WO2025090858A1 - Methods for identifying aav capsid variants with desired characteristics - Google Patents

Abstract

The present disclosure provides methods and compositions useful for screening AAV capsid libraries for desired characteristics, such as cell-type specific tropisms. Each AAV capsid library disclosed herein includes a known promoter (e.g., a ubiquitous or cell-type specific promoter) and barcode made up of silent mutations, allowing multiple AAV capsid variant libraries to be pooled and administered in a single experiment.

Description

METHODS FOR IDENTIFYING AAV CAPSID VARIANTS WITH DESIRED CHARACTERISTICS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Serial No. 63/546,120, filed October 27, 2023, the entirety of which is incorporated herein by reference.

BACKGROUND

[0002] Adeno-associated virus (AAV) vectors have become a leading platform for in vivo delivery of gene therapies for a diverse array of human diseases. AAVs can be engineered to deliver a heterologous nucleic acid of interest (e.g., a gene encoding a therapeutic protein, an antisense nucleic acid molecule, ribozyme, miRNA, siRNA, or a nucleic acid encoding a CRISPR/Cas system) to specific cells of a patient. Cell-type specificity of an AAV is determined, in part, by the AAV capsid protein sequence. Although many approaches have been used to identify novel AAV capsids with cell-type specificity, existing methods suffer from multiple disadvantages. Thus, to enable development of safer and more effective gene therapies, there remains a need for robust methods to identify novel AAV variants with cell-type specificity and other desired characteristics.

SUMMARY

[0003] The present disclosure encompasses, inter alia, a method of rapidly, efficiently, and precisely identifying AAV capsid variants with desired characteristics, including but not limited to cell-type specific and/or tissue- specific tropisms. The present disclosure is based, in part, on the discovery that silent mutations incorporated into the genomes of an AAV capsid valiant library (e.g., within a nucleic acid sequence encoding a capsid) may serve as a barcode, which when associated with a gene regulatory element (e.g., a promoter), allows for the simultaneous administration and screening of two or more AAV capsid variant libraries for celltype specific and/or tissue- specific tropisms.

[0004] In one aspect, the disclosure provides a method of identifying an adeno- associatcd virus (AAV) capsid variant with a desired characteristic, comprising:

(a) transducing a cell population or tissue with a plurality of AAV capsid variant libraries, wherein each member library within the plurality of AAV capsid variant libraries comprises a plurality of AAV capsid variants, and wherein each AAV capsid variant within a member library comprises a nucleic acid sequence comprising:

(i) a gene regulatory element chosen from:

(1) a cell-type specific and/or tissue-specific gene regulatory element, or

(2) a ubiquitous gene regulatory element; and

(ii) a nucleic acid sequence encoding a variant capsid protein and comprising a barcode, wherein the gene regulatory element is operatively linked to the nucleic acid sequence encoding the variant capsid protein and the gene regulatory element and the barcode are the same for each AAV capsid variant within a member library, wherein each member library has a different barcode from each other member library within the plurality of AAV capsid variant libraries, and wherein the barcode of one or more member libraries comprises one or more silent mutations;

(b) recovering AAV capsid variants from the cell population or tissue; and

(c) identifying an AAV capsid variant with the desired characteristic.

[0005] In certain embodiments of the method, at least two member libraries have a different gene regulatory element from each other member library within the plurality of AAV capsid variant libraries.

[0006] In some embodiments of the method, each member library has a different gene regulatory element from each other member library within the plurality of AAV capsid variant libraries.

[0007] In some embodiments of the method, a first set of at least two member libraries arc paired in that they have the same gene regulatory clement and different barcodes.

[0008] In some embodiments of the method, a second set of at least two member libraries are paired in that they have the same gene regulatory element and different barcodes, wherein the gene regulatory element of the second set is different from the gene regulatory element of the first set.

[0009] In some embodiments of the method, a barcode of one or more member librar ies comprises one or more silent mutations in at least one codon of the sequence encoding the variant capsid protein.

[0010] In some embodiments of the method, at least one codon encodes leucine, serine, and/or arginine.

[0011] In some embodiments of the method, each AAV capsid variant comprises one or more additional barcodes within the sequence encoding the variant capsid protein.

[0012] In some embodiments of the method, one or more additional barcodes comprise one or more silent mutations, wherein each member library has a different one or more additional barcodes from each other member library within the plurality of AAV capsid variant libraries.

[0013] In some embodiments of the method, a barcode or one or more additional barcodes is located within 2208 nucleotides of a sequence encoding a hypcrvariablc region and/or surface-exposed loop of the variant capsid protein.

[0014] In some embodiments of the method, a barcode and/or one or more additional barcodes comprises one or more nucleic acids.

[0015] In some embodiments of the method, a barcode and/or one or more additional barcodes comprises a hamming distance of one or more nucleic acids.

[0016] In some embodiments of the method, a barcode and/or one or more additional barcodes comprises nine nucleic acids and a minimum hamming distance of five nucleic acids.

[0017] In some embodiments of the method, a barcode and/or one more additional barcodes is encoded by the same region of the AAV capsid variant within each member library.

[0018] In some embodiments of the method, at least one member library within the plurality of AAV capsid variant libraries comprises a ubiquitous gene regulatory element.

[0019] In some embodiments of the method, at least one ubiquitous gene regulatory element is or comprises hybrid chicken beta actin (CBh) promoter, CAG promoter, simian virus 40 (SV40) promoter, cytomegalovirus (CMV) promoter, ubiquitin C (UBC) promoter, elongation factor- 1 alpha (EFl A) promoter, phosphoglycerate kinase 1 (PGK1) promoter, phosphoglycerate kinase (PGK) promoter, human beta-actin promoter, beta-actin long (BActL) promote, chimeric CMV-chicken beta-actin promoter (CBA) promoter, TRE promoter, U6 promoter, Hl promoter, 7SK promoter, or GUSb promoter, or any non-naturally occurring ubiquitous gene regulatory element.

[0020] In some embodiments of the method, a cell-type specific and/or tissue-specific gene regulatory element is or comprises a neuron- specific promoter, neuron subtype-specific promoter, microglia and/or macrophage-specific promoter, ionocyte- specific promoter, astrocyte-specific promoter, oligodendrocyte-specific promoter, muscle- specific promoter, retina- specific promoter, photoreceptor- specific promoter, rod- specific promoter, cone- specific promoter, heart-specific promoter, lung-specific promoter, liver- specific promoter, or kidneyspecific promoter.

[0021] In some embodiments of the method, a cell-type specific and/or tissue-specific gene regulatory element is a human synapsin I (hSynl) promoter, glial fibrillary acidic protein (GFAP) promoter, MHCK7 promoter, calcium/calmodulin-dependent protein kinase II (CaMKII) promoter, cerebellar Purkinje cell-specific L7-6 promoter, synapsin I with a minimal CMV sequence (Synl-minCMV) promoter, homeobox Dlx5/6, glutamate receptor 1 (GluRl) promoter, preprotachykinin 1 (Tael) promoter, dopaminergic receptor 1 (Drdla) promoter, tubulin alpha I promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor beta chain promoter, glutamic acid decarboxylase (GAD67) promoter, photoreceptor- specific rhodopsin kinase (RK) promoter, L-opsin promoter, human GRM6 gene-derived (hGRM6)

promoter, alpha-subunit of cone transducin (TaC) promoter, CK8 promoter, desmin promoter, tMCK promoter, dMCK promoter, CK6, promoter, SPc5-12 promoter, D-sitc binding protein (DBP) promoter, CCAAT enhancer binding protein a or P (C/EBP) promoter; hepatocyte nuclear factor (HNF) promoter, liver-specific IL-6-dependent DNA binding protein (IL-6DBP), or any non-naturally occurring cell-type specific and/or tissue-specific gene regulatory element.

[0022] In some embodiments of the method, each member library comprises one or more additional gene regulatory elements that are operatively linked to the sequence encoding the variant capsid protein.

[0023] In some embodiments of the method, AAV capsid variants within each member library differ from each other based on the presence of one or more of the following modifications within the variant capsid protein:

(i) a peptide inserted into a hypervariable region and/or surface-exposed loop of the variant capsid protein,

(ii) a point mutation, and

(iii) a deletion.

[0024] In some embodiments of the method, AAV capsid variants within each member library each include a different peptide inserted into a hypervariable and/or surface-exposed loop within the variant capsid protein.

[0025] In some embodiments of the method, a plurality of AAV capsid variant libraries comprises three or more AAV capsid variant libraries.

[0026] In some embodiments of the method, a recovering step comprises isolating a desired cell-type from the cell population or a desired tissue-type from the tissue.

[0027] In some embodiments of the method, an identifying step comprises:

(i) isolating mRNA from the cell population or tissue,

(ii) converting the mRNA to cDNA,

(iii) amplifying the cDNA to generate cDNA amplicons, and

(iv) sequencing the cDNA amplicons.

[0028] In some embodiments of the method, a sequencing step (iv) comprises identifying the sequence of both the barcode and the modification.

[0029] In some embodiments of the method, a converting step (ii) and/or amplifying step (iii) comprises using a primer that binds specifically to the barcode or to a nucleic acid sequence upstream or downstream of the barcode.

[0030] In some embodiments of the method, sequencing of the cDNA amplicons comprises next-generation sequencing.

[0031] In some embodiments of the method, cDNA amplicons are each less than about 500 base pairs in length.

[0032] In some embodiments of the method, sequencing encompasses a sequence encoding a hypervariable and/or surface-exposed loop of the variant capsid protein.

[0033] In some embodiments of the method, at least one desired characteristic is a celltype specific and/or tissue- specific tropism.

[0034] In some embodiments of the method, at least one desired characteristic is the absence of a cell-type specific and/or tissue-specific tropism.

[0035] In some embodiments of the method, AAV capsid variants are AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAVrh.74, or AAV12 capsid variants.

[0036] In some embodiments of the method, a cell population or tissue is transduced in vitro.

[0037] In some embodiments of the method, a cell population or tissue is transduced in vivo.

[0038] In some embodiments of the method, a plurality of AAV capsid variant libraries are pooled and transduced substantially simultaneously.

[0039] In some embodiments of the method, a transducing step comprises administration to a mammal.

[0040] In some embodiments of the method, a mammal is a non-human primate.

[0041] In another aspect, the disclosure provides a screening system comprising a plurality of AAV capsid variant libraries, wherein each member library within the plurality of AAV capsid variant libraries comprises a plurality of AAV capsid variants, and wherein each AAV capsid variant within a member library comprises a nucleic acid sequence comprising:

(i) a gene regulatory element chosen from:

(1) a cell-type specific and/or tissue-specific gene regulatory element, or

(2) a ubiquitous gene regulatory element; and

(ii) a sequence encoding a variant capsid protein and comprising a barcode, wherein the gene regulatory element is operatively linked to the sequence encoding the variant capsid protein and the gene regulatory element and the barcode are the same for each AAV capsid variant within a member library, wherein each member library has a different barcode from each other member library within the plurality of AAV capsid variant libraries, and wherein the barcode of one or more member libraries comprises one or more silent mutations.

[0042] Other features, objects, and advantages of the present disclosure are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the disclosure will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWING

[0043] FIG. 1 illustrates an exemplary genomic configuration of an AAV capsid variant within a member library of a plurality of AAV capsid variant libraries as described herein. Sequence elements shown include inverted tandem repeats (ITRs), a first gene regulatory

element which may be ubiquitous or cell-type specific and/or tissue-specific (e.g., a cell-type specific and/or tissue-specific promoter), a second gene regulatory clement, used to drive expression of a capsid protein during AAV library production (e.g., AAV2-derived p40 or AAV5-derived p41 promoter), a variant capsid protein including a region encoding modifications within the variant capsid protein (e.g., comprising an insertion site for a peptide display library) and a barcode immediately upstream thereof, as well as a polyadenylation signal and second ITR.

[0044] FIG. 2 illustrates an exemplary genomic configuration of two AAV capsid variant member libraries, as described herein. A first member library, denoted as “Promoter construct 1” comprises the ubiquitous CBh promoter. A second member library, denoted as “Promoter construct 2” comprises the neuron- specific hSynl promoter. For both AAV capsid variant member libraries, the DNA sequence of a promoter barcode is underlined and the sequence encoding modifications within a variant capsid protein (7mer peptide insert) is shown in bold. A promoter barcode is used to identify the promoter that initiated transcription of the mRNA strand, and consists of silent mutations that do not alter the amino acid sequence of a capsid variant. For both AAV capsid variant member libraries, the barcodes have a Hamming distance of 8 to enable robust error detection and correction during sequencing.

[0045] FIG. 3 illustrates an exemplary amplicon recovery strategy, as described herein, used to capture both barcode and modifications within a variant capsid protein. Following recovery of mRNA and conversion to cDNA, transcripts may be analyzed by sequencing. Prior to sequencing, forward and reverse primers are designed to bind upstream of a promoter barcode and downstream of modifications within a capsid variant to be evaluated, respectively. Resulting amplicons are then used for preparation of next-generation sequencing libraries and sequenced by any number of methods or platforms known in the art, including, e.g., Illumina high-depth short read sequencing or Sanger sequencing.

[0046] FIG. 4 illustrates two exemplary amplicon recovery strategies, as described herein, to obtain either total amplicons of all member libraries, or only those transcripts expressed from a specific promoter (i.e., a specific AAV capsid variant member library). To

obtain a nucleic acid sequence encoding modifications within a capsid variant from all member libraries, a forward and reverse primer (denoted as “F primer 1” and “R primer”, respectively) are used that are complementary to conserved nucleic acid sequences shared across all member libraries. To obtain transcript sequences expressed by a single promoter (i.e., a specific AAV capsid variant member library), a forward primer (denoted as “F primer”) may be designed to be complementary to one or more nucleotides that correspond to a barcode.

[0047] FIG. 5 illustrates an exemplary evaluation of AAV capsid variant performance from Example 1, herein. For both CBh (ubiquitous promoter) or MHCK7 (muscle- specific promoter) AAV capsid variant member libraries, AAV capsid variant performance is shown as log2 fold-change in expression (i.e., transcript abundance) of each AAV capsid variant normalized by AAV9 input library (i.e., Log2(AAV capsid variant abundance post-transduction / AAV capsid variant abundance pre-transduction, in AAV9 input library). FC, fold-change.

[0048] FIG. 6 illustrates an exemplary configuration of each AAV capsid variant member library as described in Example 2 herein, to identify AAV capsid variants with tropism specific to central nervous system (CNS) cells (top) and retina cells (bottom). To identify AAV capsid variants with CNS-specific tropism, methods described herein will include AAV capsid variant member libraries with a ubiquitous CBh promoter, neuron- specific hSynl promoter and astrocyte-specific GFAP promoter, each with a unique barcode. To identify AAV capsid variants with retina-specific tropism, methods described herein will include AAV capsid variant member libraries with a ubiquitous CBh promoter, photoreceptor- specific hGRKl promoter and RPE-specific VMD2 promoter, each with a unique barcode.

DEFINITIONS

[0049] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the disclosure and to provide additional detail regarding its practice are hereby incorporated by reference.

[0050] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; and (iii) where ranges are provided, endpoints are included.

[0051] About or approximately’. As used herein, the terms “about” or “approximately” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0052] Adeno-associated virus (AAV): As used herein, the terms “Adeno-associated virus” and “AAV” refer to a virus of family Parvoviridae and genus Dependoparvovirus. AAV is a small replication-defective, non-enveloped virus. AAV may include, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3 A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAV serotype rhlO, AAV serotype rh74, AAV from the HSC 1-17 series, AAV from the CBr, CLv or CLg series, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any variant of any of the foregoing. AAV may also include engineered or chimeric versions of a wild-type AAV that include one or more insertions, deletions and/or substitutions within one or more Cap polypeptides that affect one or more properties of the wildtype AAV serotype, including without limitation tropism and evasion of neutralizing antibodies (e.g., AAV-DJ, AAV-PHP.B, AAV-PHP.N, AAV.CAP-B1 to AAV.CAP-B25, and variants thereof). Wild-type AAV is replication deficient and requires co-infection of cells by a helper virus (e.g., adenovirus, herpes, or vaccinia virus) or supplementation of helper viral genes in order to replicate.

[0053] AAV Capsid Variant: As used herein, the term “AAV capsid variant” refers to an individual AAV virion with one or more modifications (e.g., one or more changes in amino acid sequence) in a capsid protein relative to a capsid protein of a reference AAV virion, such as a wild-type AAV. Such modifications within a variant capsid protein can comprise one or more of: (1 ) a peptide inserted into a hypervariable region and/or surface-exposed loop of a variant

capsid protein, (2) a point mutation, and (3) a deletion. Such modifications within a variant capsid protein can be located within an engineered site. In some embodiments, AAV capsid variant refers to an individual AAV virion within an AAV peptide display library.

[0054] Barcode: As used herein, the term “barcode” refers to a nucleic acid sequence within genomes of all AAV capsid variants of an AAV capsid variant member library. In some embodiments, a barcode comprises silent mutations. In some embodiments, a barcode may be a wild-type AAV nucleic acid sequence. In some embodiments, a barcode is associated with a gene regulatory element in an AAV capsid variant member library. In some embodiments, a barcode is located within a nucleic acid sequence encoding an AAV capsid. In some embodiments, a barcode is located in close proximity to one or more modifications within a capsid variant.

[0055] Cell-type: As used herein, the term “cell-type” refers to a cell or population of cells with distinct form, and/or biology. Cell-type may be identified using various characteristics, including, for example: gene expression profile, epigenetic profile, non-coding RNA profile, protein expression profile, cell surface markers, differentiation potential, proliferative capacity, response to stimuli or signals, anatomical location, morphology, staining profiles, and/or timing of appearance during development, and/or any combination of the foregoing. In some embodiments, a cell-type is defined based on a specific characteristic or combination of characteristics. For example, in some embodiments, a cell-type is defined based on expression of a specific gene or combination of genes. In some embodiments, a cell-type can be defined by tissue from which it was sourced or originated, e.g., connective tissue, muscular tissue, nervous tissue, or epithelial tissue.

[0056] Comprising: A method described herein as “comprising” or “including” one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any method described as “comprising” (or which “comprises”) one or more named elements or steps also describes the corresponding, more limited method “consisting essentially of” (or which “consists essentially of”) the same named

elements or steps, meaning that the method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the method. It is also understood that any method described herein as “comprising” or “consisting essentially of’ one or more named elements or steps also describes the corresponding, more limited, and closed-ended method “consisting of’ (or “consists of’) the named elements or steps to the exclusion of any other unnamed element or step. In any method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0057] Desired Characteristic: As used herein, the term “desired characteristic” refers to any number of identifiable features of an AAV capsid variant using the methods described herein, including, e.g., transduction in target tissues/cells (i.e., tropism), and/or reduced uptake in non-target tissues/cells, and/or increased immune evasion of circulating neutralizing factors, and/or AAV variant mRNA expression and/or stability.

[0058] Gene Regulatory Element: As used herein, the term “gene regulatory element” refers to a nucleic acid sequence that controls or affects expression of neighboring genes. In some embodiments, the gene regulatory element is a promoter. In some embodiments, the gene regulatory element is an enhancer. In some embodiments, the gene regulatory element is a silencer.

[0059] Hamming Distance: As used herein, the term “Hamming distance” refers to the number of positions at which two nucleic acid sequences of equal length differ (i.e., the minimum number of substitutions required to change one nucleic acid sequence into a different nucleic acid sequence). For example, a Hamming distance between sequences TTTGGGTTT and TTTAAATTT is 3.

[0060] Member Library: As used herein, the term “member library” is synonymous with “AAV member library” or “AAV capsid library” and refers to an individual AAV capsid variant library within a plurality of AAV capsid variant libraries. In some embodiments, each AAV member library comprises at least one a barcode that is unique to the AAV member library.

[0061] Nucleic acid: The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and singlestranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as “intemucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. In accordance with the methods and compositions described herein, in some embodiments, an RNA comprises a short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR/Cas guide RNA, microRNA (miRNA), and/or a precursor thereof.

[0062] Paired Member Libraries: As used herein, the term “paired member libraries” refers to any two AAV capsid variant member libraries comprising a shared gene regulatory element (e.g., a promoter) and two different barcodes.

[0063] Silent Mutations: As used herein, the term “silent mutations” refers to a change in a nucleic acid sequence that does not result in a change to the amino acid sequence encoded by that nucleic acid sequence relative to the nucleic acid sequence without that change.

[0064] Substantially: As used herein, the term “substantially” refers to a qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. The term “substantially” is therefore used herein to capture a potential lack of absoluteness inherent in many biological and/or chemical effects.

[0065] Variant: As used herein in the context of molecules, e.g., nucleic acids or proteins, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to one another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently linked components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological

activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a valiant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 insertion or deletion, and, in some embodiments, comprises no insertions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 insertions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0066] Vector: As used herein, the term “vector” refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into a cell. By way of non-limiting example, one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.

Another type of vector is a viral vector, wherein additional DNA segments may be packaged into

a viral capsid and can be transferred into another cell and/or organism. Certain vectors are capable of autonomous replication in a host cell into which they arc introduced (c.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors may be referred to herein as “expression vectors.”

[0067] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference in its entirety.

DETAILED DESCRIPTION

[0068] The present disclosure is based, in part, on the discovery that a plurality of adeno- associated virus (AAV) capsid variant libraries, comprised of member libraries, can be used (e.g., substantially simultaneously) to transduce a cell population or tissue, or incubated with an isolated protein (e.g., a receptor protein), and then screened for a desired characteristic in a single experiment by constructing each member library to include at least one unique barcode associated with at least one gene regulatory element (e.g., a promoter as described herein).

[0069] AAV-based vectors are valuable tools for clinical payload delivery (e.g., a therapeutic protein, an antisense nucleic acid molecule, a ribozyme, a miRNA, an siRNA, or a nucleic acid encoding a CRISPR/Cas system) useful in the treatment of a variety of human diseases. Although naturally-derived AAV vector capsids have demonstrated clinical efficacy

across a variety of disease states, there remains a need to identify novel AAV capsids with specific desired characteristics to enable development of safer and more effective gene therapies. Such desired characteristics may include, but are not limited to, enhancement of one or more of: transduction efficiency in a target cell population or tissue (i.e., tropism), reduced uptake in a non-target cell population or tissue, and increased immune evasion of circulating neutralizing factors.

[0070] Towards these aims, substantial efforts have been made to discover and engineer novel AAV capsid variants. Many approaches have been used to identify novel AAV capsid variants with desired characteristics, including, but not limited to, isolation of novel variants from animal tissue, insertion of targeting ligands on a capsid surface, and directed evolution approaches to select and/or screen libraries of AAV capsid mutants obtained by error-prone PCR, capsid shuffling, and insertion of short random peptide sequences into tolerant regions of AAV capsids.

[0071] Directed evolution is a popular approach to AAV capsid engineering because this method does not require prior knowledge or design of AAV capsid variants, as capsid variants with a desired characteristic are recovered following iterative rounds of selection in target cell populations, tissues, or against purified proteins (e.g., receptor proteins). In such directed evolution strategies, nucleotide sequences encoding AAV capsid proteins are commonly incorporated between the inverted terminal repeats (ITRs) such that each capsid packages its originating capsid nucleic acid sequence. Following transduction in target cell populations or tissues, the AAV-derived nucleic acid may be recovered by PCR or RT-PCR and incorporated into the next round library. This iterative process is repeated until a few enriched capsid variants emerge. At each round of selection, the recovered nucleic acid can also be analyzed by nextgeneration sequencing (NGS) technologies to evaluate capsid variant enrichment relative to an input library.

[0072] Although DNA-based recovery methods have been commonly used in the field, selection based on DNA alone does not effectively exclude AAV capsid variants that are transduction-deficient. For example, a capsid variant may be internalized into a cell, but fail to

achieve effective transduction. In addition, capsids may become trapped in interstitial space. As nucleic acid-bascd strategics typically use bulk tissue as input, DNA of these transductiondeficient capsid variants or interstitially-trapped capsid variants would still be captured, leading to noise during analysis. For that reason, selection strategies (termed herein as “transductionreadout”) based on recovery of RNA are more relevant for clinical translation, as the presence of capsid-derived RNA indicates that an originating capsid genome was delivered to a nucleus, underwent second strand synthesis, and became transcriptionally active.

[0073] Transduction readout-based directed evolution requires incorporating a promoter within an AAV genome that can drive capsid mRNA expression in vivo. Unique promoter properties, such as cell-type specific and/or tissue-specificity, can be leveraged to modulate selection pressure based on an intended target tissue- or cell-type. For directed evolution approaches, incorporation of a ubiquitous promoter (e.g., CAG) enables profiling of transduction across different tissues and organs. However, this approach is less than optimal to select for capsid variants with enhanced transduction in specific cell-types within tissues containing heterogeneous cell populations, such as the brain, which is comprised of neurons, astrocytes, oligodendrocytes, microglia, endothelial cells and various other cell populations. Furthermore, use of a ubiquitous promoter in this strategy may lead to enrichment of capsid variants with enhanced transduction in non-target cell populations. On the other hand, approaches that use cell type-specific promoters (e.g., human synapsin 1 promoter for neuronal-specific expression) may impart higher selection pressure for capsid variants that transduce target cell populations, but the ability to profile transduction in non-target cell populations is lost.

[0074] Until the present disclosure, there existed no methods or compositions that allowed for selection and/or screening of AAV capsid libraries with cell type or tissue resolution in a single experiment (e.g. in a single animal).. As described herein, we developed a novel directed evolution method, DEPICT (Directed Evolution Platform Incorporating Cell-type Tropism), and compositions useful therefor, to screen AAV capsid variant transduction with celltype resolution, in addition to selecting AAV capsid variants with desired cell-type specific and/or tissue-specific tropism. Such methods described herein achieve this goal, in part, by introducing promoter barcodes, which in some embodiments, comprise silent mutations, adjacent

to modifications within a variant capsid protein. Using methods and compositions described herein, a single capsid variant library can be inserted into AAV vectors, each with different promoters and a corresponding barcode, thereby creating multiple AAV capsid variant member libraries. This allows combining and administering multiple AAV capsid variant member libraries to a population of cells, after which mRNA transcripts resulting from each promoter can be sorted during data analysis using unique promoter barcodes located near a modification within a variant capsid protein.

AAV Capsid Variant Libraries

[0075] The present disclosure, among other things, provides compositions and methods for selecting, screening, and identifying AAV capsid variants with a desired characteristic. In some embodiments, the methods described herein comprise transducing a cell population or tissue, in vivo or in vitro, with a plurality of AAV capsid variant libraries comprising individual member libraries to identify one or more AAV capsid valiants with a specific tropism. In some embodiments, each member library comprises a plurality of AAV capsid variants, each of which comprise multiple genetic elements, including, e.g., one or more gene regulatory elements, a variant capsid protein, a barcode, a polyadenylation signal, and inverted tandem repeats (ITRs) (e.g., as shown in Figure 1). In some embodiments, an AAV capsid variant within a member library described herein comprises nucleic acid sequences corresponding to, from 5’ to 3’, a first ITR, a first gene regulatory element (e.g., a first promoter), a second gene regulatory element (e.g., a second promoter), a capsid protein (e.g., a variant capsid protein) comprising a barcode, a polyadenylation signal, and a second ITR. In some embodiments, a barcode is located within the protein coding sequence of the variant capsid protein. In some embodiments, a barcode is comprised of silent mutations.

[0076] In some embodiments, a first gene regulatory element and a barcode can be the same for each AAV capsid variant within a member library. In some embodiments, a second gene regulatory element and a barcode can be the same for each AAV capsid variant within a member library. In some embodiments, a first gene regulatory element comprises or is a cell-

type specific and/or tissue- specific gene regulatory element operatively linked to a nucleotide sequence encoding a variant capsid protein. In some embodiments, a first gene regulatory element comprises or is a ubiquitous gene regulatory element operatively linked to a nucleotide sequence encoding a variant capsid protein. In some embodiments, a second gene regulatory element (e.g., p40 or p41) is operatively linked to a nucleotide sequence encoding a variant capsid protein for AAV library production in a producer cell line.

Gene Regulatory Elements

[0077] In some embodiments, each AAV capsid variant within a member library described herein comprise at least two gene regulatory elements (e.g., promoters). In such embodiments, each AAV capsid variant within a member library described herein comprises at least one gene regulatory element and an additional gene regulatory element to drive expression of a variant capsid protein in a producer cell line for AAV library production (e.g., any gene regulatory element active in a producer cell, including but not limited to, p40 or p41). In some embodiments, each AAV capsid variant within a member library described herein comprises a cell-type specific and/or tissue-specific gene regulatory element. In some embodiments, each AAV capsid variant within a member library described herein comprise a ubiquitous gene regulatory element.

Cell-type specific and/or tissue-specific gene regulatory elements

[0078] In some embodiments, each AAV capsid variant within a member library comprises a cell-type specific and/or tissue-specific gene regulatory element (e.g., a promoter). In some embodiments, each AAV capsid variant within a member library comprises a non- naturally occurring cell-type specific and/or tissue-specific gene regulatory element. In some embodiments, a gene regulatory element is operatively linked to a nucleic acid sequence encoding a variant capsid protein.

[0079] Exemplary tissue- specific gene regulatory elements can include, but are not limited to, a neuron- specific promoter, astrocyte- specific promoter, oligodcndrocytc-spccific promoter, muscle- specific promoter, retina- specific promoter, photoreceptor- specific promoter, rod-specific promoter, cone-specific promoter, heart- specific promoter, lung-specific promoter, liver- specific promoter, and kidney- specific promoter.

[0080] Exemplary cell-type specific gene regulatory elements can include, but are not limited to, human synapsin 1 (hSynl) promoter, glial fibrillary acidic protein (GFAP) promoter, MHCK7 promoter, calcium/calmodulin-dependent protein kinase II (CaMKII) promoter, cerebellar Purkinje cell-specific L7-6 promoter, synapsin I with a minimal CMV sequence (Synl- minCMV) promoter, homeobox Dlx5/6, glutamate receptor 1 (GluRl) promoter, preprotachykinin 1 (Tael) promoter, dopaminergic receptor 1 (Drdla) promoter, tubulin alpha I promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor beta chain promoter, glutamic acid decarboxylase (GAD67) promoter, photoreceptor- specific rhodopsin kinase (RK) promoter, L-opsin promoter, human GRM6 gene-derived (hGRM6) promoter, alpha-subunit of cone transducin (TaC) promoter, CK8 promoter, desmin promoter, tMCK promoter, dMCK promoter, CK6, promoter, SPc5-12 promoter, D-site binding protein (DBP) promoter, CCAAT enhancer binding protein a or P (C/EBP) promoter; hepatocyte nuclear factor (HNF) promoter, and liver- specific IL-6-dependent DNA binding protein (IL-6DBP) promoter.

Ubiquitous gene regulatory elements

[0081] In some embodiments, each AAV capsid variant within a member library comprises a ubiquitous gene regulatory element (e.g., a promoter). In some embodiments, each AAV capsid variant within a member library comprises a non-naturally occurring ubiquitous gene regulatory element. Exemplary ubiquitous gene regulatory elements include, but are not limited to, hybrid chicken beta actin (CBh) promoter, CAG promoter, simian virus 40 (SV40) promoter, cytomegalovirus (CMV) promoter, ubiquitin C (UBC) promoter, elongation factor- 1 alpha (EF1A) promoter, phosphoglycerate kinase 1 (PGK1) promoter, phosphoglycerate kinase (PGK) promoter, human beta-actin promoter, beta-actin long (BActL) promote, chimeric CMV-

chicken beta-actin promoter (CBA) promoter, TRE promoter, U6 promoter, Hl promoter, 7SK promoter, or GUSb promoter.

AAV Capsid Variants

[0082] In some embodiments, each member library within a plurality of AAV capsid variant libraries comprises a plurality of AAV capsid variants. In some embodiments, all AAV capsid variants within a member library comprise at least one barcode unique to that member library, and differ in that all such AAV capsid variants comprise one or more different modifications within a variant capsid protein. In some embodiments, AAV capsid variants within a member library comprise at least two barcodes.

Modifications within a variant capsid protein

[0083] In some embodiments, each AAV capsid variant library as described herein comprises a plurality of capsid variants, each differing in that all such AAV variants comprise different modifications within the variant capsid protein. In some embodiments, modifications within a variant capsid protein comprise one or more non-native amino acid substitutions, deletions, and/or insertions, or combinations thereof. Methods of producing AAV capsid variants, each with differing modifications, as described herein, arc known in the art (sec, e.g., U.S. Publication No. US20130310443, which is hereby incorporated by reference in its entirety).

[0084] In some embodiments, modifications within a variant capsid protein comprise a modified nucleotide sequence encoding a V3 capsid protein. In some embodiments, modifications within a variant capsid protein comprise at least one non-native amino acid substitution in a capsid protein relative to a wild-type capsid protein. In some embodiments, modifications within a variant capsid protein comprise at least one non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid (e.g., a surface- exposed tyrosine) in a wild-type capsid protein. In some embodiments, modifications within a variant capsid protein comprise a non-tyrosine amino acid (e.g., a phenylalanine) at a position

that corresponds to a surface-exposed tyrosine amino acid in a wild-type capsid protein, a non- thrconinc amino acid (c.g., a valine) at a position that corresponds to a surface-exposed threonine amino acid in a wild-type capsid protein, a non-lysine amino acid (e.g., a glutamic acid) at a position that corresponds to a surface-exposed lysine amino acid in a wild-type capsid protein, a non-serine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed serine amino acid in a wild-type capsid protein, or a combination thereof. In some embodiments, modifications within a variant capsid protein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, modifications within a variant capsid protein comprise at least one non-native amino acid substitution and at least one peptide insertion. In some embodiments, modifications within a variant capsid protein comprise at least one nonnative amino acid substitution and at least one peptide insertion within a variable region of a capsid.

[0085] In some embodiments, modifications within a variant capsid protein comprise one or more peptide insertions within a capsid. In some embodiments, modifications within a variant capsid protein comprise one or more peptide insertions within one or more capsid variable regions (VR), including e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR- VIII and VR-IX. In some embodiments, modifications within a variant capsid protein comprise one or more peptide insertions within the following regions of VP1: (a) 262-269 (VR-I) of AAV9 or a corresponding position in a capsid protein of another serotype; (b) 327-332 (VR-II) of AAV9 or a corresponding position in a capsid protein of another serotype; (c) 382-386 (VR- III) of AAV9 or a corresponding position in a capsid protein of another serotype; (d) 452-460 (VR-IV) of AAV9 or a corresponding position in a capsid protein of another serotype; (e) 488- 505 (VR-V) of AAV9 or a corresponding position in a capsid protein of another serotype; (f) 527-539 (VR-VI) of AAV9 or a corresponding position in a capsid protein of another serotype; (g) 545-558 (VR-VII) of AAV9 or a corresponding position in a capsid protein of another serotype; (h) 581-593 (VR-V111) of AAV9 or a corresponding position in a capsid protein of another serotype; (i) 704-714 (VR-IX) of AAV9 or a corresponding position in a capsid protein of another serotype; or (j) any combination or all of (a)-(h). In some embodiments, modifications within a variant capsid protein comprise one or more peptide insertions within the

following regions of VP1 : (a) 452-460 (VR-IV) of AAV9 or a corresponding position in a capsid protein of another serotype; (b) 581-593 (VR-VIII) of AAV9 or a corresponding position in a capsid protein of another serotype; or (c) a combination of (a) and (b).

[0086] In some embodiments, modifications within a variant capsid protein further comprise one or more modifications to an amino acid sequence that is at or near a glycan binding region, e.g., a modification that reduces glycan binding, e.g., where the glycan is galactose. In some such embodiments the one or more modifications are at or between amino acids: (a) 271 and 272 of AAV9 or the corresponding position in the capsid protein of another serotype; (b) 446 of AAV9 or the corresponding position in the capsid protein of another serotype; (c) 470 of AAV9 or the corresponding position in the capsid protein of another serotype; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of AAV9 or the corresponding position in the capsid protein of another serotype; (e) 489 and 545 of VP1 of AAV9 or the corresponding position in the capsid protein of another serotype; (f) 591 and 621 of VP1 of AAV9 or the corresponding position in the capsid protein of another serotype; or (g) any combination or all of (a)-(f).

[0087] Additional methods for generating modifications within a variant capsid protein are described in, e.g., U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199; International Publication Nos. W02003/042397, W02005/033321, and W02006/ 110689; and U.S. Patent No. 7,588,772, each of which are hereby incorporated by reference in their entirety.

Barcodes

[0088] In some embodiments, each AAV capsid variant within a member library as described herein comprises one or more nucleic acid sequences comprising one or more barcodes, and one or more gene regulatory elements (e.g., a cell-type specific and/or tissuespecific gene regulatory element, or a ubiquitous gene regulatory element) (see, e.g., Figure 1), wherein the one or more barcodes are linked to the one or more gene regulatory elements such that identifying the nucleic acid sequence of the one or more barcodes also identifies the one or more gene regulatory elements driving the expression of the AAV capsid variant. In some

embodiments, all AAV capsid variants within a member library comprise the same at least one gene regulatory clement and one or more barcode. In some embodiments, each AAV capsid variant within a member library comprises at least one barcode that is different from each other AAV capsid variant within a different member library within the plurality of AAV capsid variant libraries. In some embodiments, each AAV capsid variant within a member library comprises two barcodes, and one or more gene regulatory elements (e.g., a cell-type specific and/or tissuespecific gene regulatory element, or a ubiquitous gene regulatory element). In some embodiments, two barcodes are linked to one or more gene regulatory elements described herein such that identifying a nucleic acid sequence of either of the two barcodes also identifies a nucleic acid sequence of one or more gene regulatory elements driving expression of the AAV capsid valiant.

[0089] In some embodiments, a barcode comprises a wild-type AAV nucleic acid sequence. In some embodiments, a barcode comprises one or more silent mutations, e.g., relative to a wild-type AAV nucleic acid sequence. In some embodiments, a barcode comprises one or more silent mutations in a nucleic acid sequence encoding a capsid protein. In some embodiments, a barcode comprises at least one silent mutation in at least one codon. In some embodiments, a barcode comprises at least one silent mutation in at least one codon encoding leucine. In some embodiments, a barcode comprises at least one silent mutation in at least one codon encoding serine. In some embodiments, a barcode comprises at least one silent mutation in at least one codon encoding arginine.

[0090] In some embodiments, a barcode is located within a specified number of nucleotides from one or more modifications within a variant capsid protein, as described herein. In some embodiments, a barcode is located at least 1, 2, 3, 4, 5, or more nucleotides upstream of a nucleotide sequence that encodes one or more modifications within a variant capsid protein. In some embodiments, a barcode is located no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nucleotides upstream of a nucleotide sequence that encodes one or more modifications within a variant capsid protein. In some embodiments, a barcode is located at least 1, 2, 3, 4, 5, or more nucleotides downstream of a nucleotide sequence that encodes one or more modifications within a valiant capsid protein. In some embodiments, a barcode is located no more than 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 15 or 20 nucleotides downstream of a nucleotide sequence that encodes one or more modifications within a variant capsid protein. In some embodiments, a barcode is located in a nucleotide sequence encoding a VP1 capsid protein. In some embodiments, a barcode is located in a nucleotide sequence encoding a VP2 capsid protein. In some embodiments, a barcode is located in a nucleotide sequence encoding a VP3 capsid protein.

[0091] In some embodiments, a barcode is located at least 1, 2, 3, 4, 5, or more nucleotides upstream or downstream of a nucleotide sequence encoding a variable region, including e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX. In some embodiments, a barcode is located no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nucleotides upstream or downstream of a nucleotide sequence encoding a variable region, including e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX.

[0092] In some embodiments, a barcode is located at least 1, 2, 3, 4, 5, or more nucleotides upstream or downstream of a nucleotide sequence encoding amino acids: (a) 262-269 (VR-I) of AAV9 or a corresponding position in a capsid protein of another serotype; (b) 327-332 (VR-II) of AAV9 or a corresponding position in a capsid protein of another serotype; (c) 382- 386 (VR-111) of AAV9 or a corresponding position in a capsid protein of another serotype; (d) 452-460 (VR-IV) of AAV9 or a corresponding position in a capsid protein of another serotype;

(e) 488-505 (VR-V) of AAV9 or a corresponding position in a capsid protein of another serotype; (f) 527-539 (VR-VI) of AAV9 or a corresponding position in a capsid protein of another serotype; (g) 545-558 (VR-VII) of AAV9 or a corresponding position in a capsid protein of another serotype; (h) 581-593 (VR-VIII) of AAV9 or a corresponding position in a capsid protein of another serotype; (i) 704-714 (VR-IX) of AAV9 or a corresponding position in a capsid protein of another serotype; or (j) any combination or all of (a)-(i).

[0093] In some embodiments, a barcode is located no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nucleotides upstream or downstream of a nucleotide sequence encoding amino acids: (a) 262-269 (VR-I) of AAV9 or a corresponding position in a capsid protein of another serotype; (b) 327-332 (VR-II) of AAV9 or a corresponding position in a capsid protein of another serotype; (c) 382-386 (VR-III) of AAV9 or a corresponding position in a capsid protein

of another serotype; (d) 452-460 (VR-IV) of AAV9 or a corresponding position in a capsid protein of another serotype; (c) 488-505 (VR-V) of AAV9 or a corresponding position in a capsid protein of another serotype; (f) 527-539 (VR-VI) of AAV9 or a corresponding position in a capsid protein of another serotype; (g) 545-558 (VR-VII) of AAV9 or a corresponding position in a capsid protein of another serotype; (h) 581-593 (VR-VIII) of AAV9 or a corresponding position in a capsid protein of another serotype; (i) 704-714 (VR-IX) of AAV9 or a corresponding position in a capsid protein of another serotype; or (j) any combination or all of (a)-(i).

[0094] In some embodiments, a barcode is located at least 1, 2, 3, 4, 5, or more nucleotides upstream or downstream of a nucleotide sequence encoding amino acids: (a) 271 and 272 of AAV9 or a corresponding position in a capsid protein of another serotype; (b) 446 of AAV9 or a corresponding position in a capsid protein of another serotype; (c) 470 of AAV9 or a corresponding position in a capsid protein of another serotype; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of AAV9 or a corresponding position in a capsid protein of another serotype; (e) 489 and 545 of VP1 of AAV9 or a corresponding position in a capsid protein of another serotype; (f) 591 and 621 of VP1 of AAV9 or a corresponding position in a capsid protein of another serotype; or (g) any combination or all of (a)-(f). In some embodiments, a barcode is located no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nucleotides upstream or downstream of a nucleotide sequence encoding amino acids: (a) 271 and 272 of AAV9 or a corresponding position in a capsid protein of another serotype; (b) 446 of AAV9 or a corresponding position in a capsid protein of another serotype; (c) 470 of AAV9 or a corresponding position in a capsid protein of another serotype; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of AAV9 or a corresponding position in a capsid protein of another serotype; (e) 489 and 545 of VP1 of AAV9 or a corresponding position in a capsid protein of another serotype; (f) 591 and 621 of VP1 of AAV9 or a corresponding position in a capsid protein of another serotype; or (g) any combination or all of (a)-(f).

[0095] In some embodiments, a barcode comprises one or more silent mutations sufficient to identify which member library of a plurality of AAV capsid variant libraries that a

captured mRNA transcript belongs to during sequencing analysis (e.g., NGS analysis). In some embodiments, a barcode of each member library is different from a barcode of all other member libraries within a plurality of AAV capsid variant libraries by a Hamming distance of one, two, three, four, or more nucleic acids. In some embodiments, a barcode comprises nine nucleic acids and a minimum Hamming distance of five nucleic acids relative to all other member libraries within the plurality of AAV capsid variant libraries.

Inverted Tandem Repeats (ITRs)

[0096] AAV capsid variant libraries, as described herein, may comprise ITR sequences derived from any AAV serotype (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-HSC 1-17, or variants or hybrids thereof). In some embodiments, ITR sequences are derived from one or more other serotypes, e.g., as described in US Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552;

9,163,260; 9,567,607; 4,797,368; 5,139,941; 5,252,479; 6,261,834; 7,718,424; 8,507,267;

8,846,389; 6,984,517; 7,479,554; 6,156,303; 8,906,675; 7,198,951; 10,041,090; 9,790,472; 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250;10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825;

8,628,966; 8,927,514; 9,623,120; 9,777,291; 9,783,825; 9,803,218; 9,834,789; 9,839,696;

9,585,971; or 10,519,198; U.S. Publication Nos. 2017/0166926; 2019/0015527; 2019/0054188; or 2020/0080109; or International Publication Nos. WO2018/160582, W02020/028751, or W02020/068990, each of which is hereby incorporated by reference in its entirety.

[0097] ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; and described in Kessler et al., PNAS, 93(24): 14082-7 (1996);

Machida, Methods in Molecular Medicine™, Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted

Integration by Adeno-Associated Vims; and U.S. Patent Nos. 5,139,941 and 5,962,313; each of which is hereby incorporated by reference in its entirety).

Paired Member Libraries

[0098] Two or more member libraries as described herein may be paired in having the same gene regulatory element (e.g., a cell-type specific and/or tissue-specific gene regulatory element, or a ubiquitous gene regulatory element) and different barcodes. For example, a first member library comprising gene regulatory element A and barcode X and a second member library comprising gene regulatory element A and barcode Y are said to be paired. In some embodiments, a first member library comprising gene regulatory clement A and barcode X, a second member library comprising gene regulatory element A and barcode Y, and a third member library comprising gene regulatory element A and barcode Z are paired.

Methods of identifying AAV Capsid Variants

[0099] The present disclosure, among other things, provides methods for identifying one or more AAV capsid valiants with a desired characteristic. Methods for identifying one or more AAV capsid variants can comprise the following steps: (a) constructing and producing a plurality of AAV capsid variant libraries as described herein, (b) transducing a cell population or tissue with a plurality of AAV capsid variant libraries, (c) recovering one or more AAV capsid valiants from a cell population or tissue, and (d) identifying one or more AAV capsid variants with a desired characteristic (e.g., a cell-type and/or tissue-specific tropism). In some embodiments, an identifying step comprises: (i) extracting mRNA from a cell population or tissue, conversion of mRNA to cDNA, and amplification of cDNA; (ii) cDNA amplicon sequencing; and (iii) evaluating AAV capsid variant performance as described herein using computational analysis.

Construction and production of AAV capsid variant libraries

[0100] As described herein, methods of identifying AAV capsid variants with a desired characteristic can comprise constructing and producing a plurality of AAV capsid variant libraries comprising two or more member libraries, where each member library comprises a plurality of AAV capsid variants. Methods of AAV vector construction are well known in the art. In some embodiments, constructing AAV capsid variant member libraries comprises cloning an AAV peptide display library into two or more entry vectors, each of which comprise at least one different barcode. In some embodiments, nucleic acid vectors encoding AAV capsid variant member libraries are then pooled for production of AAV capsid variant libraries by transfection of a host cell (e.g., mammalian cells, such as HEK293 cells).

[0101] In some embodiments, production of AAV capsid variant libraries comprises transient transfection. In some embodiments, a transient transfection method is a suspension transient transfection (sTT). In some embodiments, a transient transfection method is an adherent transient transfection. In some embodiments, the disclosure provides transfected host cells comprising at least one AAV capsid variant member library and one, two, three, or four vectors as described herein.

[0102] In some embodiments, production of AAV capsid variant libraries can comprise transfecting a host cell with one or more AAV capsid variant libraries and two additional vectors. In some embodiments, two additional vectors comprise: (i) a vector encoding at least one AAV Rep polypeptide (e.g., pRep2), and (ii) a vector encoding at least one Adenoviral helper polypeptide (e.g., pHelper). In some embodiments, production of AAV capsid variant libraries comprises transfecting a host cell with one or more AAV capsid variant libraries and one additional vector. In some embodiments, one additional vector comprises a vector encoding at least one adenoviral helper polypeptide and at least one AAV Rep polypeptide. In some embodiments, one or more AAV capsid variant libraries are transfected at a 10- to 100-fold lower molar ratio relative to typical triple or double transfection methods. In some embodiments, such methods reduce incidence of cross-packaged genomes and mosaic capsid formation. In some embodiments, a plurality of AAV capsid variant libraries produced as described herein are comprised predominantly of AAV capsid variants comprising an original ITR-flanked genome that encodes the variant capsid protein.

[0103] In some embodiments, production of AAV capsid variant libraries comprises transfection of nucleic acids (e.g., comprising one or more vector) with any transfection reagent known to a skilled person for introducing nucleic acid molecules into host cells (e.g., mammalian cells, such as HEK293 and/or HEK293T cells). In some embodiments, a transfection reagent comprises a lipid, a polymer, or a combination thereof. In some embodiments, a transfection reagent is a reagent that forms a complex with nucleic acids.

[0104] In some embodiments, a transfection reagent comprises a polymer, a lipid, or both a polymer and a lipid. In some embodiments, a transfection reagent is or comprises a polymer. In some embodiments, a transfection reagent is or comprises lipid. In some embodiments, a transfection reagent comprises a polymer and a lipid.

[0105] In some embodiments, a transfection reagent is or comprises a polymer, e.g., a cationic polymer. In some embodiments, a transfection reagent comprises polyethyleneimine (PEI), FectoVIR, TransIT-VirusGEN, or a combination thereof. In some embodiments, a transfection reagent is or comprises polyethyleneimine (PEI). In some embodiments, host cells are transfected with PEI. In some embodiments, host cells are transfected with a weight (wt.) ratio of DNA to transfection reagent (e.g., PEI) of about 1:1 to about 1:2, about 1:1 to about 1:5, or about 1:1 to about 1:10, e.g., about 1:0.05, about 1:1, about 1:1.25, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:6, about 1:7, about 1 :8, about 1 :9, or about 1 : 10. In some embodiments, a wt. ratio of DNA to transfection reagent is dependent on cell culture density (e.g., of adherent or suspension host cells).

Transduction of cell populations or tissues

[0106] As described herein, methods of identifying AAV capsid variants with a desired characteristic can comprise transducing a cell population or tissue with a plurality of AAV capsid variant libraries. In some embodiments, a plurality of AAV capsid variant libraries comprises 2, 3, 4, 5, or more member libraries. In some embodiments, a plurality of AAV capsid variant libraries comprises at least one member library comprising a ubiquitous gene regulatory element and barcode that is different from all other member libraries, and one or more additional member

library comprising a cell-type specific and/or tissue- specific gene regulatory element and barcode that is different from all other member libraries. In some embodiments, a plurality of AAV capsid variant libraries comprises at least one member library comprising a ubiquitous gene regulatory element and barcode that is different from all other member libraries, and one or more paired member libraries described herein. In some embodiments, a transducing step is or comprises administering a single pre-mixed composition comprising a plurality of AAV capsid variant libraries to a cell population or tissue. In some embodiments, a transducing step is or comprises sequentially administering individual member libraries within a plurality of AAV capsid variant libraries to a cell population or tissue.

[0107] In some embodiments, transducing a cell population or tissue with a plurality of

AAV capsid variant libraries is performed in vitro (e.g., using primary neuronal cultures, and/or iPCS-derived cultures) or in vivo (e.g., in a model organism, such as a mouse, rodent, or nonhuman primate).

AAV capsid variant screening using isolated proteins

[0108] As described herein, methods of identifying AAV capsid variants with a desired characteristic can comprise incubating one or more isolated proteins (e.g., one or more receptor proteins) with a plurality of AAV capsid variant libraries in vitro. In some embodiments, one or more isolated proteins can comprise any protein. In some embodiments, one or more isolated proteins can comprise a protein whose expression and/or accumulation is specific to a tissue or cell of an organism capable of AAV transduction. In some embodiments, one or more isolated proteins can comprise a known or suspected capsid-receptor protein. In some embodiments, one or more isolated proteins include one or more moieties to assist in protein purification, including, for example, a hexahistidine tag, glutathione-S-transferase tag, maltose-binding protein, calmodulin-binding protein, biotin, or streptavidin. In some embodiments, a plurality of AAV capsid variant libraries comprise at least two AAV capsid variant libraries, each with different barcodes. In some embodiments, methods of identifying AAV capsid variants with a desired characteristic include further purifying capsid- protein complexes. In some embodiments, capsid-

protein complexes are purified using a purification moiety. In some embodiments, AAV capsid variants that interact (i.c., form complexes) with an isolated protein arc recovered and sequenced as described elsewhere herein.

Recovering AAV capsid variants from a cell population or tissue

[0109] As described herein, methods of identifying AAV capsid variants with a desired characteristic can comprise recovering AAV capsid variants from a cell population or tissue. In some embodiments, recovering AAV capsid variants from a cell population or tissue is performed 1 day, 2 days, 3 days, or longer post-transduction. In some embodiments, recovering AAV capsid variants from a cell population or tissue is performed 1 week, 2 weeks, 3 weeks post-transduction. In some embodiments, a recovering step comprises isolating one or more desired cell-types (e.g., central nervous system cell-types, such as neuron and astrocyte) from a cell population or one or more desired tissue (e.g., brain, kidney, or liver) from a subject. In some embodiments, one or more isolated cell-types or tissue are further processed, including, e.g., by cell sorting (including fluorescence assisted cell sorting (FACS)), laser microdissection, density gradient centrifugation, or mechanical dissection. mRNA extraction and cDNA conversion, amplification, and sequencing methods

[0110] As described herein, a method of identifying AAV capsid valiants with a desired characteristic can comprise extracting mRNA, cDNA conversion, and amplification. Methods for extracting mRNA and cDNA conversion are well known in the art. In some embodiments, prior to amplicon sequencing, cDNA is first PCR amplified using forward and reverse primers. In some embodiments, forward and reverse primers are complementary to nucleic acid sequences of an AAV capsid variant genome flanking (i.e., upstream and downstream) both a barcode and modifications within a variant capsid protein (see, e.g., Figure 3). In some embodiments, cDNA is first amplified resulting in amplification of a specific genomic region (e.g., comprising nucleic acids encoding a barcode and modifications within a variant capsid protein) in one or more member libraries (e.g., all member libraries). In some embodiments, resulting cDNA amplicons

containing both barcode and modifications within a variant capsid protein are analyzed by next- generation sequencing (NGS). In some embodiments, one or more member libraries arc selectively amplified from eDNA using at least one primer that is complementary to one or more nucleotides within a barcode of a member library (see, e.g., Figure 4). In some embodiments, one or more member libraries are selectively amplified from eDNA using at least one primer that is complementary to all nucleotides encoding a barcode of a member library.

[0111] As described herein, methods of identifying AAV capsid variants with a desired characteristic can comprise amplicon sequencing. Multiple methods for amplicon sequencing are known in the ail. In some embodiments, amplicon sequencing is performed using any method known in the art resulting in the identification of a nucleic acid’s sequence, including, e.g., Sanger sequencing, next generation sequencing (NGS), and pyro sequencing. In some embodiments, amplicon sequencing is performed using NGS. In some embodiments, an amplicon is of sufficient length to enable sequencing on an Illumina short-read sequencing instrument by single and/or paired-end sequencing. In some embodiments, a eDNA amplicon (or amplicon resulting from amplification thereof) sequenced by NGS is < 500 bp, < 400 bp, < 300 bp, or < 200 bp in length. In some embodiments, amplicon sequencing identifies both a barcode and a nucleotide sequence encoding modifications within a variant capsid protein.

Evaluating AAV capsid variant performance

[0112] As described herein, methods of identifying AAV capsid variants with a desired characteristic can comprise evaluating AAV capsid variant performance using computational analysis to identify, for each AAV capsid variant, one or more of the following: (a) modifications within a variant capsid, (b) at least one barcode, (c) a gene regulatory element controlling expression of a variant capsid protein, and (d) AAV capsid variant transcript abundance. AAV capsid variant performance can refer to any number of metrics that indicate a difference between an AAV capsid variant and a reference AAV capsid (e.g., a wild-type AAV capsid). In some embodiments, AAV capsid variant performance includes comparing transcript abundance of variant capsid proteins driven by different gene regulatory elements as assessed by

sequencing (e.g., NGS). In some embodiments, AAV capsid variant performance relates to tropism specificity conferred by a variant capsid protein.

[0113] In some embodiments, computational analysis is achieved through any one of numerous computational methods known in the ail, which may include a separation of sequenced transcripts based on barcode and/or modifications within a variant capsid protein. In some embodiments, AAV capsid variant performance is performed, in part, by identifying a member library to which each sequenced transcript belongs to by specifying a 5’ flanking region corresponding to each barcode sequence and analyzing transcript abundance variation of each AAV capsid variant transcript grouped by barcode. In some embodiments, AAV capsid variant performance includes determining that one or more AAV capsid variants exhibit a tropism specific to one or more cell-types or tissues. In some embodiments, AAV capsid variant performance is evaluated by comparing: (a) transcript abundance of one or more AAV capsid variants from a first member library against (b) transcript abundance of the same one or more AAV capsid variants from a second member library. In some embodiments, AAV capsid variant performance is evaluated by comparing: (a) transcript abundance of one or more AAV capsid variants whose expression was driven by a cell-type specific and/or tissue- specific gene regulatory element against (b) transcript abundance of the same one or more AAV capsid variants (i.e. , comprising the same modifications within a variant capsid protein) whose expression was driven by a ubiquitous gene regulatory element (see, e.g., Figure 5). In some embodiments, AAV capsid variant performance is evaluated by comparing: (a) transcript abundance of one or more AAV capsid variants whose expression was driven by a cell-type specific and/or tissue-specific gene regulatory element against (b) transcript abundance of the same one or more AAV capsid variants (i.e., comprising the same modifications within a variant capsid protein) whose expression was driven by a different cell-type specific and/or tissuespecific gene regulatory element.

[0114] In some embodiments, transcript abundances of each AAV capsid variant are normalized prior to evaluating AAV capsid variant performance. In some embodiments, transcript abundances are normalized based on an input AAV library used in a transducing step. In some embodiments, AAV capsid valiants with an abundance metric of zero are accounted for

by adding a pseudocount of 0.1 . In some embodiments, AAV capsid variant performance includes evaluating a correlation between an abundance metric of AAV capsid variants with a cell-type specific and/or tissue- specific gene regulatory element and the same AAV capsid variants with a ubiquitous gene regulatory element.

[0115] When paired member libraries described herein were used to transduce a cell population or tissue, AAV capsid variant performance can be compared between individual AAV capsid variants, each comprising the same modifications within a variant capsid protein and driven by the same gene regulatory element, but comprising different barcodes. In such embodiments, evaluating variant performance involves a statistical test to demonstrate and/or evaluate reproducibility of abundance metrics for a given AAV capsid variant with different barcodes. In some embodiments, evaluating AAV capsid variant performance involves comparing an abundance metric of an AAV capsid variant derived from different cell-types and/or tissues. For example, in some embodiments, transcript abundance of an AAV capsid variant isolated from the central nervous system is compared with abundance metrics of the same AAV capsid variant isolated from one or more other tissues described herein.

AAV Serotypes

[0116] The present disclosure, among other things, provides methods for screening and identifying AAV capsid variants with a desired characteristic. Generally, AAV capsid variant libraries for use in methods described herein may be of any AAV serotype, or comprise one or more sequence elements derived from any AAV serotype. In some embodiments, AAV serotypes used in the methods described herein include, but not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV- PHP.N, or AAV.CAP-B1 to AAV.CAP-B25, as well as valiants or hybrids thereof. For example, in some embodiments, an AAV comprises or is an AAV2/5, AAV2/6, AAV2/8 or AAV2/9 vector (e.g., AAV6, AAV8 or AAV9 serotype having AAV2-derived ITRs).

[0117] In some embodiments, a AAV is derived from an AAV genome sequence or a variant thereof as described in US Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552; 9,163,260; 9,567,607; 4,797,368; 5,139,941; 5,252,479; 6,261,834; 7,718,424; 8,507,267; 8,846,389; 6,984,517; 7,479,554; 6,156,303; 8,906,675; 7,198,951; 10,041,090; 9,790,472; 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250;10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,927,514; 9,623,120; 9,777,291; 9,783,825; 9,803,218; 9,834,789; 9,839,696; 9,585,971; or 10,519,198; U.S. Publication Nos. 2017/0166926; 2019/0015527; 2019/0054188; or 2020/0080109; or International Publication Nos. WO2018/160582, W02020/028751, or W02020/068990, each of which is hereby incorporated by reference in its entirety.

[0118] In some embodiments, an AAV serotype may have or comprise a mutation in an AAV9 sequence (e.g., as described in Pulicherla et al., Molecular Therapy, 19(6): 1070-1078 (2011), which is hereby incorporated by reference in its entirety). AAV9 serotypes may include, but not limited to, AAV9.68, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, and AAV9.84. In certain embodiments, an AAV9 variant comprises or is AAVhu68 or a variant thereof (e.g., as described in International Publication No.

WO2018/160585, which is hereby incorporated by reference in its entirety). Other AAV vectors are described in, e.g., Sharma et al., Brain Res Bull., 81(2-3):273 (2010), which is hereby incorporated by reference in its entirety.

[0119] In some embodiments, an AAV comprises or is a naturally occurring AAV. In some embodiments, an AAV is a modified AAV or a variant of a naturally occurring AAV.

[0120] In some embodiments, AAV capsid libraries as described herein may comprise or be based on a serotype selected from any following serotypes or variants thereof including, but not limited to, AAV9.68, AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.4O, AAV 12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.l l, AAV16.3, AAV16.8/hu.l0, AAV161.1O/hu.6O, AAV161.6/hu.61, AAVl-7/rh.48, AAV1- 8/rh.49, AAV2, AAV2.5T, AAV2- 15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2- 3/rh.61, AAV24.1, AAV2-4/rh.5O, AAV2-5/rh.51, AAV27.3,

AAV29.3/bb.1 , AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-l l/rh.53, AAV3-3, AAV33.12/hu.l7, AAV33.4/hu.l5, AAV33.8/hu.l6, AAV3-9/rh.52, AAV3a, AAV3b, AAV4, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42- 6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/rl l.64, AAV4-8/rh.64, AAV4-9/rh.54, AAV5, AAV52.1/hu.2O, AAV52/hu.l9, AAV5- 22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27, AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-l/hu.l, AAVH2, AAVH-5/hu.3, AAVH6, AAVhEl.l, AAVhER1.14, AAVhErl.16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.l, AAVhu.10, AAVhu.ll, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.tl9, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG- 9/hu.39, AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11,

AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV- PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC 8, AAVpi.l, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh,13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48. E2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, B P61 AAV, B P62 AAV, B P63 AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK 16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and AAV SM 10-8.

[0121] An AAV serotype may be from any number of species. For example, an AAV may be or comprise an avian AAV (AAAV), e.g., as described in U.S. Patent No. 9,238,800, which is hereby incorporated by reference in its entirety. An AAV serotype may be or comprise a bovine AAV (BAAV), e.g., as described in U.S. Patent Nos. 9,193,769 or 7,427,396, each of which is hereby incorporated by reference in its entirety. An AAV may be or comprise a caprine AAV, e.g., as described in U.S. Patent No. 7,427,396, which is hereby incorporated by reference in its entirety. An AAV serotype may also be a variant or hybrid of any of the foregoing.

[0122] In some embodiments, an AAV capsid variant library may be or comprise a serotype generated from an AAV9 capsid library with mutations in amino acids 390 to 627 (VP1 numbering), e.g., as described in Pulicherla et al., Molecular Therapy, 19(6): 1070-1078 (2011), which is hereby incorporated by reference in its entirety. In some embodiments, an AAV serotype (with corresponding nucleotide and amino acid substitutions) may include, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K),

AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T18O5A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (CI 531 A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T153OC, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A,;G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T18O6C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L), and AAV9.95 (T1605A; F535L).

INCORPORATION BY REFERENCE

[0123] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be

used in the practice or testing of the present invention, suitable methods and materials are described herein.

EXAMPLES

[0124] The following Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

Example 1: Identifying AAV capsid variants with muscle (tissue)-specific tropism

Introduction

[0125] As described herein, Directed Evolution Platform Incorporating Cell-type Tropism (DEPICT) is a novel AAV capsid variant library screening method that allows for identification, selection, and directed evolution of AAV capsid variants with a desired tropism at cell-type resolution. DEPICT achieves this unparalleled cell-type resolution, in part, by constructing a plurality of AAV capsid variant libraries (individually, “member libraries”), each AAV capsid variant within a member library comprising a promoter and an associated barcode comprised of silent mutations (sec, c.g., Figures 1 and 2). Because each member library of AAV capsid variants contains a unique barcode associated with a specific promoter, multiple AAV libraries can be administered to a cell line, tissue, or organism. After administration, mRNA is recovered and next generation sequencing is used to simultaneously identify, for each AAV virion transcript, the sequences of both a capsid variant and its promoter-associated barcode. The barcode is then used to associate each variant capsid protein with a promoter driving its expression. Thus, methods described herein constitute a novel directed evolution strategy, by which multiple AAV capsid variant libraries can be simultaneously administered, and from which, AAV capsid variants with enhanced transduction efficiency in target tissues/cells, reduced uptake in non-target tissues/cells, and increased immune evasion of circulating neutralizing factors can be identified.

[0126] In addition, DEPICT barcodes may be used as unique molecular identifiers (UMIs). A common obstacle encountered in screening high-diversity mutant libraries is identifying true hits from noise. Noise may arise from factors such as PCR stochasticity during amplification of capsid mRNA from the target tissues or cells of interest. Thus, DEPICT barcodes may also be employed as codon replicates to increase confidence in calculated foldchange if enrichment is seen across multiple DEPICT barcodes associated with the same mutation. This can be achieved by introducing multiple DEPICT barcodes per individual promoter.

Materials and Methods

[0127] An AAV peptide display library with a diversity of IxlO⁵ to 2xl0⁵ unique capsid variant sequences was cloned into either a CBh-containing or MHCK7 -containing entry vector that included a promoter- specific barcode upstream of a cloning site. Resulting vectors were mixed and transfected into producer cells, using methods known in the art, to produce AAV capsid variant libraries. Resulting AAV capsid variant libraries were administered to cynomolgus macaques. Three weeks after administration, RNA was isolated from skeletal muscle and AAV mRNA was recovered using RT-PCR with a gene-specific RT primer followed by PCR amplification using a primer pair that flanked an upstream promoter barcode on the 5’ end (i.e., a primer complementary to the conserved sequence immediately upstream of the barcode) and a peptide insertion site on the 3’ end (see, e.g., Figure 3). The recovered amplicons, which were <200 bp in length, underwent NGS library preparation for high-depth short-read sequencing. Following NGS analysis, mRNA derived from CBh or MHCK7 promoter were separated by binning transcripts according to their promoter barcodes. Variant fold-change values were calculated following normalization to the input AAV library. To account for variants with zero reads, a pseudocount of 0.1 was added to all variants.

Results

[0128] Overall, we found high correlation in variant enrichment between CBh and MHCK7 promoter in skeletal muscle, particularly among the top performing variants (Figure 5). This result suggests that CBh and MHCK7 promoter expression has high concordance in skeletal

muscle, with relatively smaller fractions of other cell populations such as fibroblasts and endothelial cells.

Example 2: Materials and Methods

[0129] The methods described herein are used to identify AAV capsid variants with tropism to specific cell-types within the central nervous system (CNS) and retina (Figure 6). For both CNS- and retina-tropism screening, three member libraries are constructed. For each member library used to identify AAV capsid variants with CNS-specific tropisms, a highly diverse capsid variant library is cloned into an entry vector containing a promoter- specific barcode upstream of a cloning site and (i) a ubiquitous CBh promoter, (ii) neuron- specific hSynl promoter, or (iii) astrocyte-specific GFAP promoter. For each member library used to identify AAV capsid variants with retina-specific tropisms, a highly diverse capsid variant library is cloned into an entry vector containing a promoter- specific barcode upstream of a cloning site and (i) a ubiquitous CBh promoter, (ii) photoreceptor- specific hGRKl promoter, or (iii) REP-specific VMD2 promoter.

[0130] The resulting vectors are mixed and transfected into producer cells using methods known in the ail, to produce AAV capsid variant libraries. The resulting AAV capsid variant libraries are administered to cynomolgus macaques. Three weeks after administration, RNA is isolated from various tissues of each cynomolgus macaque. Specifically, for macaques that received AAV capsid variant libraries comprising retina-specific promoters, retinal tissue will be collected. Similarly, for macaques that received AAV capsid variant libraries comprising neuron- specific promoters, CNS tissues will be collected. In all cases, mRNA will be collected and analyzed as performed in Example 1 herein. In addition, cDNA from each sample will be amplified using a forward primer whose sequence is complementary to the barcode of each sample (Figure 4) to amplify each member library individually prior to analysis by NGS.

Equivalents

[0131] It is to be appreciated by those skilled in the art that various alterations, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be pail of the present disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and any invention described in the present disclosure if further described in detail by the claims that follow.

[0132] Those skilled in the art will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes described herein. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference in their entireties.

Claims

CLAIMS We claim:

1. A method of identifying an adeno-associated virus (AAV) capsid variant with a desired characteristic, comprising:

(a) transducing a cell population or tissue with a plurality of AAV capsid variant libraries, wherein each member library within the plurality of AAV capsid variant libraries comprises a plurality of AAV capsid variants, and wherein each AAV capsid variant within a member library comprises a nucleic acid sequence comprising:

(i) a gene regulatory element chosen from:

(1) a cell-type specific and/or tissue-specific gene regulatory element, or

(2) a ubiquitous gene regulatory element; and

(ii) a nucleic acid sequence encoding a variant capsid protein and comprising a barcode, wherein the gene regulatory element is operatively linked to the nucleic acid sequence encoding the variant capsid protein and the gene regulatory element and the barcode are the same for each AAV capsid variant within a member library, wherein each member library has a different barcode from each other member library within the plurality of AAV capsid variant libraries, and wherein the barcode of one or more member libraries comprises one or more silent mutations;

(b) recovering AAV capsid variants from the cell population or tissue; and

(c) identifying an AAV capsid variant with the desired characteristic.

2. The method of claim 1, wherein at least two member libraries have a different gene regulatory element from each other member library within the plurality of AAV capsid variant libraries.

3. The method of claim 1 or 2, wherein each member library has a different gene regulatory element from each other member library within the plurality of AAV capsid variant libraries.

4. The method of claim 1 or 2, wherein a first set of at least two member libraries are paired in that they have the same gene regulatory element and different barcodes.

5. The method of claim 4, wherein a second set of at least two member libraries are paired in that they have the same gene regulatory element and different barcodes, wherein the gene regulatory element of the second set is different from the gene regulatory element of the first set.

6. The method of any one of the preceding claims, wherein the barcode of one or more member libraries comprises one or more silent mutations in at least one codon of the sequence encoding the variant capsid protein.

7. The method of claim 6, wherein the at least one codon encodes leucine, serine, and/or arginine.

8. The method of any one of the preceding claims, wherein each AAV capsid variant comprises one or more additional barcodes within the sequence encoding the variant capsid protein.

9. The method of claim 8, wherein the one or more additional barcodes comprise one or more silent mutations, wherein each member library has a different one or more additional barcodes from each other member library within the plurality of AAV capsid variant libraries.

10. The method of any one of the preceding claims, wherein the barcode or one or more additional barcodes is located within 2208 nucleotides of a sequence encoding a hypervariable region and/or surface-exposed loop of the variant capsid protein.

11. The method of any one of the preceding claims, wherein the barcode and/or one or more additional barcodes comprises one or more nucleic acids.

12. The method of any one of the preceding claims, wherein the barcode and/or one or more additional barcodes comprises a hamming distance of one or more nucleic acids.

13. The method of any one of the preceding claims, wherein the barcode and/or one or more additional barcodes comprises nine nucleic acids and a minimum hamming distance of five nucleic acids.

14. The method of any one of the preceding claims, wherein the barcode and/or one more additional barcodes is encoded by the same region of the AAV capsid variant within each member library.

15. The method of any one of the preceding claims, wherein at least one member library within the plurality of AAV capsid variant libraries comprises a ubiquitous gene regulatory element.

16. The method of any one of the preceding claims, wherein the ubiquitous gene regulatory element is or comprises hybrid chicken beta actin (CBh) promoter, CAG promoter, simian virus 40 (SV40) promoter, cytomegalovirus (CMV) promoter, ubiquitin C (UBC) promoter, elongation factor- 1 alpha (EFl A) promoter, phosphoglycerate kinase 1 (PGK1) promoter, phosphoglycerate kinase (PGK) promoter, human beta-actin promoter, beta -act in long (BActL) promote, chimeric CMV-chicken beta-actin promoter (CBA) promoter, TRE promoter, U6 promoter. Hl promoter, 7SK promoter, or GUSb promoter, or any non-naturally occurring ubiquitous gene regulatory element.

17. The method of any one of the preceding claims, wherein the cell-type specific and/or tissue-specific gene regulatory clement is or comprises a neuron- specific promoter, neuron subtype- specific promoter, microglia and/or macrophage- specific promoter, ionocyte-specific promoter, astrocyte- specific promoter, oligodendrocyte- specific promoter, muscle-specific promoter, retina-specific promoter, photoreceptor- specific promoter, rod-specific promoter, cone- specific promoter, heart- specific promoter, lung- specific promoter, liver- specific promoter, or kidney- specific promoter.

18. The method of claim 17, wherein the cell-type specific and/or tissue-specific gene regulatory element is a human synapsin I (hSynl) promoter, glial fibrillary acidic protein (GFAP) promoter, MHCK7 promoter, calcium/calmodulin-dependent protein kinase II (CaMKII) promoter, cerebellar Purkinje cell-specific L7-6 promoter, synapsin I with a minimal CMV sequence (Synl-minCMV) promoter, homeobox Dlx5/6, glutamate receptor 1 (GluRl) promoter, preprotachykinin 1 (Tael) promoter, dopaminergic receptor 1 (Drdla) promoter, tubulin alpha I promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor beta chain promoter, glutamic acid decarboxylase (GAD67) promoter, photoreceptor- specific rhodopsin kinase (RK) promoter, L-opsin promoter, human GRM6 gene-derived (hGRM6) promoter, alpha-subunit of cone transducin (TaC) promoter, CK8 promoter, desmin promoter, tMCK promoter, dMCK promoter, CK6, promoter, SPc5-12 promoter, D-site binding protein (DBP) promoter, CCAAT enhancer binding protein a or (C/EBP) promoter; hepatocyte nuclear factor (HNF) promoter, liver- specific IL-6-dependent DNA binding protein (IL-6DBP), or any non-naturally occurring cell-type specific and/or tissue-specific gene regulatory element.

19. The method of any one of the preceding claims, wherein each member library comprises one or more additional gene regulatory elements that are operatively linked to the sequence encoding the variant capsid protein.

20. The method of any one of the preceding claims, wherein the AAV capsid variants within each member library differ from each other based on the presence of one or more of the following modifications within the variant capsid protein:

(i) a peptide inserted into a hypervariable region and/or surface-exposed loop of the variant capsid protein,

(ii) a point mutation, and

(iii) a deletion.

21. The method of claim 20, wherein the AAV capsid variants within each member library each include a different peptide inserted into a hypervariable and/or surface-exposed loop within the variant capsid protein.

22. The method of any one of the preceding claims, wherein the plurality of AAV capsid variant libraries comprises three or more AAV capsid variant libraries.

23. The method of any one of the preceding claims, wherein the recovering step comprises isolating a desired cell-type from the cell population or a desired tissue from the tissue.

24. The method of any one of the preceding claims, wherein the identifying step comprises:

(i) isolating mRNA from the cell population or tissue,

(ii) converting the mRNA to cDNA,

(iii) amplifying the cDNA to generate cDNA amplicons, and

(iv) sequencing the cDNA amplicons.

25. The method of claim 24, wherein the sequencing step (iv) comprises identifying the sequence of both the barcode and the modification.

26. The method of claim 24 or 25, wherein the converting step (ii) and/or amplifying step (iii) comprises using a primer that binds specifically to the barcode or to a nucleic acid sequence upstream or downstream of the barcode.

27. The method of any one of claims 24-26, wherein the sequencing of the cDNA amplicons comprises next-generation sequencing.

28. The method of any one of claims 24-27, wherein the cDNA amplicons are each less than about 500 base pairs in length.

29. The method of any one of claims 24-28, wherein the sequencing encompasses a sequence encoding a hypervariable and/or surface-exposed loop of the variant capsid protein.

30. The method of any one of the preceding claims, wherein the desired characteristic is a cell-type specific and/or tissue- specific tropism.

31. The method of any one of the preceding claims, wherein the desired characteristic is the absence of a cell-type specific and/or tissue-specific tropism.

32. The method of any one of the preceding claims, wherein the AAV capsid variants are AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV 11, AAVrh.74, or AAV 12 capsid variants.

33. The method of any one of the preceding claims, wherein the cell population or tissue is transduced in vitro.

34. The method of any one of the preceding claims, wherein the cell population or tissue is transduced in vivo.

35. The method of any one of the preceding claims, wherein the plurality of AAV capsid variant libraries arc pooled and transduced substantially simultaneously.

36. The method of any one of the preceding claims, wherein the transducing step comprises administration to a mammal.

37. The method of claim 36, wherein the mammal is a non-human primate.

38. A screening system comprising a plurality of AAV capsid variant libraries, wherein each member library within the plurality of AAV capsid variant libraries comprises a plurality of AAV capsid variants, and wherein each AAV capsid variant within a member library comprises a nucleic acid sequence comprising:

(i) a gene regulatory element chosen from:

(1) a cell-type specific and/or tissue-specific gene regulatory element, or

(2) a ubiquitous gene regulatory element; and

(ii) a sequence encoding a variant capsid protein and comprising a barcode, wherein the gene regulatory element is operatively linked to the sequence encoding the variant capsid protein and the gene regulatory element and the barcode are the same for each AAV capsid variant within a member library, wherein each member library has a different barcode from each other member library within the plurality of AAV capsid variant libraries, and wherein the barcode of one or more member libraries comprises one or more silent mutations.