AAV VP1 amino acid sequence examples are of serotype AAVrh74, AAVHSC16, AAV3B, AAV6, AAV8, and AAV9, wherein the positions in double underline-only are comprised in VR- I, the positions in dashed underline-only are comprised in VR-II, the positions in bold-only are comprised in VR-III, the positions in italics-only are comprised in VR-IV, the positions underline-only are comprised in VR-V, the positions in both italics and bold are comprised in VR-VI, the positions in both bold and underline are comprised in VR-II, the positions in both italics and underline are comprised in VR-III, and the positions in bold, italics, and underline are comprised in VR-IX. In some embodiments of the methods described herein, a variant position(s) of interest is an amino acid position(s) in a VR of AAVrh74 VP1, AAVHSC16 VP1, AAV3B VP1, AAV6 VP1, AAV8 VP1, or AAV9 VP1. However, in some embodiments, a variant position(s) of interest is a position(s) in a VR of VP1 from serotype AAV1, AAV2, AAV4, AAV5, AAV7, AAVrh.10, or AAV44.9. In some embodiments, a variant position(s) of - 9 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 interest is a position(s) in a VR of VP1 from a serotype selected from: AAVrh74, AAVHSC16, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, and AAV44.9, and the variant position(s) of interest correspond(s) to a position(s) indicated in the VP1 sequence below from a different serotype selected from: AAVrh74, AAVHSC16, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, and AAV44.9 (see, e.g., the alignment of the wildtype VP1 amino acid sequences from AAV1, AAV8, AAVrh10, and AAVrh74 in FIG. 3, and the alignment of the wild-type VP1 amino acid sequences from serotypes AAVHSC16, AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAVrh10, and AAV44.9 in FIG. 15). In some embodiments, a variant amino acid position of interest is at one or more of the following amino acid positions in VP1 of AAVrh74 or at a corresponding position(s) in a VP1 of a different AAV serotype described herein: position 292, position 293, position 300, position 301, position 302, position 304, position 305, position 306, position 314, position 315, position 316, position 317, position 321, position 325, position 331, position 332, position 337, position 338, position 339, position 340, position 341, position 346, position 347, position 348, position 349, position 350, position 351, position 352, position 353, position 354, position 355, position 356, position 357, position 375, position 376, position 377, position 378, position 379, position 380, position 381, position 382, position 383, position 384, position 385, position 386, position 387, position 388, position 391, position 392, position 393, position 394, position 395, position 396, position 397, position 398, position 453, position 454, position 455, position 456, position 457, position 458, position 459, position 460, position 461, position 462, position 463, position 464, position 465, position 466, position 467, position 468, position 469, position 470, position 471, position 472, position 473, position 496, position 497, position 498, position 505, position 506, position 507, position 508, position 509, position 510, position 511, position 518, position 519, position 520, position 521, position 522, position 523, position 524, position 525, position 526, position 527, position 528, position 529, position 535, position 536, position 537, position 538, position 539, position 540, position 541, position 542, position 543, position 544, position 555, position 556, position 557, position 558, position 559, position 560, position 579, position 580, position 581, position 582, position 583, position 584, position 585, position 586, position 587, position 588, position 590, position 591, position 592, position 593, position 594, position 595, position 596, or position 597. AAVrh74 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESP VKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAA GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNT - 10 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQV FTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSY NFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYR QQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDN VDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKI PHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL* (SEQ ID NO: 1) AAVHSC16 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEA AKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASG GGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVF TDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSY EFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQ RVSTTVTQNNNSEIAWPRASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDAD KVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTD GNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE IQYTSNYCKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL* (SEQ ID NO: 112) AAV3B VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNE ADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAA KTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGG GAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDS EYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFE DVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQ RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAEL DNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHT DGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNP EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL* (SEQ ID NO: 113) AAV6 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEG AKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASG GGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVF SDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYT FEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQ QRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTAL DNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHT DGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNP EVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL* (SEQ ID NO: 114) AAV8 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG AKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAA GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNT YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQV FTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFT YTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPC YRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDN ADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKI PHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL* (SEQ ID NO: 115) AAV9 VP1 amino acid sequence: - 11 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEA AKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASG GGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVF TDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSY EFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQ RVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDA DKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHT DGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWN PEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL* (SEQ ID NO: 116) AAV1 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG AKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASG GGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVF SDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYT FEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYR QQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTAL DNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPH TDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWN PEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (SEQ ID NO: 118) AAV2 VP1 amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEAD AAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKT APGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSG APMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYS TPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFED VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV MITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG HFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQ YTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 119) AAV4 VP1 amino acid sequence: MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQQRLQGDTSFGGNLGRAVFQAKKRVLEPLGLVEQAG ETAPGKKRPLIESPQQPDSSTGIGKKGKQPAKKKLVFEDETGAGDGPPEGSTSGAMSDDSEMRAAAGGAA VEGGQGADGVGNASGDWHCDSTWSEGHVTTTSTRTWVLPTYNNHLYKRLGESLQSNTYNGFSTPWGYF DFNRFHCHFSPRDWQRLINNNWGMRPKAMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSSYELPY VMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGNTSQQQTDRNAFYCLEYFPSQMLRTGNNFEITYSFEKVP FHSMYAHSQSLDRLMNPLIDQYLWGLQSTTTGTTLNAGTATTNFTKLRPTNFSNFKKNWLPGPSIKQQGFS KTANQNYKIPATGSDSLIKYETHSTLDGRWSALTPGPPMATAGPADSKFSNSQLIFAGPKQNGNTATVPGT LIFTSEEELAATNATDTDMWGNLPGGDQSNSNLPTVDRLTALGAVPGMVWQNRDIYYQGPIWAKIPHTD GHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFSSTPVNSFITQYSTGQVSVQIDWEIQKERSKRWNPEVQ FTSNYGQQNSLLWAPDAAGKYTEPRAIGTRYLTHHL (SEQ ID NO: 120) AAV5 VP1 amino acid sequence: MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADE VAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTA PTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGAD GVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNR FHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGN GTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPS QNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSA FATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPV NRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGF - 12 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 GLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQF VDFAPDSTGEYRTTRPIGTRYLTRPL (SEQ ID NO: 121) AAV7 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG AKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSSVGSGTVAA GGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTNDNTY FGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTIQVF SDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNFEFSYSF EDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQAKNWLPGPCFR QQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGKTGATNKTTL ENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQGALPGMVWQNRDVYLQGPIWAKIPH TDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWN PEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL (SEQ ID NO: 122) AAV44.9 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG AKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPDPQPLGEPPAAPSGLGPNTMASG GGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTY FGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSY TFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMASQARNWVPGPSYR QQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDEDRFFPSSGVLIFGKQGAGNDGV DYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 125) AAVrh10 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG AKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAA GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNT YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQV FTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSY QFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCY RQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDN VDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWA KIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL (SEQ ID NO: 124) In some embodiments, in act 110, targets comprise membrane proteins, or fragments thereof, on the surface of a cell. Non-limiting examples of membrane proteins include integral membrane proteins and peripheral membrane proteins, such as, enzymes, receptors, cell-cell junction proteins, cell adhesion proteins, ion channels, transporters, chimeric antigen receptors, and lineage-specific cell surface antigens. In some embodiments, membrane proteins are present on the surface of a mammalian cell (e.g., a human cell), such as, a cell located in the blood, liver, spleen, lymph, brain, muscle, bone, lungs, etc. In some embodiments, in act 110, targets comprise wild-type proteins. In some embodiments, targets comprise cell surface proteins that are specific to a cell type and / or tissue. In some embodiments, targets comprise cell surface proteins that are expressed by cells of blood tissue, liver tissue, splenic tissue, lymph tissue, brain tissue, muscle tissue, bone tissue, lung - 13 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 tissue, etc. In some embodiments, targets comprise cell surface proteins that are specific to stem cells or progenitor cells. In some embodiments, targets comprise cell surface proteins specific to totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, embryonic stem cells, skin stem cells, etc. In some embodiments, targets comprise mutant or variant forms of a corresponding wild- type protein. In some embodiments, targets comprise membrane proteins, or fragments thereof, implicated in a disease or disorder, such as, ones that are overexpressed and / or mutated in a disease. In some embodiments, targets comprise membrane proteins that are normally not expressed on the surface of normal cells in a target tissue but are expressed in a target tissue on the surface of diseased cells. In some embodiments, target comprise mutant or variant form of a corresponding wild-type protein, wherein the mutant or variant form is associated with a disease, disorder, or condition described herein. In some embodiments, in act 110, targets comprise hetero-multimeric complexes of the cell surface. In some embodiments, targets comprise at least one KCNQ1, KCNE1, and CALM1. In some embodiments, targets comprise a complex comprising KCNQ1, KCNE1, and CALM1.In some embodiments, target comprise the wild-type form of at least one of KCNQ1, KCNE1, and CALM1. In some embodiments, target comprise the wild-type form of a complex comprising KCNQ1, KCNE1, and CALM1. In some embodiments, target comprise a mutant form of at least one of KCNQ1, KCNE1, and CALM1. In some embodiments, target comprise a mutant form of a complex comprising KCNQ1, KCNE1, and CALM1. In some embodiments, in act 120, the positions of variation identified in act 110 may be chosen to retain the corresponding wild-type codon or changed to a heterologous codon. In some embodiments, a heterologous codon is a codon encoding an amino acid at a corresponding variant position of interest, such as a codon comprising a non-synonymous mutation, or a codon comprising a different nucleotide sequence from the parent sequence but encodes the same amino acid, such as a codon comprising a synonymous mutation). In some embodiments, wherein a heterologous codon encodes the amino acid found in the variant position of interest, the heterologous codon may comprise the corresponding codon in the foster sequence or a different codon which encodes the same amino acid as the corresponding codon in the foster sequence. In some embodiments, all positions of variation identified in act 110 may be changed to heterologous codons. In some embodiments, some positions of a variant identified in act 110 are changed to heterologous codons and some retain the wild-type codon. Accordingly, in some embodiments, a combination of codons are used, wherein each codon in the combination is - 14 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 selected from: a wild-type codon, a heterologous codon encoding the same amino acid found in the wild-type protein sequence, and a heterologous codon encoding variant amino acid. In some embodiments, a single heterologous codon is chosen to encode an amino acid corresponding to variant positions of interest identified in act 110 (e.g., introducing a plurality of a first codon encoding a first amino acid into an engineered sequence, such as by using a plurality of a TCT codon in an engineered sequence to encode serine at a plurality of positions). In some embodiments, more than one heterologous codon is chosen to encode an amino acid corresponding to variant positions of interest identified in act 110 (e.g., introducing one or more of a first codon encoding a first amino acid and a second codon encoding the first amino acid into an engineered sequence, such as by using one or more TCT codons and one or more TCC codons in an engineered sequence to encode serine at a plurality of positions). In some embodiments, a heterologous codon is chosen based on codon usage bias of an expression system (e.g., the expression system that will be contacted with the AAV library during positive / negative selection described below). In some embodiments, a heterologous codon is chosen based on the codon usage bias of a mammalian expression system. In some embodiments, the codon usage bias is that of rat, mouse, rabbit, goat, sheep, donkey, dog, cat, monkey, or human. In some embodiments, codon usage bias is taken from a web-based prediction tool or table. In some embodiments, a codon less frequently used by a given expression system may be chosen. In some embodiments, a heterologous codon or a wild-type codon encodes an amino acid corresponding to a variant position of interest that is located in a VR of an AAV capsid protein (e.g., a variant position of interest in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, or VR-IX of an AAV VP1). In some embodiments, a heterologous codon or a wild-type codon corresponds to an amino acid at a variant position of interest that is in a VR of an AAV VP1, wherein the AAV VP1 is of a serotype selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC16, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2- BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8-C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, AAV44.9, and AAVr3.45. In some embodiments, the serotype is AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAVrh.74, or AAV44.9. Examples - 15 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 of nucleotide sequences in the AAV genome (e.g., nucleotide sequences comprising wild-type codons of the wild-type VP1 gene) can be found at the following GenBank accession numbers: NC_002077.1 (AAV1), NC_001401.2 (AAV2), NP_043941.1 (AAV3), NC_001829.1 (AAV4), NC_006152.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AY530579.1 (AAV9), MQ286650.1 (AAV44.9) AY243015.1 (AAVrh10), and LP899424.1 (AAVrh74). In some embodiments, in act 130, nucleic acid library synthesis comprises the use of trimer nucleotide fragments as building blocks. In some embodiments, the trimer nucleotide fragments are used to produce protein variants comprising heterologous codons at the positions identified in act 120. In some embodiments, trimer nucleotide fragments are used to produce protein variants comprising wild-type codons at one or more of the positions identified in act 120 (e.g., positions that will be comprised in the encoded protein variants and comprise the same residue as the corresponding position in the wild-type counterpart, such as positions in a protein variant that are unchanged relative to a parent sequence). In some embodiments, library synthesis comprises de novo gene synthesis. In some embodiments, de novo gene synthesis comprises use of trimer nucleotide fragments as building blocks. A non-limiting example of a method for library synthesis comprising de novo gene synthesis using trimer nucleotide fragments as building blocks is described in Virnekäs B et al. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 1994 Dec 25;22(25):5600-7 which is incorporated by reference herein. In some embodiments, the library comprises a complexity (e.g., the number of distinct engineered sequences in the library, such as the number of variants in the library or the number of variants in the library in addition to the number of foster sequences and / or the parent sequence in the library) of 2-10, 10-100, 100-200, 200-500, 500-1,000, 1,000-2,000, 2,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-1,000,000, or more than 1,000,000 library members (e.g., 1 million-2 million, 2 million-5 million, 5 million-10 million, or 10 million-100 million library members). In some embodiments, in act 130, the protein variants corresponding to the sequences of the synthesized library comprise engineered AAV capsid proteins. In some embodiments, the engineered AAV capsid proteins comprise variants of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC16, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), - 16 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8-C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, AAV44.9, or AAVr3.45. In some embodiments, in act 130, the engineered AAV capsid variants comprise one or more amino acid substitutions in one or more VRs of an AAV capsid protein. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in VR I, VR II, VR III, VR IV, VR V, VR VI, VR VII, VR-VIII, and / or VR-IX. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in a VR (e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and / or VR-IX) of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV- HSC16, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV- HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8- C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, AAV44.9, or AAVr3.45. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in a VR (e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and / or VR-IX) of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAVrh.74, or AAV44.9. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in VR IV, VR V, VR VI, VR VII, and / or VR VIII of AAVrh.10. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in VR IV, VR V, VR VI, VR VII, and / or VR VIII of AAV2. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in VR IV, VR V, VR VI, VR VII, and / or VR VIII of AAV8. In some embodiments, the engineered AAV capsid variants comprise one or more amino acid substitutions in VR IV, VR V, VR VI, VR VII, and / or VR VIII of AAVrh.74. Sequences shown in FIG. 13 and FIG. 14 represent a portion of an AAV capsid protein (e.g., positions 261-520 of a variant AAV-rh74 VP1 sequence in FIG. 13, or positions 201-600 of a variant AAV-rh74 VP1 sequence in FIG. 14). In some embodiments, recombinant full length VP1 sequences can - 17 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 comprise these variant regions in the context of an N-terminal portion (e.g., N-terminal amino acids 1-260 or 1-200 of AAV-rh74 VP1) and / or C-terminal portion (e.g., amino acids C-terminal to position 520 or position 600 of AAV-rh74 VP1) of an AAV VP1 protein. In some embodiments, the N-terminal and / or C-terminal portions are from AAV-rh74. However, in some embodiments the N-terminal and / or C-terminal portions can be corresponding portions from one or more other AAV serotypes. In some embodiments, one or more of the amino acid substitutions identified according to the methods described in this application can be introduced into variable regions of other AAV serotypes (e.g., at positions corresponding to the positions shown in FIGs. 13 and 14). In some embodiments, the engineered AAV capsid variants comprise one or more variant amino acid positions of clones 1-3, 2-4, 6-4, or 11-3 described herein (see, e.g., FIG. 14). In some embodiments, the engineered AAV capsid variants comprise one or more variant amino acid positions of clones 1-3, 2-4, 6-4, or 11-3 and one or more other variant amino acid positions in the wild-type VP1 protein of serotype AAVrh.74 (see, e.g., FIG. 14). In some embodiments, the engineered AAV capsid variants comprise the variant amino acid positions of clones 1-3, 2-4, 6-4, or 11-3 described herein (see, e.g., FIG. 14). In some embodiments, an engineered nucleic acid comprises a nucleotide sequence encoding a variant AAV capsid amino acid sequence (e.g., comprising one or more of the amino acid substitutions shown in FIGs. 13 and 14). In some embodiments, in act 130, the engineered AAV capsid variants comprises a variant VP1, wherein the variant VP1 comprises one or more variant amino acid positions. In some embodiments, the one or more variant amino acid positions comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or more than 100 amino acid positions. In some embodiments, a VP1 variant comprises an amino acid substitution, an amino acid deletion, or an amino acid insertion at one or more variant amino acid positions. In some embodiments, a VP1 variant comprises an amino acid substitution, an amino acid deletion, or an amino acid insertion at one or more corresponding positions in a VP1 protein of a different AAV serotype (e.g., corresponding positions located in a VR, such as VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and / or VR-IX). In some embodiments, the engineered AAV capsid variants comprises a variant VP1, wherein the variant VP1 comprises a variant amino acid at a position corresponding to one or more of the following amino acid positions in AAVrh74 VP1 or the corresponding position(s) in a VP1 of a different AAV serotype: position 292, position 293, position 300, position 301, position 302, position 304, position 305, position 306, position 314, position 315, position 316, position 317, position 321, position 325, position 331, position 332, - 18 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 position 337, position 338, position 339, position 340, position 341, position 346, position 347, position 348, position 349, position 350, position 351, position 352, position 353, position 354, position 355, position 356, position 357, position 375, position 376, position 377, position 378, position 379, position 380, position 381, position 382, position 383, position 384, position 385, position 386, position 387, position 388, position 391, position 392, position 393, position 394, position 395, position 396, position 397, position 398, position 453, position 454, position 455, position 456, position 457, position 458, position 459, position 460, position 461, position 462, position 463, position 464, position 465, position 466, position 467, position 468, position 469, position 470, position 471, position 472, position 473, position 496, position 497, position 498, position 505, position 506, position 507, position 508, position 509, position 510, position 511, position 518, position 519, position 520, position 521, position 522, position 523, position 524, position 525, position 526, position 527, position 528, position 529, position 535, position 536, position 537, position 538, position 539, position 540, position 541, position 542, position 543, position 544, position 555, position 556, position 557, position 558, position 559, position 560, position 579, position 580, position 581, position 582, position 583, position 584, position 585, position 586, position 587, position 588, position 590, position 591, position 592, position 593, position 594, position 595, position 596, or position 597. In some embodiments, the variant VP1 is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC16, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8-C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, AAV44.9, or AAVr3.45. In some embodiments, a variant VP1 (e.g., a variant VP1 from a library described herein, for example comprising one or more variable region amino acid substitutions) is modified to further comprise a variant amino acid (e.g., an amino acid substitution) at one or more positions that correspond to amino acid position 346, amino acid position 501, amino acid position 505, and / or amino acid position 706 in the VP1 of serotype AAVHSC16 (e.g., a variant VP1 comprising one or more of: a threonine (T) at an amino acid position corresponding to position 346 in AAVHSC16 VP1, an isoleucine (I) at an amino acid position corresponding to position 501 in AAVHSC16 VP1, an arginine (R) at an amino acid corresponding to position 505 in AAVHSC16 VP1, or a cysteine (C) at an amino acid position - 19 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 corresponding to position 706 in AAVHSC16 VP1, such as to generate an AAV variant capsid that has reduced liver targeting). In some embodiments, in act 130, an amino acid substitution in a VR of an AAV capsid protein is a conservative substitution. For example, in some embodiments, a conservative substitution replaces a first hydrophilic amino acid residue (e.g., a hydrophilic amino acid in a parent sequence) with a second hydrophilic amino acid residue (e.g., a hydrophilic amino acid in a foster sequence). In other embodiments, a conservative substitution replaces a first hydrophobic amino acid residue (e.g., a hydrophobic amino acid in a parent sequence) with a second hydrophobic amino acid residue (e.g., a hydrophobic amino acid in a foster sequence). In some embodiments, when a conservative substitution is made, a hydrophilic amino acid is selected from: asparagine (N), glutamine (Q), serine (S), threonine (T), cysteine (C), aspartate (D), glutamine (E), arginine (R), lysine (K), or histidine (H), and a hydrophobic amino acid is selected from: glycine (G), alanine (A), valine (V), leucine (L), methionine (M), isoleucine (I), phenylalanine (F), tyrosine (Y), tryptophan (Y), and proline (P). In some embodiments, a conservative amino acid substitution replaces a residue at a corresponding amino acid position (e.g., a residue at a corresponding amino acid position in a parent sequence) with a residue (e.g., a residue in a foster sequence) having a side chain of a similar charge and / or shape. Non-limiting examples of substitutions that can replace amino acids with residues having a similar charge and / or shape include replacement of a glycine for an alanine, replacement of a glutamate for an aspartate, replacement of a lysine for an arginine, replacement of a leucine for an isoleucine, replacement of a phenylalanine for a tryptophan, replacement of an asparagine for a glutamine, and replacement of a serine for a threonine. In some embodiments, in act 130, an amino acid substitution in a VR of an AAV capsid protein is a non-conservative substitution. For example, in some embodiments, a non- conservative substitution replaces a hydrophilic amino acid residue (e.g., a hydrophilic amino acid in a parent sequence) with a hydrophobic amino acid residue (e.g., a hydrophobic amino acid in a foster sequence). In some embodiments, when a non-conservative substitution is made, a hydrophilic amino acid is selected from: asparagine (N), glutamine (Q), serine (S), threonine (T), cysteine (C), aspartate (D), glutamine (E), arginine (R), lysine (K), or histidine (H), and a hydrophobic amino acid is selected from: glycine (G), alanine (A), valine (V), leucine (L), methionine (M), isoleucine (I), phenylalanine (F), tyrosine (Y), tryptophan (Y), and proline (P). In some embodiments, a non-conservative amino acid substitution replaces a residue at a corresponding amino acid position (e.g., a residue at a corresponding amino acid position in a parent sequence) with a residue (e.g., a residue in a foster sequence) having a side chain of a - 20 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 different charge and / or shape. Non-limiting examples of substitutions that can replace amino acids with residues having a different charge and / or shape include replacement of a glycine for a threonine, replacement of a glutamate for an arginine, replacement of a lysine for an aspartate, replacement of an asparagine for an isoleucine, replacement of a phenylalanine for a cysteine, replacement of a leucine for a glutamine, and replacement of a serine for an alanine. In some embodiments, in act 140, production of the vector library comprises one or more methods known in the art for nucleic acid engineering. In some embodiments, in act 140, production of the vector library comprises in vitro amplification (e.g., using PCR) of DNA. In some embodiments, production of the vector library comprises recombinant cloning techniques (e.g., using restriction enzymes). Non-limiting examples of such techniques, and suitable enzymes for such techniques (e.g., restriction enzymes, ligases, DNA polymerases, etc.) have been previously described, for example, see Sambrook et al., Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover) IREL Press, Oxford, (1985); each of which is incorporated herein by reference. In some embodiments, in act 140, vectors comprise single-stranded and / or double- stranded nucleic acids. In some embodiments, vectors are linear. In some embodiments, vectors are circular. In some embodiments, vectors comprise plasmids, such as, circular plasmids, nanoplasmids, and minicircle plasmids. In some embodiments, vectors are self-complementary. In some embodiments, vectors comprise antibiotic-resistance genes. In some embodiments, in act 140, vectors comprise sequences found in viral genomes. In some embodiments, vectors comprise sequences found in AAV genomes. In some embodiments, vectors of the library comprise an AAV rep gene and an AAV cap gene flanked by AAV inverted terminal repeats (ITRs). In some embodiments, vectors comprise an AAV rep gene and an AAV cap gene corresponding to an engineered AAV capsid protein produced in process 100, wherein said rep and cap gene are flanked by AAV ITRs. In some embodiments, vectors of the library comprise other AAV genes and / or helper genes described herein. In some embodiments, the AAV rep and AAV cap genes, or AAV rep and engineered AAV cap gene, and flanking AAV ITRs are sufficiently small to be effectively packaged into an AAV particle. In some embodiments, the AAV rep and AAV cap genes, or AAV rep and engineered AAV cap gene, and flanking AAV ITRs comprise a sequence of no more than 5-6 kb in length. In some embodiments, in act 150, the host cell is a mammalian cell (e.g., a human cell), a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. In some embodiments, the - 21 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 host cell is a HEK293 cell. Suitable transfection techniques have been previously described. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. In some embodiments, in act 150, production of viral particle libraries comprises production of an AAV particle library. In some embodiments, production of an AAV particle library comprises co-transfection with two or more vectors (e.g., one plasmid of the library and a second plasmid). In some embodiments, production of an AAV particle library comprises the use of a helper nucleic acid. In some embodiments, the helper nucleic acid comprises one or more helper genes (e.g., E1, E2A, E4, and VA) which function in trans for productive AAV replication and encapsidation. Helper nucleic acids, and methods of making said nucleic acids, have been previously described and are commercially available. For example, see pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany, other products and services available from: Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini , Journal of Virology, Vol. 79, 5296- 5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adenoassociated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). In some embodiments, a helper nucleic acid is provided by a helper virus. In some embodiments, the helper virus is an adenovirus, such as, adenovirus 5 (Ad5). In some embodiments, the host cell is stably transfected with a helper nucleic acid. In some embodiments, one or more additional acts may be performed. For example, in some embodiments a sequencing reaction may be performed to determine the sequence variant present at one or more steps in the process. In some embodiments, the nucleic acid library produced in act 130 is sequenced. In some embodiments, the vector library produced in act 140 is sequenced. In some embodiments, nucleic acids of the viral particle library produced in act 150 are sequenced. - 22 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 In some embodiments, a protein variant, or a nucleic acid encoding said protein variant, produced by process 100 may serve as the parent sequence in a subsequent execution of process 100. In some embodiments, at least one protein variant, or nucleic acid encoding said protein variant, produced by process 100 may serve as the one or more foster sequences in a subsequent execution of process 100. FIG. 2 illustrates a non-limiting embodiment of a process 200 for identifying and / or isolating viral particles of interest from a viral particle library (e.g., to identify and / or isolate viral particles that preferentially bind to one or more cell surface proteins of interest). In act 210, a library of viral particles (e.g., AAV particles comprising capsid protein variants) is contacted to a preparation of a first host cell lineage that is engineered to express one or more surface proteins of interest. In some embodiments, this contacting is performed under conditions that promote transduction of the host cell by viral particles that interact with the host cell (e.g., by binding to the one or more cell surface proteins). In some embodiments, viral particles that transduce the host cell can then replicate to produce more viral particles within the host cell. In act 220, positive selection is performed, wherein viral particles that transduced the first host cell lineage expressing one or more surface proteins of interest are obtained. In act 230, the viral particles obtained in act 220 are contacted to a preparation of the first host cell lineage that does not express the one or more surface proteins of interest (also referred to as a naïve host cell lineage). In act 240, negative selection is performed, wherein viral particles that do not interact with the naïve host cell lineage of act 230 are isolated. In some embodiments, the viral particles isolated in act 240 can be further processed via one or more subsequent rounds of acts 210-240 in optional act 250 (e.g., using one or more different host cell lineages). Subsequently, viral particles from act 240 (or from one or more additional cycles of act 250) can be isolated and / or identified in act 260. In some embodiments, in act 210, two host cell populations prepared from the same cell lineage are used wherein a first population comprises host cells that express a target protein, or fragment thereof, and a second cell population does not comprise the target. In some embodiments, the first population of host cells express a target comprising a binding site that is known to bind a protein encoded by the one or more foster sequences (e.g., a target protein that can bind to an AAV VP1, such as a VP1 of serotype AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAVrh.74, or AAV44.9). In some embodiments, the first population of host cells express a target comprising a binding site that putatively binds to a protein variant, described herein or produced by the methods described herein. In some embodiments, the target naturally localizes to the cell surface of the first host cell population and - 23 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 the binding site is exposed on the extracellular side of the membrane. In some embodiments, the first host cell population expresses a target, or fragment thereof, encoded by a vector (e.g., a plasmid). In some embodiments, the first host cell population expresses a target, or fragment thereof, encoded by a gene located in the cellular genome. In some embodiments, the host cell lineage is negative for the target and the first host cell population prepared from said lineage is engineered to express the target protein sequence. In some embodiments, the first host cell population is engineered to comprise a vector. In some embodiments, the vector is integrated into the cellular genome. In some embodiments, the host cell lineage is positive for the target and the second host cell population prepared from said lineage is engineered to comprise a knockout of the gene encoding the target protein. In some embodiments, the target protein is a variant of the wild-type form of the target protein, such as a variant target protein comprising one or more mutations that are associated with a disease, disorder, or condition described herein and / or a variant target protein comprising one or more mutations that do not effect binding to the protein encoded by the protein encoded by the one or more foster sequences. In some embodiments, in act 210, host cells mammalian cells. In some embodiments, mammalian host cells comprise primary cells. In some embodiments, host cells comprise cells derived from a cell line. Non-limiting examples of cell lines that may be used as host cells include HEK293 cells, COS cells, HeLa cells, HeLaS3, BHK cells, CHO cells or PER.C6® (see, e.g., ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-2.2, ATCC® CCL-10™, or ATCC® CCL-61™. In some embodiments, host cells are at least one of HuH7 (hepatocarcinoma), AC-16 (cardiomyocytes), HEK 293 (embryonic kidney), and HeLa (cervix). In some embodiments, in act 210, contacting the viral particle library with host cells comprises transduction of the first host cell population comprising the target protein. In some embodiments, the first host cell population replicates and produces viral particles that successfully transduce the first host cell population. In some embodiments, the first host cell population is contacted with a helper nucleic acid before, during, or after the host cells are contacted with the viral particle library. In some embodiments, the helper nucleic acid is a helper for AAV particle production. In some embodiments, the helper nucleic acid comprises one or more helper genes for AAV production (e.g., E1, E2A, E4, and VA). In some embodiments, the helper nucleic acid is provided as a helper virus. In some embodiments, the helper virus is an adenovirus (e.g., Ad5). In some embodiments, the host cells are stably transfected with a helper nucleic acid. - 24 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 In some embodiments, in act 210, the viral particle library comprises AAV particles. In some embodiments, the AAV particles of the library comprise engineered AAV capsid protein described herein or are produced by methods described herein. In some embodiments, the viral particle library is produced by process 100. In some embodiments, in act 220, obtaining the viral particles that transduced the host cells comprises isolation and / or purification of the viral particles. In some embodiments, isolation and / or purification comprises centrifugation. In some embodiments, isolation and / or purification comprises filtration. In some embodiments, isolation and purification comprises tangential flow filtration (TFF), dialysis membrane filtration, centrifugation using centrifugation filtration devices (e.g., at 150kD Molecular weight cut-off (MWCO) membrane filter devices; see, e.g., products from Orbital Bioscience and Millipore), gradient ultracentrifugation, affinity purification, size-exclusion chromatography, ion-exchange chromatography, or any combination thereof. In some embodiments, in act 230, the second host cell population is not contacted with a helper nucleic acid. In some embodiments, the second host cell population is contacted with a helper nucleic acid before, during, or after the host cells are contacted with the viral particle library. In some embodiments, the second host cell population is stably transfected with a helper nucleic acid. In some embodiments, in act 240, the viral particles that do not bind the second host cell population are isolated and / or purified. In some embodiments, the viral particles that do not bind and transduce the second host cell population are isolated and / or purified. In some embodiments, isolation and / or purification comprises centrifugation. In some embodiments, isolation and / or purification comprises filtration. In some embodiments, isolation and purification comprises tangential flow filtration (TFF), dialysis membrane filtration, centrifugation using centrifugation filtration devices (e.g., at 150kD Molecular weight cut-off (MWCO) membrane filter devices; see, e.g., products from Orbital Bioscience and Millipore), gradient ultracentrifugation, affinity purification, size-exclusion chromatography, ion-exchange chromatography, or any combination thereof. In some embodiments, in act 250, the cycles of act 210-240 are repeated using first and second cell populations prepared from a different cell lineage. In some embodiments, the cycles of act 210-240 are repeated once. In some embodiments, the cycles of act 210-240 are repeated more than once wherein each repetition of said cycle does not use host cells of the same lineage more than once. In some embodiments, the cycles of act 210-240 are repeated twice. In some embodiments, the cycles of act 210-240 are repeated three times. In some embodiments, the - 25 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 cycles of act 210-240 are repeated four times. In some embodiments, the cycles of act 210-240 are repeated five or more times. In some embodiments, the cycles of act 210-240 are repeated six, seven, eight, nine, ten or more times. In some embodiments, in act 260, the viral particles that do not bind the second host cell population in act 240 are isolated and / or purified. In some embodiments, isolation and purification comprises tangential flow filtration (TFF), dialysis membrane filtration, centrifugation using centrifugation filtration devices (e.g., at 150kD Molecular weight cut-off (MWCO) membrane filter devices; see, e.g., products from Orbital Bioscience and Millipore), gradient ultracentrifugation, affinity purification, size-exclusion chromatography, ion-exchange chromatography, or any combination thereof. In some embodiments, the viral particles isolated in act 230 (e.g., after one or more rounds of processing) can be evaluated. In some embodiments, nucleic acids of these viral particles are sequenced, for example, to determine the sequence of the encoded capsid protein. In some embodiments, acts 210-250 comprise positive / negative selection. In some embodiments, positive / negative selection comprises providing a first population of HEK293 cells expressing the target protein on the cell surface and contacting said cells with an AAV particle library and the Ad5 helper virus. In some embodiments, after incubating the first population of HEK 293 cells in contact with the AAV particle library and Ad5, produced viral particles are isolated and / or purified using methods described herein, thereby completing one round of positive selection. In some embodiments, AAV particles that were obtained during the previous positive selection round are then contacted with a second population of HEK 293 cells that do not express the target protein on the cell surface. In some embodiments, AAV particles that do not bind to the second population of HEK 293 cells are isolated and / or purified using methods described herein, thereby completing one round of negative selection. In some embodiments, one or more additional cycles (e.g., three additional cycles yielding a total of four cycles) of positive / negative selection are used employing different host cell types, wherein each cycle comprises the use of its own respective host cell type. In some embodiments, it may be desirable to use different cell types with different cell surface protein types (other than the target) to ensure representation of multiple cell surface receptors for positive / negative selection purposes. In some embodiments, the second cycle may involve HuH7 cells. In some embodiments, the third cycle comprises use of AC-16 cells. In some embodiments, the fourth cycle comprises use of HeLa cells. In some embodiments, four rounds of positive / negative selection are performed using HEK293 cells in the first round, HuH7 cells in the second round, AC-16 cells in the third round, and HuH7 cells in the fourth round. In some embodiments, four - 26 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 rounds of positive / negative selection are performed wherein the target expressed by the first population of each respective host cell lineage comprises a hetero-multimeric complex comprising KCNQ1, KCNE1, and CALM1. In some embodiments, positive / negative comprises the method shown in FIG. 12. In some embodiments, following process 200, a nucleic acid encoding the protein variants that bind the target and present on the surface of the viral particles isolated and / or identified in act 260 is produced. In some embodiments, the nucleic acid is cloned into a vector. In some embodiments, the nucleic acid, or vector thereof, are transfected into cells. In some embodiments, a recombinant protein corresponding encoded by the nucleic acid is produced. In some embodiments, the recombinant protein is added to a pharmaceutical composition. In some embodiments, the nucleic acid is used to produce an rAAV particle, or recombinant viral genome thereof. As used herein, an “rAAV particle” comprises at least one AAV capsid protein and a “recombinant AAV genome” comprising a heterologous nucleic acid flanked by AAV ITRs. In some embodiments, the at least one AAV capsid protein comprises a variant VP1 described herein. In some embodiments, rAAV genomes may be linear or circular, single-stranded or double-stranded, and / or self-complementary. ITR sequences are about 145 bp in length. While the entire sequences encoding the ITRs are commonly used in engineering rAAVs, modification of these sequences is permissible and may be done using standard techniques (see, e.g., texts such as such as, Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520532 (1996)). For example, artificial ITRs may be engineered for tissue specificity. The components (e.g., a nucleic acid comprising the heterologous nucleic acid and flanking AAV ITRs, a packaging nucleic acid comprising AAV rep and AAV cap sequences, and a helper nucleic acid) cultured in a cell to package a rAAV genome in a capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components may be provided by a cell which has been engineered to contain one or more of the required components. Such a cell will contain the required component(s) under the control of either an inducible promoter or a constitutive promoter. In some embodiments, rAAV particles may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650 incorporated by reference herein). Typically, the rAAV particles are produced by transfecting a cell with a vector (comprising a heterologous nucleic acid flanked by ITR elements) to be packaged into rAAV particles, and at least one AAV helper or packaging nucleic acid. In some embodiments, two nucleic acids are used which include a helper nucleic acid and a packaging - 27 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 nucleic acid. Said packaging nucleic acid comprises AAV rep and AAV cap sequences. In some embodiments, the helper nucleic acid and the packaging nucleic acid are provided on the same vector. In some embodiments, the packaging nucleic acid comprises a cap sequence corresponding to a capsid protein of an AAV serotype described herein. In some embodiments, the packaging nucleic acid comprises a cap sequence corresponding to a protein variant produced by the methods described herein. In some embodiments, the protein variant is an engineered AAV capsid protein of any one of clones 1-3, 2-4, 6-4, or 11-3. In some embodiments, one or more engineered AAV capsid proteins identified from a library described in this application can be used to target viral particles to a cell or tissue type of interest. In some embodiments, rAAV particles comprising one or more of these engineered AAV capsid proteins and a recombinant genome comprising a heterologous nucleic acid of interest flanked by AAV ITRs may be used to deliver a heterologous nucleic acid to target cells or tissues. In some embodiments, rAAV particles comprising the heterologous nucleic acid and an engineered AAV capsid protein identified from the library described herein assist in the treatment of a disease or condition. In some embodiments, rAAV particles described herein are used to produce a pharmaceutical composition. In some embodiments, a heterologous nucleic acid is an engineered nucleic acid sequence. In some embodiments, a heterologous nucleic acid is a naturally occurring nucleic acid sequence (e.g., a non-engineered sequence) that is not normally found between AAV ITRs. In some embodiments, a heterologous nucleic acid comprises contain segments of DNA taken from different organisms. In some embodiments, a heterologous nucleic acid may comprise any combination of naturally-occurring and engineered DNA sequences. In some embodiments, a heterologous nucleic acid comprises at least one transgene. As used herein, “transgene” refers to a DNA sequence which encodes an RNA to be expressed in a cell. In some embodiments, a heterologous nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transgenes. Examples of sequences found in transgenes include sequences encoding interfering RNAs (e.g., shRNAs, siRNAs, miRNAs, ncRNAs, piRNAs, pro-siRNAs, etc.), exon- skipping RNAs, RNA gapmers, enzymatic RNAs, guide RNAs or gRNAs (e.g., sgRNAs) of a CRISPR / Cas editing system (e.g., Cas9-based genome editing and derivatives thereof such as, base editing and prime editing), small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and mRNAs. In some embodiments, a transgene comprises a cDNA sequence. In some embodiments, RNA expressed from a transgene is not translated into a protein. In some embodiments, a transgene encodes an mRNA that is translated into a protein. In some embodiments, a transgene may comprise multiple DNA sequences (e.g., encodes multiple - 28 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 RNA species). In some embodiments, heterologous nucleic acids comprise a plurality of sequences wherein at least one sequence (e.g., a transgene) of the plurality are expressed in a cell and at least one other sequence of the plurality are not expressed in the cell (e.g., repetitive elements, repair templates, etc.). In some embodiments, a transgene encodes a selectable marker. In some embodiments, a selectable marker is used to assay heterologous nucleic acid expression in a cell or subject and / or sort transduced host and / or target cells. Examples of selectable markers include cell surface proteins (e.g., an antibody or antigen-binding fragment thereof, receptors, membrane proteins which become glycosylated upon expression in a cell, etc.) and fluorescent markers. In some embodiments, a transgene encodes a therapeutic RNA and / or protein. As used herein, a “therapeutic RNA” or “therapeutic protein” leads to a physiological change that is associated with or expected to at least partially, if not fully, remedy at least one symptom associated with a disease, disorder, or condition. A therapeutic RNA refers to an RNA expressed from a transgene that is therapeutic as an RNA upon expression in a target cell and without being translated into a protein. A therapeutic protein refers to any proteinaceous molecule that is translated from an RNA expressed from a transgene which is therapeutic upon translation in a target cell. The therapeutic RNA or protein may be therapeutic for any disease, disease, or condition described herein upon administration to a subject in need thereof. Non-limiting examples of therapeutic RNAs include any antisense RNA (e.g., shRNA, miRNA, etc.), exon-skipping RNA, RNA gapmer, enzymatic RNA, gRNA, snRNA, rRNA, or tRNA. In some embodiments, a therapeutic RNA modulates the expression of a gene implicated in a disease, disorder, or condition. Non-limiting examples of therapeutic proteins include enzymes (such as, proteases, signaling proteins, transcriptional regulators, Cas9, base editors, prime editors, etc.), enzymatic domains, enzyme substrates, hormones, receptors (e.g., chimeric antigen receptors), components of gene editing ribonucleoprotein complexes (e.g., Cas9, base editors, prime editors, etc), peptibodies, growth factors, clotting factors, cytokines, chemokines, activating or inhibitory peptides acting on cell surface receptors or ion channels, cell-permeable peptides targeting intracellular processes, thrombolytics, bone morphogenetic proteins, Fc- fusion proteins, anticoagulants, and antibodies or antigen-binding fragments thereof. In some embodiments, a therapeutic RNA or protein is selected for the purposes of gene replacement therapy. In some embodiments, the therapeutic protein is selected for the purposes of vaccine production against a human pathogen (e.g., a protein, or fragment thereof, comprising an antigen of a human pathogen). In some embodiments, the therapeutic RNA or protein is selected for anti-cancer therapy. - 29 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 In some embodiments, a transgene comprises gene sequences (e.g., those coding for interfering RNAs, mRNAs, etc.) operably linked to at least one regulatory sequence described herein. In some embodiments, a transgene comprises at least one regulatory sequence located at the 5’ end such that it is located upstream of a gene sequence (e.g., in the 5’ untranslated region (UTR)). In some embodiments, a transgene comprises at least one regulatory sequence located at the 3' end such that it is located downstream of the gene sequence (e.g., in the 3’ UTR). In some embodiments, a transgene comprises at least one regulatory at the 5’ end and at least one regulatory sequence located at the 3’ end such that regulatory sequences are located upstream and downstream of the gene sequence. In some embodiments, regulatory sequences comprise constitutive promoters, inducible promoters, tissue-specific promoters, promoters comprising exons and / or introns, polyadenylation sequences, enhancer elements, splice donor / acceptor sequences, introns, or any combination thereof. However, in some embodiments, a heterologous nucleic acid is not transcribed into an RNA and / or promotes synthesis of a corresponding protein. In some embodiments, a heterologous nucleic acid comprises at least one sequence that is not expressed in a cell. In some embodiments, a heterologous nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sequences not expressed in a cell. In some embodiments, a heterologous nucleic acid comprises a regulatory sequence described herein. In some embodiments, a heterologous nucleic acid comprises a regulatory sequence not operably linked to a gene sequence encoding an RNA. In some embodiments, a heterologous nucleic acid comprises a nucleic acid used as a substrate for genetic engineering (e.g., a repair template used for homology-directed repair mechanisms). In some embodiments, the rAAV particle can be delivered to a subject via any suitable route. In some embodiments, administration is performed subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intracranially, intrathecally, orally, intraperitoneally, or by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs. In some embodiments, compositions are administered to a subject through only one administration route. In some embodiments, multiple administration routes may be exploited (e.g., serially, or simultaneously) for administration of the composition to a subject. Treatment methods involving rAAV particles are also known in the art (see, e.g., Adeno-Associated Virus Vectors in Clinical Trials. Barrie J. Carter. Human Gene Therapy. May 2005, 16(5): 541-550. doi:10.1089 / hum.2005.16.541. Published in Volume: 16 Issue 5: May 25, 2005; Neuropharmacology. 2013 Jun;69: 82-8. doi: 10.1016 / j.neuropharm.2012.03.004. Epub 2012 Mar 17.; Adeno-associated virus (AAV) gene therapy for neurological disease. Weinberg MS1, Samulski RJ, McCown TJ.Gene therapy for - 30 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 lysosomal storage disorders. Yew NS, Cheng SH. Pediatr Endocrinol Rev. 2013 Nov;11 Suppl 1:99-109; Directed evolution of novel adeno-associated viruses for therapeutic gene delivery. Bartel MA, Weinstein JR, Schaffer DV.Gene Ther. 2012 Jun;19(6):694-700. doi: 10.1038 / gt.2012.20. Epub 2012 Mar 8; Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Mingozzi F, High KA. Nat Rev Genet. 2011 May;12(5):341-55. doi: 10.1038 / nrg2988). In some embodiments, a subject can be a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In some embodiments, the non- human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as, chicken, duck, goose, or turkey)). In some embodiments, the non-human animal is a fish, reptile, or amphibian. In some embodiments, the subject has, is suspected of having, is at risk of having, or is expected to develop a disease, disorder, or condition. In some embodiments, administration of an rAAV particle described herein is used to treat a disease, disorder, or condition. In some embodiments, the disease, disorder, or condition comprises a genetic disease, cancer, inflammatory disease or an inflammatory condition, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, a kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or other disease, or any combination thereof. EXAMPLES As a universal source of AAV vectors for targeted transduction of human organs, tissues, and cell types with potentially attenuated pre-existing neutralizing antibody profile, a new AAV capsid library was designed based on recently discovered rhesus monkey AAV variants 74 (library “parent sequence”, a.k.a. “scaffold”) [1]. As the contributor variants (“foster sequences”) for the library design, one additional rhesus monkey serotype – 10 (rh10) and two human AAV variants (1 and 8) (FIG. 3) were selected. An example of the AAV rh74 sequence is provided at WO 2016 / 025339 A2 which is incorporated by reference herein. Additionally, an example of the 738 amino acid long AAV rh74 VP1 amino acid sequence is shown below. - 31 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 VP1 AAV-rh74 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLV NPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQ YGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL (SEQ ID NO: 1) Human or evolutionary close to human AAV serotypes serve as reliable source of mutational pool with high affinity to human organs. For library design and production, a novel strategy of trimer / codon synthesis was used for precise tuning of a library composition [3]. Previous library design efforts were based on The International Union of Pure and Applied Chemistry (IUPAC) nomenclature system [4]. The methods disclosed herein significantly improve library assembly. A virtual family shuffling protocol (VSF) with IUPAC-encoded oligonucleotide synthesis [5] was used. To generate an AAV library using VFS approach, AAV capsid sequences for multiple known AAV serotypes were aligned against the selected AAV parent sequence (FIG. 3). To increase an assembly fitness of potential of AAV capsid variants within the library, mutants exclusively within so-called variable regions (VRs) were incorporated. These VRs are characterized by a significant evolutionary diversity between multiple serotypes and demonstrate the capacity to be modified without significant effect of capsid’s assembly fitness. Using foster-to-parent capsid sequence alignment, a pool of possible mutations for each amino acid (AA) residue position in the variable regions were identified. Then, the choice of AA residues in a given position to already present in the known foster parents’ pool was restricted to increase the library’s fitness. Example 1 This Example describes library design and production using IUPAC-encoded gene sequences. In an IUPAC approach [5], to encode the AA variability in each position, IUPAC nucleotide coding was used which allowed the encoding of a degenerate nucleotide without - 32 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 specifying all four nucleotide residues A, G, T, or C (FIG. 4). For example, Y encodes either C or T, W encodes either A or T etc. The IUPAC encoding partially mitigated exponential growth of the library complexity. In case of degenerate nucleotide coding, the complexity grows with the rate 20X(where X is a length of AA sequence) given that oligonucleotide synthesis is carried out with all four A, C, G and T bases per each position (NNN). Although the IUPAC encoding allowed narrowing down the complexity compared to degenerate NNN encoding, the problem has not been completely solved as the designed library still incorporated uncalled-for mutations. In order to mathematically quantify redundant mutations in IUPAC encoding approach, a python script that used IUPAC-encoded library design was used which emulated oligonucleotide synthesis with subsequent virtual peptide sequence translation. For example, for introduction of certain mutations in 5, 8 or 12 AA positions using IUPAC nomenclature, this pattern had the following designed mutant complexity: 5 positions – 288 total possible combinations, 8 positions – 576, and 12 position – 9216 variants (FIG. 5, middle bars in each group of three bars). This is designated as “explicit” complexity. Instead, the IUPAC encoding leads to generation of 288, ~1.1E+5, and ~1.1E+8 variants which was significantly higher than the intended designed complexity (herein, referred to as “implicit” complexity). Generation of the uncalled-for extra mutants in the context of the full-length library had significant negative consequences. For example, the large theoretical un-warranted complexity negated the power of structural prediction of VSF approach leading to the excess production of dead-end assembly variants. Furthermore, the accumulation of variants with low assembly fitness significantly affected library yield and also creates a selective advantage for wild type- like capsids, thus extending the overall number of selection rounds in subsequent directed evolution. Under the IUPAC encoding scheme, single VR regions sub-libraries were assembled first in order to recover intra-VR-compatible residues. Once all VR were separately mutagenized, assembled, and viral DNA from the sub-libraries isolated, single VRs were multiplexed in a full-length master library. Frequently, combinations of single VR regions led to the generation of the wild type sequences due to accumulation of non-target mutations in one or more VR regions. Additionally, said protocol often forced re-design of VRs and re-assembly of sub-libraries. As such, IUPAC-based VFS approach was time-consuming and laborious. Example 2 - 33 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 This Example describes a novel trimer / codon synthesis that utilized tri-nucleotides as building blocks [3]. This method allowed control of the mutational profile of AA composition and design of a library with only intended mutations. Furthermore, substituting AA residues complexity (explicit) for nucleotide complexity (implicit) allowed for utilizing the codons with the highest codon usage complexities in human cells which also positively impacts the yield of the libraries. In the trimer-based VFS approach, nucleotide fragments that included up to three VRs at once were synthetically generated. To verify the applicability of the trimer / codon synthesis for accelerated library assembly, DNA from packaged AAV sub-library A that included VR IV, V and VI were isolated and Sanger sequenced. This library was built by direct overlapping of trimer synthesized fragments of VR IV, V, VI without individual VRs’ pre-assembly. The sequencing of pooled encapsidated viral DNA from sub-library A validated significant variability in all designed VR regions (FIG. 7). Similarly, using trimer synthesis, sub-library B that included VR-VII and VR-VIII was produced. For the design of sub-libraries A and B, the following stipulation was used: the total complexity of each sub-library should not exceed a practical limitation of the down-stream plasmid library production (~5E+9 clones). Subsequently, DNA isolated from sub-libraries A and B were multiplexed in a full-length master library – AAV-rh74-AB library. To validate the variability of capsid composition of the library, capsid regions of the sub- library A and selected sequenced several distinct clones (65 clones) were sub-cloned. Among the sequenced clones, the same sequence were not detected twice and the mutation profiles displayed wide distributions: from 11 to 33 substitutions (average is 23.4±5.5) out of 64 theoretical mutation insertion positions in parental AAVrh74 serotype (FIG. 8). The phylogenetic tree representation based on point accepted mutation (PAM) clustering of sequenced AAV-rh74-AB library capsids vs. parental AAV-rh74 built by Multalin software [6] is shown in FIG. 9. The final full length AAVrh74 based library demonstrated an exceptional virus yield (>10E+13 AAV vector genomes / mL, an average of ten-fold increase compared to IUPAC-encoded libraries). The Weblogo-built AA sequence diversities (analyzed for only 58 sequenced capsid variants, with shorter / longer variants excluded from analysis) isolated from AAV-rh74-AB library are shown in FIG. 10. Thus, utilizing the trimer VSF approach allowed generation of full length AAVrh74 library with inclusion VR domains IV, V, VI, VII, VIII in a short period of time featuring the intended explicit complexities across the whole degenerate length of the scaffold capsid genome. - 34 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 As a non-limiting example of an application of the method, additional modifications of the AAV-rh74-AB library were assembled that included VR1 from AAV1: AAV-rh74-AB1 in order to minimize potential intra-liver distribution of AAV rh74 based library. Thus, library AAV-rh74-AB1 contained variable regions IV, V, VI, VII and VIII from AAVrh74 with multiple mutations based on VFS approach and stable VR1 from AAV1. After assembly of AAV-rh74-AB library, AB fragment (from assembled virus) were PCR-amplified and combined with VRI from AAV1 (FIGs. 11A-11B). Example 3 This Example describes an example of a positive / negative selection method for AAV library screening using a supervised directed evolution approach. Voltage-gated potassium (Kv) channels facilitate dynamic cellular repolarization during an action potential, opening in response to membrane depolarization to facilitate K+ efflux. In mammals, this hetero-multimeric complex is required for rhythmic beating of the heart. Moreover, targeting Kv channel will allow AAV-mediated cardiomyocytes-specific gene delivery. Sequential positive / negative selection in established human cell lines of different lineages, and expressing targeted membrane protein (e.g., hetero-multimers) resulted in isolating a capsid variant utilizing the protein as a heretofore unknown receptor. The library was used to isolate variants targeting a hetero-multimeric complex of KCNQ1 (Potassium voltage-gated channel subfamily Q member 1), KCNE1 (Potassium voltage-gated channel subfamily E regulatory subunit 1), and CALM1 (Calmodulin 1). Four sequential rounds of positive / negative selections were conducted using the following established human cell lines: HuH7 (hepatocarcinoma), AC-16 (cardiomyocytes), HEK 293 (embryonic kidney), and HeLa (cervix) using AAV-rh74-AB library (FIG. 12). After Round 4, 16 random clones were sequenced, and their respective amino-acid sequences were aligned to the parent scaffold AAV-rh74 (FIG. 13). Four clones were selected for further analyses (FIG. 14). Two clones (1-3 and 2-4) were identical across the sequenced regions, while clones 6-4 and 11-3 were significantly different in VR-I, VR-IV, and VR-V. Clones 1-3, 6-4, and 11-3 were vectorized, the respective AAV-Antares vectors were packaged, and tested in HeLa cells expressing KCNQ1, KCNE1, and CALM1, side by-side with parental AAV-rh74 (MOI of 10e4 vg / cell). While AAV-rh74, and clone 11-3 failed to transduce HeLa cells by any appreciable measure, clones 1-3, and 6-4 transduced cells at a significant rate. - 35 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 References For Examples 1-3 1. Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis. 2009 Feb 1;199(3):381-90. 2. https: / / patentscope.wipo.int / search / en / detail.jsf?docId=WO2016025339 3. Virnekäs B, Ge L, Plückthun A, Schneider KC, Wellnhofer G, Moroney SE. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 1994 Dec 25;22(25):5600-7. 4. Johnson AD. An extended IUPAC nomenclature code for polymorphic nucleic acids. Bioinformatics. 2010 May 15;26(10):1386-9. 5. Marsic D, Govindasamy L, Currlin S, Markusic DM, Tseng YS, Herzog RW, Agbandje- McKenna M, Zolotukhin S. Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants. Mol Ther. 2014 Nov;22(11):1900-9. 6. F. Corpet. Multiple sequence alignment with hierarchical clustering. 1988, Nucl. Acids Res., 16 (22), 10881-10890. EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, - 36 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element - 37 - 12443610.1 UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”. - 38 - 12443610.1

Claims

UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 CLAIMS What is claimed is:

1. A method comprising: (a) identifying variant amino acid positions of interest relative to a parent sequence; (b) designing engineered nucleic acid sequences encoding protein variants of the parent sequence, wherein the engineered nucleic acid sequence comprises either a wild-type codon or a heterologous codon substituted for the wild-type codon, relative to the parent sequence, corresponding to each variant amino acid position of interest identified in step (a); and (c) assembling a nucleic acid library comprising one or more engineered nucleic acids, wherein assembly comprises linking trimer nucleotide fragments as building blocks to produce one or more engineered nucleic acids comprising the engineered nucleic acid sequences designed in step (b).

2. The method of claim 1, further comprising cloning the library into a plurality of vectors, thereby producing a vector library.

3. The method of claim 1 or 2, further comprising sequencing the library.

4. The method of claim 2 or 3, further comprising replicating the vector library in a cell population.

5. The method of any one of claims 2-4, further comprising transfecting the vector library into host cells and thereby producing a viral particle library, optionally wherein transfecting further comprises contacting host cells with a helper nucleic acid.

6. The method of claim 5, wherein the viral particles comprise adeno-associated virus (AAV) particles.

7. The method of any one of claims 1-6, wherein the parent sequence comprises or encodes viral surface proteins. - 39 - 12443610.1UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 8. The method of any one of claims 1-7, wherein the parent sequence comprises or encodes adeno-associated virus (AAV) capsid proteins.

9. The method of claim 8, wherein the AAV capsid proteins comprise at least one variable region (VR) of AAV capsid proteins.

10. The method of claim 8 or 9, wherein one or more of the variant amino acid positions of interest are in a variable region (VR) of an AAV capsid protein.

11. The method of claim 9 or 10, wherein the AAV capsid proteins are of a mammalian serotype.

12. The method of claim 11, wherein (a) the serotype of the parent sequence is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, or AAVrh.74; and (b) the variant amino acid position is found in at least one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, or AAVrh.74, wherein the serotypes of (a) and (b) are different.

13. A protein variant produced by the method of any one of claims 1-12, or a nucleic acid encoding said protein variant.

14. The protein variant, of claim 13, wherein the protein variant is an engineered viral capsid protein, or a nucleic acid encoding said engineered viral capsid protein.

15. The protein variant, of claim 14, wherein the engineered viral capsid protein is an engineered AAV capsid protein, or a nucleic acid encoding said engineered AAV capsid protein.

16. The protein variant of claim 15, wherein the engineered AAV capsid protein comprises at least one of clones 1-3, 2-4, 6-4, or 11-3, or a nucleic acid encoding at least one of said clones.

17. A cell, or cell population thereof, comprising the protein variant, or nucleic acid encoding said protein variant, of any one of claims 13-16. - 40 - 12443610.1UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 18. A library produced by the method of any one of claims 1-12.

19. A cell population comprising the library produced by the method of any one of claims 1- 12.

20. The cell or cell population of claim 17 or 19, wherein the cells are mammalian cells.

21. The mammalian cells of claim 20, wherein the mammalian cells are human cells.

22. A method of screening a viral particle library comprising: (a) providing a viral particle library comprising different proteins of interest; (b) contacting the viral particle library with a first host cell population prepared from a first host cell lineage, wherein the first host cell population comprises a target protein on the surface of the cells to which the proteins of interest putatively bind, optionally wherein contacting further comprises contacting the first host cell population with a helper nucleic acid; (c) obtaining viral particles that transduce the first host cell population; (d) contacting the viral particles obtained in step (c) with a second host cell population prepared from the first host cell lineage, wherein the second host cell population does not comprise the target protein; (e) obtaining viral particles that do not bind to second host cell population; and (f) optionally repeating steps (a)-(e) one or more times with first and second populations prepared from a host cell lineage different from the first host cell lineage, wherein each repetition of steps (a)-(e) does not use host cells of the same lineage more than once.

23. The method of claim 22, wherein the viral particle library is produced by the method of any one of claims 5-12 and / or at least one of the proteins of interest comprise the protein variant of any one of claims 13-16.

24. The method of claim 22or 23, wherein the host cells are mammalian.

25. The method of claim 24, wherein the host cells are human.

26. A viral particle isolated by the method of any one of claims 22-25. - 41 - 12443610.1UF Ref. No.: UFTT19069 WGS Ref. No.: U1196.70108WO00 27. The viral particle of claim 26, wherein the viral particle is an AAV particle.

28. A cell, or cell population thereof, transduced with the viral particle of claim 26 or 27.

29. The cell of claim 28, wherein cell is a mammalian cell.

30. The mammalian cell of claim 29, wherein the cell is a human cell.

31. An engineered AAV capsid protein comprising any one of clones 1-3, 2-4, 6-4, or 11-3, or a nucleic acid encoding any one of said clones.

32. An rAAV particle comprising: (a) a heterologous nucleic acid flanked by AAV inverted terminal repeats (ITRs); and (b) at least one AAV capsid protein comprising an engineered AAV capsid protein produced by the method of any one of claims 1-12 or screened by the method of any one of claims 22-25.

33. An rAAV particle comprising (a) a heterologous nucleic acid flanked by AAV ITRs; and (b) at least one AAV capsid protein, wherein the at least one AAV capsid protein comprises the engineered AAV capsid protein of claim 31.

34. The rAAV particle of claim 32 or 33, wherein the heterologous nucleic acid encodes a therapeutic RNA or a therapeutic protein.

35. A method comprising administering the rAAV particle of any one of claims 32-34 to a subject.

36. The method of claim 35, wherein the subject has, is suspected of having, or is at risk of developing, a disease or disorder.

37. The method of claim 36, wherein the heterologous nucleic acid encodes a therapeutic RNA or a therapeutic protein which is therapeutic for the disease or disorder. - 42 - 12443610.1

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