Methods for identifying AAV capsid variants with desired characteristics

Abstract

This disclosure provides methods and compositions for screening AAV capsid libraries for desired characteristics such as cell type-specific tropism. Each AAV capsid library disclosed herein contains a known promoter (e.g., ubiquitous or cell type-specific promoter) and a barcode consisting of silent mutations, allowing multiple AAV capsid variant libraries to be combined and administered in a single experiment.

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Application Serial No. 63 / 546,120, filed October 27, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0002] Adeno-associated virus (AAV) vectors have become a leading platform for in vivo delivery of gene therapies for a wide variety of human diseases. AAVs can be engineered to deliver heterologous nucleic acids of interest (e.g., genes encoding therapeutic proteins, antisense nucleic acid molecules, ribozymes, miRNAs, siRNAs, or nucleic acids encoding the CRISPR / Cas system) to specific cells of a patient. The cell-type specificity of AAVs is partly determined by the AAV capsid protein sequence. Although many methods have been used to identify novel AAV capsids with cell-type specificity, existing methods have several drawbacks. Therefore, robust methods remain needed to identify novel AAV variants with cell-type specificity and other desired characteristics in order to develop safer and more effective gene therapies. Summary of the Invention

[0003] This disclosure particularly covers a method for rapidly, efficiently, and accurately identifying AAV capsid variants with desired characteristics, including but not limited to cell type specificity and / or tissue specificity. This disclosure is partly based on the finding that silent mutations incorporated into the genome of an AAV capsid variant library (e.g., within the nucleic acid sequence encoding the capsid) can act as barcodes, allowing the simultaneous administration and screening of two or more AAV capsid variant libraries to obtain cell type specificity and / or tissue specificity when associated with gene regulatory elements (e.g., promoters).

[0004] In one aspect, this disclosure provides a method for identifying adeno-associated virus (AAV) capsid variants having desired characteristics, comprising: (a) Transducing cell populations or tissues using multiple AAV capsid variant libraries. Each member library within the plurality of AAV capsid variant libraries contains a plurality of AAV capsid variants, and Each AAV capsid variant within the member library contains a nucleic acid sequence comprising: (i) Selected from the following gene regulatory elements: (1) Cell type-specific and / or tissue-specific gene regulatory elements, or (2) Pervasive gene regulatory elements; and (ii) A nucleic acid sequence that encodes a variant capsid protein and contains a barcode. 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 identical for each AAV capsid variant within the member library. Within the plurality of AAV capsid variant libraries, each member library has a barcode distinct from all other member libraries, and The barcodes in one or more member libraries contain one or more silent mutations; (b) Recovering the AAV capsid variant from the cell population or tissue; and (c) Identify AAV capsid variants that have the desired characteristics.

[0005] In some implementations of this method, within multiple AAV capsid variant libraries, at least two member libraries have gene regulatory elements that are different from each of the other member libraries.

[0006] In some implementations of this method, within multiple AAV capsid variant libraries, each member library has gene regulatory elements that are different from each other member library.

[0007] In some implementations of this method, the first set of at least two member libraries are paired because they have the same gene regulatory elements and different barcodes.

[0008] In some implementations of this method, at least two member libraries in the second group are paired because they have the same gene regulatory elements and different barcodes, wherein the gene regulatory elements in the second group are different from those in the first group.

[0009] In some embodiments of the method, the barcodes of one or more member libraries contain 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 implementations of this method, each AAV capsid variant includes one or more additional barcodes within the sequence encoding the variant capsid protein.

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

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

[0014] In some implementations of this method, the barcode and / or one or more additional barcodes contain one or more nucleic acids.

[0015] In some implementations of this method, the barcode and / or one or more additional barcodes contain the Hamming distance of one or more nucleic acids.

[0016] In some implementations of this method, the barcode and / or one or more additional barcodes contain the minimum Hamming distance between nine and five nucleic acids.

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

[0018] In some implementations of this method, at least one member library within a plurality of AAV capsid variant libraries contains pervasive gene regulatory elements.

[0019] In some embodiments of this method, at least one ubiquitous gene regulatory element is or comprises a hybrid chicken β-actin (CBh) promoter, a CAG promoter, a simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter, a ubiquitin C (UBC) promoter, an elongation factor-1α (EF1A) promoter, a phosphoglycerate kinase 1 (PGK1) promoter, a phosphoglycerate kinase (PGK) promoter, a human β-actin promoter, a β-actin long (BActL) promoter, a chimeric CMV-chicken β-actin promoter (CBA) promoter, a TRE promoter, a U6 promoter, an H1 promoter, a 7SK promoter, or a GUSb promoter, or any non-naturally occurring ubiquitous gene regulatory element.

[0020] In some embodiments of this method, cell type-specific and / or tissue-specific gene regulatory elements are or include neuron-specific promoters, neuron subtype-specific promoters, microglia and / or macrophage-specific promoters, ionocyte-specific promoters, astrocyte-specific promoters, oligodendrocyte-specific promoters, muscle-specific promoters, retina-specific promoters, photoreceptor-specific promoters, rod-specific promoters, cone-specific promoters, heart-specific promoters, lung-specific promoters, liver-specific promoters, or kidney-specific promoters.

[0021] In some embodiments of this method, cell type-specific and / or tissue-specific gene regulatory elements are human synaptic protein I (hSyn1) promoter, glial fibrillary acidic protein (GFAP) promoter, MHCK7 promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, cerebellar Purkinje cell-specific L7-6 promoter, synaptic protein I with minimal CMV sequence (SynI-minCMV) promoter, homeobox Dlx5 / 6, glutamate receptor 1 (GluR1) promoter, protakininogen 1 (Tacl) promoter, dopaminergic receptor 1... (Drdla) promoter, tubulin αI promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor β chain promoter, glutamate decarboxylase (GAD67) promoter, photoreceptor-specific rhodopsin kinase (RK) promoter, L-opsin promoter, human GRM6 gene-derived (hGRM6) promoter, cone cell transduction α subunit (TαC) promoter, CK8 promoter, desmin promoter, tMCK promoter, dMCK promoter, CK6 promoter, SPc5-12 promoter, D-site binding protein (DBP) promoter, CCAAT enhancer binding protein α or β (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 implementations of this method, each member library contains one or more additional gene regulatory elements operatively linked to a sequence encoding a variant capsid protein.

[0023] In some implementations of this method, each AAV capsid variant within a member library is distinguished from one another based on the presence of one or more of the following modifications within the variant capsid protein: (i) Peptides inserted into the hypervariable region and / or surface-exposed loop of the said variant capsid protein, (ii) point mutations, and (iii) Missing.

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

[0025] In some implementations of this method, the multiple AAV capsid variant libraries contain three or more AAV capsid variant libraries.

[0026] In some embodiments of this method, the recovery step includes separating the desired cell type from the cell population or the desired tissue type from the tissue.

[0027] In some implementations of this method, the identification step includes: (i) Isolate mRNA from the cell population or tissue. (ii) Convert the mRNA into cDNA. (iii) Amplify the cDNA to generate a cDNA amplicon, and (iv) Sequencing the cDNA amplicon.

[0028] In some implementations of this method, the sequencing step (iv) includes identifying the barcode and modifying the sequences of both.

[0029] In some embodiments of the method, the transformation step (ii) and / or amplification step (iii) include using primers that specifically bind to nucleic acid sequences upstream or downstream of the barcode.

[0030] In some implementations of this method, the sequencing of cDNA amplicon includes next-generation sequencing.

[0031] In some implementations of this method, the cDNA amplicon is less than about 500 base pairs in length.

[0032] In some implementations of this method, sequencing covers sequences encoding variant capsid proteins and / or surface-exposed loops.

[0033] In some embodiments of the method, at least one desired feature is cell type specificity and / or tissue specificity.

[0034] In some embodiments of the method, at least one desired feature is the absence of cell type specificity and / or tissue specificity.

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

[0036] In some implementations of this method, cell populations or tissues are transduced in vitro.

[0037] In some implementations of this method, cell populations or tissues are transduced in vivo.

[0038] In some implementations of this method, multiple AAV capsid variant libraries are merged and transduced substantially simultaneously.

[0039] In some embodiments of this method, the transduction step includes administration to a mammal.

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

[0041] On the other hand, this disclosure provides a screening system that includes multiple AAV capsid variant libraries. Each member library within the plurality of AAV capsid variant libraries contains a plurality of AAV capsid variants, and Each AAV capsid variant within the member library contains a nucleic acid sequence comprising: (i) Selected from the following gene regulatory elements: (1) Cell type-specific and / or tissue-specific gene regulatory elements, or (2) Pervasive gene regulatory elements; and (ii) A sequence encoding a variant capsid protein and containing a barcode. The gene regulatory element is operatively linked to the sequence encoding the variant capsid protein, and the gene regulatory element and the barcode are identical for each AAV capsid variant within the member library. Within the plurality of AAV capsid variant libraries, each member library has a barcode distinct from all other member libraries, and The barcode of one or more member libraries contains one or more silent mutations.

[0042] Other features, objects, and advantages of this disclosure will become apparent in the following detailed description. However, it should be understood that while embodiments of this disclosure are shown, the detailed description is given by way of illustration only and not limitation. Various changes and modifications within the scope of this disclosure will become apparent to those skilled in the art based on the detailed description. Attached Figure Description

[0043] Figure 1 Exemplary genomic configurations of AAV capsid variants within member libraries of multiple AAV capsid variant libraries as described herein are illustrated. The sequence elements shown include inverted tandem repeats (ITRs), a first gene regulatory element (e.g., a cell type-specific and / or tissue-specific promoter) that may be ubiquitous or cell type-specific and / or tissue-specific, a second gene regulatory element (e.g., an AAV2-derived p40 or AAV5-derived p41 promoter) for driving capsid protein expression during AAV library production, a region encoding modifications within the variant capsid protein (e.g., an insertion site containing a peptide display library), a barcode of the variant capsid protein immediately upstream thereof, and a polyadenylation signal and a second ITR.

[0044] Figure 2 Exemplary genomic configurations of two AAV capsid variant member libraries as described herein are shown. The first member library, referred to as “Promoter Construct 1,” contains the pervasive CBh promoter. The second member library, referred to as “Promoter Construct 2,” contains the neuron-specific hSyn1 promoter. For both AAV capsid variant member libraries, the DNA sequence of the promoter barcode is underlined, and the sequence encoding modifications within the variant capsid protein (7-peptide insert) is shown in bold. The promoter barcode is used to identify the promoter that initiates transcription of the mRNA chain and consists of silent mutations that do not alter the amino acid sequence of the capsid variant. For both AAV capsid variant member libraries, the Hamming distance of the barcodes is 8 to enable robust error detection and correction during sequencing.

[0045] Figure 3 An exemplary amplicon recovery strategy, as described herein, is illustrated for capturing barcodes and modifications within variant capsid proteins. After recovering mRNA and converting it to cDNA, the transcript can be analyzed by sequencing. Prior to sequencing, forward and reverse primers are designed to bind upstream of the promoter barcode and downstream of the modification within the capsid variant to be evaluated, respectively. The resulting amplicon is then used to prepare next-generation sequencing libraries and sequenced using many methods or platforms known in the art, including, for example, Illumina high-depth short-read sequencing or Sanger sequencing.

[0046] Figure 4 Two exemplary amplicon recovery strategies, as described herein, are illustrated to obtain total amplicon from all member libraries, or only those transcripts expressed by a specific promoter (i.e., a specific AAV capsid variant member library). To obtain the nucleic acid sequence encoding the modification within the capsid variant from all member libraries, forward and reverse primers (denoted as "F primer 1" and "R primer," respectively) are used, which are complementary to a conserved nucleic acid sequence shared by all member libraries. To obtain the transcript sequence expressed by a single promoter (i.e., a specific AAV capsid variant member library), a forward primer (denoted as "F primer") can be designed to be complementary to one or more nucleotides corresponding to the barcode.

[0047] Figure 5 An exemplary evaluation of the AAV capsid variant performance of Example 1 of this document is shown. For both CBh (ubiquitous promoter) and MHCK7 (muscle-specific promoter) AAV capsid variant member libraries, AAV capsid variant performance is shown as the log2 fold change (i.e., Log2(AAV capsid variant post-transduction abundance / AAV capsid variant pre-transduction abundance) of the expression of each AAV capsid variant normalized to the AAV9 input library. FC, fold change.

[0048] Figure 6 Exemplary configurations of AAV capsid variant member libraries as described in Example 2 herein are shown to identify AAV capsid variants having tropism specificity for central nervous system (CNS) cells (top) and retinal cells (bottom). To identify AAV capsid variants with CNS-specific tropism, the methods described herein will comprise AAV capsid variant member libraries having a pervasive CBh promoter, a neuron-specific hSyn1 promoter, and an astrocyte-specific GFAP promoter, each promoter having a unique barcode. To identify AAV capsid variants with retinal-specific tropism, the methods described herein will comprise AAV capsid variant member libraries having a pervasive CBh promoter, a photoreceptor-specific hGRK1 promoter, and an RPE-specific VMD2 promoter, each promoter having a unique barcode.

[0049] definition To facilitate understanding of this disclosure, certain terms are defined below. Further definitions of the following and other terms are set forth throughout the specification. Publications and other references cited herein to describe the background of this disclosure and to provide additional details regarding its practice are hereby incorporated by reference.

[0050] In this application, unless the context clearly indicates otherwise, (i) the term “a” is to be understood as “at least one”; (ii) the term “or” is to be understood as “and / or”; and (iii) when a range is provided, the endpoints are included.

[0051] about or approximately As used herein, the terms “about” or “approximately” with respect to numerical values ​​are generally considered to include values ​​that fall within the range of 5%, 10%, 15%, or 20% in either direction (greater than or less than), unless otherwise stated or obvious from the context (unless the value is less than 0% or more than 100% of the possible value).

[0052] Adeno-associated virus (AAV):As used herein, the terms “adeno-associated virus” and “AAV” refer to viruses belonging to the family Parvoviridae and the 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 3A 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 rh10, 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, horse AAV, sheep AAV, goat AAV, shrimp AAV, and any variant of the foregoing. AAVs may also include engineered or chimeric forms of wild-type AAVs containing one or more insertions, deletions, and / or substitutions within one or more Cap polypeptides that affect one or more properties of the wild-type AAV serotype, including but not limited to the attraction 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 AAVs are replication-deficient and require co-infection of cells with helper viruses (e.g., adenovirus, herpesvirus, or vaccinia virus) or supplementation with helper virus genes for replication.

[0053] AAV capsid variants: As used herein, the term "AAV capsid variant" refers to an individual AAV virion that has one or more modifications (e.g., one or more changes in the amino acid sequence) in its capsid protein relative to a reference AAV virion (such as wild-type AAV). Such modifications within the variant capsid protein may include one or more of the following: (1) a peptide inserted into a hypervariable region and / or a surface-exposed loop of the variant capsid protein, (2) a point mutation, and (3) a deletion. Such modifications within the variant capsid protein may be located within engineered sites. In some embodiments, an AAV capsid variant refers to a single AAV virion within an AAV peptide display library.

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

[0055] Cell type: As used herein, the term "cell type" refers to cells or groups of cells that have different forms and / or biological characteristics. A variety of characteristics can be used to identify cell types, including, for example: gene expression profiles, epigenetic profiles, non-coding RNA profiles, protein expression profiles, 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, cell types are defined based on specific characteristics or combinations of characteristics. For example, in some embodiments, cell types are defined based on the expression of specific genes or combinations of genes. In some embodiments, cell types may be defined by the tissue of their origin or source (e.g., connective tissue, muscle tissue, nervous tissue, or epithelial tissue).

[0056] Include: Methods described herein as “comprising” or “including” one or more specified elements or steps are open-ended, meaning that the specified elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid verbosity, it should also be understood that any method described as “comprising” (or “comprises”) one or more specified elements or steps also describes a corresponding, more limited method as “consisting substantially of the same specified elements or steps” (or “consisting substantially of the same specified elements or steps”), meaning that the method includes the specified essential elements or steps and may also include additional elements or steps that do not materially affect the essential and novel features of the method. It should also be understood that any method described herein as “comprising one or more specified elements or steps” or “consisting substantially of” also describes a corresponding, more limited, and closed method as “consisting of the specified elements or steps” (or “consisting of”) to exclude any other unspecified elements or steps. In any method disclosed herein, a known or disclosed equivalent of any specified essential element or step may replace that element or step.

[0057] Required features:As used herein, the term “desired characteristic” refers to any number of identifiable characteristics of an AAV capsid variant using the methods described herein, including, for example, transduction (i.e., tropism) in target tissues / cells, 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 elements: As used herein, the term "gene regulatory element" refers to a nucleic acid sequence that controls or influences the expression of a neighboring gene. In some embodiments, a gene regulatory element is a promoter. In some embodiments, a gene regulatory element is an enhancer. In some embodiments, a gene regulatory element is a silencer.

[0059] Hamming distance: As used in this article, the term "Hamming distance" refers to the number of different positions between two nucleic acid sequences of the same length (i.e., the minimum number of substitutions required to change one nucleic acid sequence into a different nucleic acid sequence). For example, the Hamming distance between the sequences TTTGGGTTT and TTTAAATTT is 3.

[0060] Member's Library: As used herein, the term "member library" is synonymous with "AAV member library" or "AAV capsid library" and refers to a single AAV capsid variant library within a plurality of AAV capsid variant libraries. In some embodiments, each AAV member library contains at least one barcode unique to the AAV member library.

[0061] Nucleic acid:The term “nucleic acid” includes any nucleotide, its analogues, and their polymers. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. These terms include RNA or DNA analogues made from nucleotide analogues and modified polynucleotides (such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides) as equivalents. The term covers polynucleotides or oligonucleotides (RNA) and polydeoxynucleotides or oligodeoxynucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleotides and / or modified nucleotides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus bridges (also referred to herein as “nucleotide internucleotide bonds”). This term encompasses nucleic acids containing any combination of nucleotides, modified nucleotides, sugars, modified sugars, phosphate bridges, or modified phosphorus bridges. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing both ribose and modified ribose moieties. In some embodiments, the prefix "poly" refers to nucleic acids 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 nucleic acids containing 2 to about 200 nucleotide monomer units. According to the methods and compositions described herein, in some embodiments, the RNA includes short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR / Cas guide RNA, microRNA (miRNA), and / or precursors thereof.

[0062] Pairing Member Library: As used herein, the term “paired member library” refers to any two AAV capsid variant member libraries that contain a shared gene regulatory element (e.g., a promoter) and two distinct barcodes.

[0063] Silent mutation: As used in this article, the term "silent mutation" refers to a change in a nucleic acid sequence that does not result in a change in the amino acid sequence encoded by that nucleic acid sequence, relative to a nucleic acid sequence without the change.

[0064] basically: As used herein, the term “substantially” refers to a qualitative condition that exhibits the overall or near-overall degree or extent to which the characteristic or property of interest is expressed. Thus, the term “substantially” is used herein to capture the potential lack of absoluteness inherent in many biological and / or chemical effects.

[0065] Variants:As used herein in the context of molecules (e.g., nucleic acids or proteins), the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, for example, in the presence or absence or level of one or more chemical motifs compared to the reference entity. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of its structural identity with the reference molecule. As understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one respect. To cite just a few examples, a polypeptide may have a characteristic sequence element consisting of multiple amino acids at designated positions relative to each other in linear or three-dimensional space and / or contributing to a specific structural motif and / or biological function; a nucleic acid may have a characteristic sequence element consisting of multiple nucleotide residues at designated positions relative to each other in linear or three-dimensional space. In some embodiments, variant peptides or nucleic acids may differ from reference peptides or nucleic acids due to one or more differences in the amino acid or nucleotide sequence and / or one or more differences in the chemical portion (e.g., carbohydrate, lipid, phosphate group) that serves as a covalent linking component of the peptide or nucleic acid (e.g., linked to the peptide or nucleic acid backbone). In some embodiments, variant peptides or nucleic acids exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference peptide or nucleic acid. In some embodiments, variant peptides or nucleic acids do not share at least one characteristic sequence element with the reference peptide or nucleic acid. In some embodiments, the reference peptide or nucleic acid has one or more biological activities. In some embodiments, variant peptides or nucleic acids have one or more biological activities of the reference peptide or nucleic acid. In some embodiments, variant peptides or nucleic acids lack one or more biological activities of the reference peptide or nucleic acid. In some embodiments, variant peptides or nucleic acids exhibit a reduced level of one or more biological activities compared to the reference peptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a “variant” of the reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference, but with minor sequence changes at specific positions. Typically, compared to the reference, the variant has 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 residues substituted, inserted, or deleted. In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference.Typically, compared to a reference, the variant polypeptide or nucleic acid contains a very small number (e.g., less 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 involved in a specific biological activity). In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 insertion or deletion compared to the reference, and in some embodiments, no insertion or deletion is included. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains less 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, or about 6 insertions or deletions, and typically less than about 5, about 4, about 3, or about 2 insertions or deletions. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature.

[0066] Carrier: As used herein, the term "vector" refers to a molecule containing nucleic acid molecules, wherein the vector is capable of transporting the nucleic acid molecules into a cell. As a non-limiting example, one type of vector is a "plasmid," which refers to a circular double-stranded DNA loop capable of attaching an additional DNA segment. Another type of vector is a viral vector, wherein the additional DNA segment can be packaged into a viral capsid and can be transferred to another cell and / or organism. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors). Other vectors (e.g., non-augmented mammalian vectors) can integrate into the host cell's genome upon introduction and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. In this document, such vectors may be referred to as "expression vectors."

[0067] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), the entire text of which is incorporated herein by reference. Detailed Implementation

[0068] This disclosure is partly based on the finding that multiple adeno-associated virus (AAV) capsid variant libraries consisting of member libraries can be used (e.g., substantially simultaneously) to transduce cell populations or tissues, or to incubate with isolated proteins (e.g., receptor proteins), and then screen for desired features 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., the promoter described herein).

[0069] AAV-based vectors are valuable tools for delivering clinically effective payloads (e.g., therapeutic proteins, antisense nucleic acid molecules, ribozymes, miRNAs, siRNAs, or nucleic acids encoding the CRISPR / Cas system) for the treatment of a variety of human diseases. While naturally derived AAV vector capsids have demonstrated clinical efficacy for multiple disease states, there is still a need to identify novel AAV capsids with specific desired characteristics to develop safer and more effective gene therapies. Such desired characteristics may include, but are not limited to, enhanced transduction efficiency (i.e., tropism) in target cell populations or tissues, reduced uptake in non-target cell populations or tissues, and increased immune evasion of circulating neutralizing factors.

[0070] To achieve these goals, significant efforts have been made to discover and engineer novel AAV capsid variants. Numerous methods have been used to identify novel AAV capsid variants with desired characteristics, including but not limited to isolating novel variants from animal tissues, inserting targeting ligands into the capsid surface, and directed evolution methods for selecting and / or screening AAV capsid mutant libraries obtained through error-prone PCR, capsid shuffling, and the insertion of short random peptide sequences into the tolerance region of the AAV capsid.

[0071] Directed evolution is a popular approach for AAV capsid engineering because it does not require prior knowledge or design of AAV capsid variants, as variants with the desired characteristics are recovered after several rounds of iterative selection in target cell populations, tissues, or for purified proteins (e.g., receptor proteins). In such directed evolution strategies, the nucleotide sequence encoding the AAV capsid protein is typically incorporated between inverted terminal repeats (ITRs), such that each capsid encapsulates its original capsid nucleic acid sequence. After transduction in target cell populations or tissues, the AAV-derived nucleic acids can be recovered by PCR or RT-PCR and incorporated into the next round of libraries. This iterative process is repeated until some enriched capsid variants emerge. In each round of selection, the recovered nucleic acids can also be analyzed using next-generation sequencing (NGS) technology to assess capsid variant enrichment relative to the input library.

[0072] Although DNA-based recovery methods are widely used in this field, DNA-based selection alone cannot effectively exclude transduction-deficient AAV capsid variants. For example, capsid variants may be internalized into cells but fail to achieve efficient transduction. Furthermore, the capsid may be trapped in interstitial spaces. Because nucleic acid-based strategies typically use large amounts of tissue as input, the DNA of these transduction-deficient or mesenchymal-trapping capsid variants will still be captured, leading to noise during analysis. Therefore, RNA-based recovery selection strategies (referred to here as “transduction-readout”) are more relevant to clinical translation, as the presence of capsid-derived RNA indicates that the original capsid genome has been delivered to the nucleus, undergoes second-strand synthesis, and becomes transcriptionally active.

[0073] Directed evolution based on transduction readout requires the incorporation of promoters into the AAV genome that can drive capsid mRNA expression in vivo. Unique promoter properties (such as cell type specificity and / or tissue specificity) can be utilized to modulate selection pressure based on the intended target tissue or cell type. For directed evolution approaches, incorporation of ubiquitous promoters (e.g., CAG) enables atlasing of transduction across different tissues and organs. However, this approach is not optimal for selecting capsid variants that transduce enhanced variants in specific cell types within tissues containing heterogeneous cell populations, such as the brain, which consists of neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, and various other cell populations. Furthermore, using ubiquitous promoters in this strategy may lead to the enrichment of capsid variants transduced in non-target cell populations with enhanced transduction. On the other hand, using cell type-specific promoters (e.g., the human synaptic protein 1 promoter for neuron-specific expression) can impose higher selection pressure on capsid variants transducing target cell populations, but sacrifices the ability to atlas transduce in non-target cell populations.

[0074] Prior to this disclosure, no method or composition existed that allowed for the 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, in addition to selecting AAV capsid variants with desired cell type specificity and / or tissue specificity tropism, we have developed a novel directed evolution approach, DEPICT (Directed Evolution Platform Incorporating Cell Type Tropicism), and compositions useful thereto for screening AAV capsid variant transduction at cell type resolution. Such methods described herein achieve this goal in part by introducing promoter barcodes, which in some embodiments contain a silent mutation adjacent to a modification within the variant capsid protein. Using the methods and compositions described herein, a single capsid variant library can be inserted into an AAV vector, each vector having a different promoter and a corresponding barcode, thereby generating multiple AAV capsid variant member libraries. This allows multiple AAV capsid variant member libraries to be combined and applied to a cell population, after which the mRNA transcripts generated by each promoter can be sorted during data analysis using the unique promoter barcode located near the modification within the variant capsid protein.

[0075] AAV Capsid Variants Library This disclosure provides, in particular, compositions and methods for selecting, screening, and identifying AAV capsid variants with desired characteristics. In some embodiments, the methods described herein include transducing cell populations or tissues in vivo or in vitro with a library of multiple AAV capsid variants comprising a single member library to identify one or more AAV capsid variants exhibiting specific tropism. In some embodiments, each member library comprises multiple AAV capsid variants, each variant comprising multiple genetic elements, including, for example, one or more gene regulatory elements, variant capsid proteins, barcodes, polyadenylation signals, and inverted tandem repeats (ITRs) (e.g., such as...). Figure 1 (As shown in the diagram). In some embodiments, the AAV capsid variants within the member library described herein comprise nucleic acid sequences from 5' to 3' corresponding to 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 containing a barcode (e.g., a variant capsid protein), a polyadenylation signal, and a second ITR. In some embodiments, the barcode is located within the protein-coding sequence of the variant capsid protein. In some embodiments, the barcode is constituted by a silent mutation.

[0076] In some embodiments, the first gene regulatory element and barcode may be identical for each AAV capsid variant within the member library. In some embodiments, the second gene regulatory element and barcode may be identical for each AAV capsid variant within the member library. In some embodiments, the 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, the 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, the second gene regulatory element (e.g., p40 or p41) is operatively linked to a nucleotide sequence encoding a variant capsid protein for use in generating an AAV library in a production cell line.

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

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

[0079] Exemplary tissue-specific gene regulatory elements may include, but are not limited to, neuron-specific promoters, astrocyte-specific promoters, oligodendrocyte-specific promoters, muscle-specific promoters, retina-specific promoters, photoreceptor-specific promoters, rod-specific promoters, cone-specific promoters, heart-specific promoters, lung-specific promoters, liver-specific promoters, and kidney-specific promoters.

[0080] Exemplary cell type-specific gene regulatory elements may include, but are not limited to, the human synaptic protein I (hSyn1) promoter, the glial fibrillary acidic protein (GFAP) promoter, the MHCK7 promoter, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, the cerebellar Purkinje cell-specific L7-6 promoter, the synaptic protein I (SynI-minCMV) promoter with a minimal CMV sequence, homeobox Dlx5 / 6, the glutamate receptor 1 (GluR1) promoter, the protakininogen 1 (Tacl) promoter, and the dopaminergic receptor 1. (Drdla) promoter, tubulin αI promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor β chain promoter, glutamate decarboxylase (GAD67) promoter, photoreceptor-specific rhodopsin kinase (RK) promoter, L-opsin promoter, human GRM6 gene-derived (hGRM6) promoter, cone cell transduction α subunit (TαC) promoter, CK8 promoter, desmin promoter, tMCK promoter, dMCK promoter, CK6 promoter, SPc5-12 promoter, D-site binding protein (DBP) promoter, CCAAT enhancer binding protein α or β (C / EBP) promoter; hepatocyte nuclear factor (HNF) promoter and liver-specific IL-6-dependent DNA-binding protein (IL-6DBP) promoter.

[0081] ubiquitous gene regulatory elements In some embodiments, each AAV capsid variant within the member library contains a pervasive gene regulatory element (e.g., a promoter). In some embodiments, each AAV capsid variant within the member library contains a non-naturally occurring pervasive gene regulatory element. Exemplary pervasive gene regulatory elements include, but are not limited to, the hybrid chicken β-actin (CBh) promoter, the CAG promoter, the simian virus 40 (SV40) promoter, the cytomegalovirus (CMV) promoter, the ubiquitin C (UBC) promoter, the elongation factor-1α (EF1A) promoter, the phosphoglycerate kinase 1 (PGK1) promoter, the phosphoglycerate kinase (PGK) promoter, the human β-actin promoter, the β-actin long (BActL) promoter, the chimeric CMV-chicken β-actin promoter (CBA) promoter, the TRE promoter, the U6 promoter, the H1 promoter, the 7SK promoter, or the GUSb promoter.

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

[0083] Modifications within variant capsid proteins In some embodiments, each AAV capsid variant library described herein contains multiple capsid variants, each variant differing in that all such AAV variants contain different modifications within the variant capsid protein. In some embodiments, the modifications within the variant capsid protein comprise one or more non-natural amino acid substitutions, deletions, and / or insertions, or combinations thereof. As described herein, methods for generating AAV capsid variants, each with different modifications, are known in the art (see, for example, U.S. Publication No. US20130310443, the entire contents of which are incorporated herein by reference).

[0084] In some embodiments, the modification within the variant capsid protein comprises a modified nucleotide sequence encoding the V3 capsid protein. In some embodiments, the modification within the variant capsid protein comprises a substitution of at least one non-natural amino acid relative to the wild-type capsid protein. In some embodiments, the modification within the variant capsid protein comprises at least one non-natural amino acid substitution at a position corresponding to a surface-exposed amino acid (e.g., surface-exposed tyrosine) in the wild-type capsid protein. In some embodiments, the modification within the variant capsid protein comprises a non-tyrosine amino acid (e.g., phenylalanine) at a position corresponding to a surface-exposed tyrosine amino acid in the wild-type capsid protein, a non-threonine amino acid (e.g., valine) at a position corresponding to a surface-exposed threonine amino acid in the wild-type capsid protein, a non-lysine amino acid (e.g., glutamic acid) at a position corresponding to a surface-exposed lysine amino acid in the wild-type capsid protein, a non-serine amino acid (e.g., valine) at a position corresponding to a surface-exposed serine amino acid in the wild-type capsid protein, or a combination thereof. In some embodiments, the modification within the variant capsid protein comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions. In some embodiments, the modification within the variant capsid protein comprises at least one non-natural amino acid substitution and at least one peptide insertion. In some embodiments, the modification within the variant capsid protein comprises at least one non-natural amino acid substitution and at least one peptide insertion within the variable region of the capsid.

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

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

[0087] Additional methods for generating modifications within the variant capsid protein are described, for example, in U.S. Patent Nos. 7,790,449; 7,282,199; International Publications Nos. WO2003 / 042397, WO2005 / 033321 and WO2006 / 110689; and U.S. Patent No. 7,588,772, each of which is incorporated herein by reference in its entirety.

[0088] barcode In some embodiments, each AAV capsid variant within the member library described herein comprises one or more nucleic acid sequences, said nucleic acid sequences comprising one or more barcodes and one or more gene regulatory elements (e.g., cell type-specific and / or tissue-specific gene regulatory elements, or ubiquitous gene regulatory elements) (see, for example, Figure 1In some embodiments, all AAV capsid variants within a member library contain the same at least one gene regulatory element and one or more barcodes. In some embodiments, each AAV capsid variant within a member library contains at least one barcode that is different from each AAV capsid variant in a different member library. In some embodiments, each AAV capsid variant within a member library contains two barcodes and one or more gene regulatory elements (e.g., cell type-specific and / or tissue-specific gene regulatory elements, or ubiquitous gene regulatory elements). In some embodiments, two barcodes are linked to one or more gene regulatory elements described herein, such that the nucleic acid sequence identifying either barcode also identifies the nucleic acid sequence of one or more gene regulatory elements driving the expression of the AAV capsid variant.

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

[0090] In some embodiments, the barcode is located within a specific number of nucleotides of one or more modifications within the variant capsid protein, as described herein. In some embodiments, the barcode is located upstream of at least one, two, three, four, five, or more nucleotides encoding one or more modifications within the variant capsid protein. In some embodiments, the barcode is located upstream of no more than one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty nucleotides encoding one or more modifications within the variant capsid protein. In some embodiments, the barcode is located downstream of at least one, two, three, four, five, or more nucleotides encoding one or more modifications within the variant capsid protein. In some embodiments, the barcode is located downstream of no more than one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty nucleotides encoding one or more modifications within the variant capsid protein. In some embodiments, the barcode is located in the nucleotide sequence encoding the VP1 capsid protein. In some embodiments, the barcode is located in the nucleotide sequence encoding the VP2 capsid protein. In some embodiments, the barcode is located in the nucleotide sequence encoding the VP3 capsid protein.

[0091] In some embodiments, the barcode is located at at least one, two, three, four, five, or more nucleotides upstream or downstream of the nucleotide sequence encoding the variable region, said variable region including, for example, VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and VR-IX. In some embodiments, the barcode is located at no more than one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty nucleotides upstream or downstream of the nucleotide sequence encoding the variable region, said variable region including, for example, VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and VR-IX.

[0092] In some embodiments, the barcode is located at at least one, two, three, four, five, or more nucleotides upstream or downstream of the nucleotide sequence encoding an amino acid: (a) the corresponding position in the capsid protein of AAV9 at 262-269 (VR-I) or another serotype; (b) the corresponding position in the capsid protein of AAV9 at 327-332 (VR-II) or another serotype; (c) the corresponding position in the capsid protein of AAV9 at 382-386 (VR-III) or another serotype; (d) the corresponding position in the capsid protein of AAV9 at 452-460 (VR-IV) or another serotype; (e) the corresponding position in the capsid protein of AAV9 at 488-505 (VR-V) or another serotype; (f) the corresponding position in the capsid protein of AAV9 at 527-539 (VR-VI) or another serotype; (g) the corresponding position in the capsid protein of AAV9 at 545-558. (VR-VII) or the corresponding position in the capsid protein of another serotype; (h) 581-593 (VR-VIII) of AAV9 or the corresponding position in the capsid protein of another serotype; (i) 704-714 (VR-IX) of AAV9 or the corresponding position in the capsid protein of another serotype; or (j) any combination or all of (a)-(i).

[0093] In some embodiments, the barcode is located upstream or downstream of the nucleotide sequence encoding the amino acid at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleotides: (a) the corresponding position in the capsid protein of AAV9 at 262-269 (VR-I) or another serotype; (b) the corresponding position in the capsid protein of AAV9 at 327-332 (VR-II) or another serotype; (c) the corresponding position in the capsid protein of AAV9 at 382-386 (VR-III) or another serotype; (d) the corresponding position in the capsid protein of AAV9 at 452-460 (VR-IV) or another serotype; (e) the corresponding position in the capsid protein of AAV9 at 488-505 (VR-V) or another serotype; (f) the corresponding position in the capsid protein of AAV9 at 527-539. (g) The corresponding position in the capsid protein of AAV9 at (VR-VI) or another serotype; (h) The corresponding position in the capsid protein of AAV9 at (VR-VII) or another serotype; (i) The corresponding position in the capsid protein of AAV9 at (VR-VIII) or another serotype; or (j) Any combination or all of (a)-(i).

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

[0095] In some implementations, the barcode contains one or more silencing mutations sufficient to identify, during sequencing analysis (e.g., NGS analysis), which member library the captured mRNA transcript belongs to among the multiple AAV capsid variant libraries. In some implementations, the barcode for each member library differs from the barcodes of all other member libraries within the multiple AAV capsid variant libraries by a Hamming distance of one, two, three, four, or more nucleic acids. In some implementations, the barcode contains nine nucleic acids, and the minimum Hamming distance relative to all other member libraries within the multiple AAV capsid variant libraries is five nucleic acids.

[0096] Inverted tandem repeat (ITR) sequence As described herein, AAV capsid variant libraries may contain ITR sequences derived from any AAV serotype (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAVrh74, AAV-HSC1-17, or variants or hybrids thereof). In some embodiments, the ITR sequence is derived from one or more other serotypes, for example, as described in the following documents: U.S. 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, 8,50 7,267, 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, WO2020 / 028751, or WO2020 / 068990, the full text of each of these documents is incorporated herein by reference.

[0097] ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see, for example, 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™, ViralVectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201 ©Humana Press Inc. 2003. Chapter 10, Targeted Integration by Adeno-Associated Virus; and products and services in U.S. Patent Nos. 5,139,941 and 5,962,313; the entire contents of each of these documents are hereby incorporated by reference).

[0098] Pairing Member Library Two or more member libraries as described herein can be paired in terms of having the same gene regulatory elements (e.g., cell type-specific and / or tissue-specific gene regulatory elements, or pervasive gene regulatory elements) and different barcodes. For example, a first member library containing gene regulatory element A and barcode X and a second member library containing gene regulatory element A and barcode Y are considered paired. In some embodiments, a first member library containing gene regulatory element A and barcode X, a second member library containing gene regulatory element A and barcode Y, and a third member library containing gene regulatory element A and barcode Z are paired.

[0099] Methods for identifying AAV capsid variants This disclosure provides, in particular, a method for identifying one or more AAV capsid variants having desired characteristics. The method for identifying one or more AAV capsid variants may include the steps of: (a) constructing and generating a plurality of AAV capsid variant libraries as described herein; (b) transducing a cell population or tissue with the plurality of AAV capsid variant libraries; (c) recovering one or more AAV capsid variants from the cell population or tissue; and (d) identifying one or more AAV capsid variants having desired characteristics (e.g., cell type and / or tissue-specific tropism). In some embodiments, the identification steps include: (i) extracting mRNA from the cell population or tissue, converting the mRNA to cDNA, and amplifying the cDNA; (ii) sequencing the cDNA amplicon; and (iii) evaluating the performance of the AAV capsid variants as described herein using computational analysis.

[0100] Construction and generation of AAV capsid variant libraries As described herein, methods for identifying AAV capsid variants with desired characteristics may include constructing and generating multiple AAV capsid variant libraries comprising two or more member libraries, wherein each member library contains multiple AAV capsid variants. Methods for constructing AAV vectors are well known in the art. In some embodiments, constructing an AAV capsid variant member library includes cloning an AAV peptide display library into two or more entry vectors, each containing at least one distinct barcode. In some embodiments, the nucleic acid vector encoding the AAV capsid variant member library is then incorporated by transfecting a host cell (e.g., mammalian cells, such as HEK293 cells) to generate the AAV capsid variant library.

[0101] In some embodiments, the generation of the AAV capsid variant library includes transient transfection. In some embodiments, the transient transfection method is suspension transient transfection (sTT). In some embodiments, the transient transfection method is adherent transient transfection. In some embodiments, this disclosure provides a transfected host cell containing at least one AAV capsid variant member library and one, two, three, or four vectors as described herein.

[0102] In some embodiments, the generation of an AAV capsid variant library may include transfecting a host cell with one or more AAV capsid variant libraries and two additional vectors. In some embodiments, the two additional vectors include: (i) a vector encoding at least one AAV Rep polypeptide (e.g., pRep2), and (ii) a vector encoding at least one adenovirus helper polypeptide (e.g., pHelper). In some embodiments, the generation of an AAV capsid variant library includes 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 adenovirus helper polypeptide and at least one AAV Rep polypeptide. In some embodiments, one or more AAV capsid variant libraries are transfected at a molar ratio 10 to 100 times lower than typical triple or double transfection methods. In some embodiments, such methods reduce the incidence of cross-packaging genomes and chimeric capsid formation. In some embodiments, the multiple AAV capsid variant libraries generated as described herein consist primarily of AAV capsid variants containing flanking genomes of the original ITR encoding variant capsid proteins.

[0103] In some embodiments, the generation of an AAV capsid variant library includes transfecting nucleic acids (e.g., containing one or more vectors) with any transfection reagent known to those skilled in the art for introducing nucleic acid molecules into host cells (e.g., mammalian cells, such as HEK293 and / or HEK293T cells). In some embodiments, the transfection reagent comprises lipids, polymers, or combinations thereof. In some embodiments, the transfection reagent is a reagent that forms a complex with the nucleic acid.

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

[0105] In some embodiments, the transfection reagent is or contains a polymer, such as a cationic polymer. In some embodiments, the transfection reagent contains polyethyleneimine (PEI), FectoVIR, TransIT-VirusGEN, or combinations thereof. In some embodiments, the transfection reagent is or contains polyethyleneimine (PEI). In some embodiments, host cells are transfected with PEI. In some embodiments, host cells are transfected with a DNA to transfection agent (e.g., PEI) weight (wt) ratio of about 1:1 to about 1:2, about 1:1 to about 1:5, or about 1:1 to about 1:10, such as 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 implementations, the weight ratio of DNA to transfection agent depends on the cell culture density (e.g., the density of adherent or suspended host cells).

[0106] Transduction of cell populations or tissues As described herein, methods for identifying AAV capsid variants with desired characteristics may include transducing cell populations or tissues with multiple AAV capsid variant libraries. In some embodiments, the multiple AAV capsid variant libraries comprise two, three, four, five, or more member libraries. In some embodiments, the multiple AAV capsid variant libraries comprise at least one member library containing pervasive gene regulatory elements and barcodes different from all other member libraries, and one or more additional member libraries containing cell type-specific and / or tissue-specific gene regulatory elements and barcodes different from all other member libraries. In some embodiments, the multiple AAV capsid variant libraries comprise at least one member library containing pervasive gene regulatory elements and barcodes different from all other member libraries and one or more paired member libraries as described herein. In some embodiments, the transduction step is or includes applying a single premixed composition containing multiple AAV capsid variant libraries to a cell population or tissue. In some embodiments, the transduction step is or includes sequentially applying individual member libraries within the multiple AAV capsid variant libraries to a cell population or tissue.

[0107] In some implementations, cell populations or tissues are transduced with multiple AAV capsid variant libraries in vitro (e.g., using primary neuron cultures and / or iPCS-derived cultures) or in vivo (e.g., in model organisms such as mice, rodents, or non-human primates).

[0108] Screening for AAV capsid variants using isolated proteins As described herein, methods for identifying AAV capsid variants with desired characteristics may include incubating one or more isolated proteins (e.g., one or more receptor proteins) together with a plurality of AAV capsid variant libraries in vitro. In some embodiments, the one or more isolated proteins may comprise any protein. In some embodiments, the one or more isolated proteins may comprise proteins whose expression and / or accumulation are specific to the tissues or cells of an organism capable of AAV transduction. In some embodiments, the one or more isolated proteins may comprise a known or suspected capsid receptor protein. In some embodiments, the one or more isolated proteins comprise one or more portions that facilitate protein purification, including, for example, a hexahistine tag, a glutathione S-transferase tag, a maltose-binding protein, a calmodulin-binding protein, biotin, or streptavidin. In some embodiments, the plurality of AAV capsid variant libraries comprise at least two AAV capsid variant libraries, each with a different barcode. In some embodiments, methods for identifying AAV capsid variants with desired characteristics include further purification of the capsid-protein complex. In some embodiments, the capsid-protein complex is purified using a purification portion. In some implementations, as described elsewhere in this document, AAV capsid variants that interact with isolated proteins (i.e., form complexes) are recovered and sequenced.

[0109] Recover AAV capsid variants from cell populations or tissues As described herein, methods for identifying AAV capsid variants with desired characteristics may include recovering AAV capsid variants from cell populations or tissues. In some embodiments, recovery of AAV capsid variants from cell populations or tissues is performed 1 day, 2 days, 3 days, or longer after transduction. In some embodiments, recovery of AAV capsid variants from cell populations or tissues is performed 1 week, 2 weeks, or 3 weeks after transduction. In some embodiments, the recovery step includes isolating one or more desired cell types (e.g., central nervous system cell types, such as neurons and astrocytes) from cell populations or one or more desired tissues (e.g., brain, kidney, or liver) from a subject. In some embodiments, further processing of one or more isolated cell types or tissues includes, for example, cell sorting (including fluorescence-assisted cell sorting (FACS)), laser microdissection, density gradient centrifugation, or mechanical dissection.

[0110] mRNA extraction and cDNA transformation, amplification and sequencing methods As described herein, methods for identifying AAV capsid variants with desired characteristics may include mRNA extraction, cDNA transformation, and amplification. Methods for mRNA extraction and cDNA transformation are well known in the art. In some embodiments, cDNA is first amplified by PCR using forward and reverse primers prior to amplicon sequencing. In some embodiments, the forward and reverse primers are complementary to the nucleic acid sequences of the AAV capsid variant genome located flanking (i.e., upstream and downstream) the barcode and modification within the variant capsid protein (see, for example, ...). Figure 3 In some embodiments, cDNA is first amplified, resulting in the amplification of a specific genomic region (e.g., containing a barcode encoding a variant capsid protein and a modified nucleic acid) within one or more member libraries (e.g., all member libraries). In some embodiments, the resulting cDNA amplicon containing both the barcode and the modification within the variant capsid protein is analyzed by next-generation sequencing (NGS). In some embodiments, one or more member libraries are selectively amplified from cDNA using at least one primer complementary to one or more nucleotides within the barcode of a member library (see, for example, ...). Figure 4 In some implementations, one or more member libraries are selectively amplified from cDNA using at least one primer that is complementary to all nucleotides of the barcode encoding the member library.

[0111] As described herein, methods for identifying AAV capsid variants with desired characteristics may include amplicon sequencing. Various methods for amplicon sequencing are known in the art. In some embodiments, amplicon sequencing is performed using any method known in the art that leads to nucleic acid sequence identification, including, for example, Sanger sequencing, next-generation sequencing (NGS), and pyro sequencing. In some embodiments, amplicon sequencing is performed using NGS. In some embodiments, the amplicon is of sufficient length to be sequenced on an Illumina short-read sequencer by single-end and / or paired-end sequencing. In some embodiments, the cDNA amplicon (or amplicon amplified therefrom) sequenced by NGS is <500 bp, <400 bp, <300 bp, or <200 bp in length. In some embodiments, amplicon sequencing identifies both the barcode and nucleotide sequence encoding modifications within the variant capsid protein.

[0112] Evaluation of AAV capsid variant performance As described herein, methods for identifying AAV capsid variants with desired characteristics may include using computational analyses to assess AAV capsid variant performance to identify one or more of the following for each AAV capsid variant: (a) modifications within the variant capsid, (b) at least one barcode, (c) a gene regulatory element controlling the expression of the variant capsid protein, and (d) the abundance of AAV capsid variant transcripts. AAV capsid variant performance can refer to any number of measures indicative of the 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 the transcript abundance of variant capsid proteins driven by different gene regulatory elements assessed by sequencing (e.g., NGS). In some embodiments, AAV capsid variant performance involves the tropism specificity conferred by the variant capsid protein.

[0113] In some embodiments, computational analysis is performed using any of the numerous computational methods known in the art, which may include the separation of sequenced transcripts based on barcodes and / or modifications within the variant capsid protein. In some embodiments, AAV capsid variant performance is performed in part by identifying the member library to which each sequenced transcript belongs, said identification being performed by specifying a 5' flanking region corresponding to each barcode sequence and analyzing transcript abundance variations for 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 assessed by comparing (a) the transcript abundance of one or more AAV capsid variants from a first member library with (b) the transcript abundance of the same one or more AAV capsid variants from a second member library. In some implementations, AAV capsid variant performance is assessed by comparing (a) the transcript abundance of one or more AAV capsid variants expressed by cell type-specific and / or tissue-specific gene regulatory elements with (b) the transcript abundance of the same one or more AAV capsid variants expressed by ubiquitous gene regulatory elements (i.e., containing the same modifications within the variant capsid protein) (see, for example, Figure 5 In some implementations, AAV capsid variant performance is assessed by comparing (a) the transcript abundance of one or more AAV capsid variants expressed by cell type-specific and / or tissue-specific gene regulatory elements with (b) the transcript abundance of the same one or more AAV capsid variants expressed by different cell type-specific and / or tissue-specific gene regulatory elements (i.e., containing the same modifications within the variant capsid protein).

[0114] In some embodiments, the transcript abundance of each AAV capsid variant is normalized before evaluating AAV capsid variant performance. In some embodiments, transcript abundance is normalized based on the input AAV library used in the transduction step. In some embodiments, AAV capsid variants with an abundance metric of zero are indicated by adding a pseudo-count of 0.1. In some embodiments, AAV capsid variant performance includes assessing the correlation between the abundance metric of AAV capsid variants with cell type-specific and / or tissue-specific gene regulatory elements and the same AAV capsid variants with ubiquitous gene regulatory elements.

[0115] When the paired member libraries described herein are used to transduce cell populations or tissues, AAV capsid variant performance can be compared between individual AAV capsid variants, each containing the same modifications within the variant capsid protein and driven by the same genetic regulatory elements, but containing different barcodes. In such embodiments, evaluating variant performance includes statistical tests to demonstrate and / or assess the reproducibility of abundance measures of a given AAV capsid variant with different barcodes. In some embodiments, evaluating AAV capsid variant performance includes comparing abundance measures of AAV capsid variants from different cell types and / or tissues. For example, in some embodiments, the transcript abundance of an AAV capsid variant isolated from the central nervous system is compared with abundance measures of the same AAV capsid variant isolated from one or more other tissues described herein.

[0116] AAV serotype This disclosure provides, in particular, methods for screening and identifying AAV capsid variants having desired characteristics. Typically, the AAV capsid variant library used in the methods described herein can have any AAV serotype or contain one or more sequence elements derived from any AAV serotype. In some embodiments, the AAV serotypes used in the methods described herein include, but are not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAVrh74, AAV-HSC1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP-B1 to AAV.CAP-B25, and their variants or hybrids. For example, in some implementations, AAV includes or is an AAV2 / 5, AAV2 / 6, AAV2 / 8 or AAV2 / 9 vector (e.g., AAV6, AAV8 or AAV9 serotype having an ITR derived from AAV2).

[0117] In some implementations, AAV is derived from the AAV genomic sequence or a variant thereof, as described in the following documents: 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, and 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, WO2020 / 028751, or WO2020 / 068990, the full text of each of these documents is incorporated herein by reference.

[0118] In some embodiments, the AAV serotype may have or contain mutations in the AAV9 sequence (e.g., as described in Pulicherla et al., Molecular Therapy, 19(6):1070-1078 (2011), the entire text of which is incorporated herein by reference). The AAV9 serotype may include, but is 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 some embodiments, AAV9 variants include or are variants of AAVhu68 (e.g., as described in International Publication No. WO2018 / 160585, the entire text of which is incorporated herein by reference). Other AAV vectors are described, for example, in Sharma et al., Brain Res Bull., 81(2-3):273 (2010), the entire text of which is incorporated herein by reference.

[0119] In some implementations, AAV includes or is naturally occurring AAV. In some implementations, AAV is a modified AAV or a variant of naturally occurring AAV.

[0120] In some implementations, the AAV capsid library as described herein may comprise or be based on serotypes selected from any of the following serotypes or variants thereof, including but not limited to: AAV9.68, AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.40, 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.11, AAV16.3, AAV16.8 / hu.10, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV1-7 / 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.50, AAV2-5 / rh.51, AAV27.3, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV2G9, AAV-2-p re-miRNA-101, AAV3, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-11 / rh.53, AAV3-3, AAV33.12 / hu.17, AAV33.4 / hu.1 5. AAV33.8 / hu.16, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV 42-12, AAV42-13, AAV42-15, AAV42-1b, 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 / r11.64, AAV4-8 / rh.64, AAV4-9 / rh.54, AAV5, AAV52.1 / hu.20, AAV52 / hu.19、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、A AV7、AAV7.2、AAV7.3 / hu.7、AAV8、AAV-8b、AAV-8h、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9. AV9.24、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.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1 、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAV-h、AAVH-1 / hu.1、AAVH2 、AAVH-5 / hu.3、AAVH6、AAVhE1.1、AAVhER1.14、AAVhEr1.16、AAVhEr1.18、AAVhER1.23、AAVhEr1.35、AAVhE r1.36、AAVhEr1.5、AAVhEr1.7、AAVhEr1.8、AAVhEr2.16、AAVhEr2.29、AAVhEr2.30、AAVhEr2.31、AAVhEr2.3 6、AAVhEr2.4、AAVhEr3.1、AAVhu.1、AAVhu.10、AAVhu.11、AAVhu.12、AAVhu.13、AAVhu.14 / 9、AAVhu.15、AA Vhu.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.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、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.t1 9、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-LK07 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-LK19 PAEC12、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC 8、AAVpi.1、AAVpi.2、AAVpi.3、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.19 AVrh.2、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.2R、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh. h.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.43、AAVrh.44、AAVrh.45、AAV rh.46、AAVrh.47、AAVrh.48、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.50、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.64R1, A AVrh.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, BP63 AAV, Bovine AAV, Goat AAV, Japanese AAV10, True 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] AAV serotypes can originate from any number of species. For example, an AAV can be or include avian AAV (AAAV), as described, for example, in U.S. Patent No. 9,238,800, the entire contents of which are incorporated herein by reference. An AAV serotype can be or include bovine AAV (BAAV), as described, for example, in U.S. Patent Nos. 9,193,769 or 7,427,396, the entire contents of which are incorporated herein by reference. An AAV can be or include goat AAV, as described, for example, in U.S. Patent No. 7,427,396, the entire contents of which are incorporated herein by reference. An AAV serotype can also be a variant or hybrid of any of the foregoing.

[0122] In some implementations, the AAV capsid variant library may be or comprise a serotype generated from an AAV9 capsid library having mutations in amino acids 390 to 627 (VP1 number), for example as described in Pulicherla et al., Molecular Therapy, 19(6):1070-1078 (2011), the full text of which is incorporated herein by reference. In some implementations, AAV serotypes (with corresponding nucleotide and amino acid substitutions) may include, but are 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 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, C179 4A, 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, T1805A; 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, T1530C, 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, T1806C; 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).

[0123] By incorporating references All publications, patent applications, patents, and other references (including GenBank accession numbers) mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described 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 pertains. While similar or equivalent methods and materials may be used in the practice or testing of this invention, suitable methods and materials are described herein.

[0124] Example The following embodiments are provided to provide a complete disclosure and description of how to perform and use the invention for those skilled in the art, and are not intended to limit the scope of the invention as the inventors believe, nor are they intended to represent all or only the experiments performed.

[0125] Example 1: Identification of AAV capsid variants with muscle (tissue) specific tropism introduction As described in this paper, the Directed Evolution Platform Incorporating Cell Type Tendency (DEPICT) is a novel method for screening AAV capsid variant libraries that allows for the identification, selection, and directed evolution of AAV capsid variants with desired tropisms at cell type resolution. DEPICT achieves this unprecedented cell type resolution in part by constructing multiple AAV capsid variant libraries (individually, “member libraries”), each AAV capsid variant within a member library containing a promoter and an associated barcode consisting of silent mutations (see, for example, ...). Figure 1 and Figure 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 cell lines, tissues, or organisms. Following administration, mRNA is recovered, and next-generation sequencing is used to simultaneously identify both the capsid variant and its promoter-associated barcode for each AAV virosomal transcript. The barcode is then used to associate each variant capsid protein with the promoter driving its expression. Therefore, the method described herein constitutes a novel directed evolution strategy that allows for the simultaneous administration of multiple AAV capsid variant libraries, thereby identifying 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.

[0126] Furthermore, DEPICT barcodes can be used as unique molecular identifiers (UMIs). A common obstacle encountered when screening highly diverse mutant libraries is identifying true hits from noise. Noise can be caused by factors such as the randomness of PCR during capsid mRNA amplification in the target tissue or cell of interest. Therefore, if enrichment is observed on multiple DEPICT barcodes associated with the same mutation, DEPICT barcodes can also be used as codon repeats to increase the confidence of calculated fold changes. This can be achieved by introducing multiple DEPICT barcodes for each promoter.

[0127] Materials and methods Will have 1×10 5 Up to 2×10 5A unique capsid variant sequence-diverse AAV peptide display library was cloned into an entry vector containing CBh or MHCK7, the vector including a promoter-specific barcode upstream of the cloning site. The resulting vectors were mixed and transfected into production cells using methods known in the art to generate an AAV capsid variant library. The resulting AAV capsid variant library was administered to cynomolgus monkeys. Three weeks after administration, RNA was isolated from skeletal muscle and AAV mRNA was recovered by RT-PCR using gene-specific RT primers, followed by PCR amplification using primer pairs flanked by an upstream promoter barcode (i.e., a primer complementary to a conserved sequence immediately upstream of the barcode) and flanked by a peptide insertion site (see, for example, ...). Figure 3 Recovered amplicones <200 bp in length underwent NGS library preparation for high-depth short-read sequencing. Following NGS analysis, mRNAs from the CBh or MHCK7 promoter were binned transcripts according to their promoter barcodes. Variant fold change values ​​were calculated based on normalization of the input AAV library. To account for variants with zero reads, a spurious count of 0.1 was added to all variants.

[0128] result In summary, we found a high correlation in variant enrichment between the CBh and MHCK7 promoters in skeletal muscle, particularly in the best-performing variants. Figure 5 This result indicates that CBh and MHCK7 promoter expression is highly consistent in skeletal muscle, while its proportion is relatively small in other cell populations such as fibroblasts and endothelial cells.

[0129] Example 2: Materials and Methods The methods described in this article are used to identify AAV capsid variants that are tropistic towards specific cell types in the central nervous system (CNS) and retina. Figure 6 For CNS and retinal tropism screening, three member libraries were constructed. For each member library used to identify AAV capsid variants with CNS-specific tropism, a highly diverse capsid variant library was cloned into an entry vector containing a promoter-specific barcode upstream of the cloning site and (i) the CBh promoter, (ii) the neuron-specific hSyn1 promoter, or (iii) the astrocyte-specific GFAP promoter. For each member library used to identify AAV capsid variants with retinal-specific tropism, a highly diverse capsid variant library was cloned into an entry vector containing a promoter-specific barcode upstream of the cloning site and (i) the CBh promoter, (ii) the photoreceptor-specific hGRK1 promoter, or (iii) the REP-specific VMD2 promoter.

[0130] The resulting vectors were mixed and transfected into production cells using methods known in the art to generate an AAV capsid variant library. The resulting AAV capsid variant library was administered to cynomolgus monkeys. Three weeks after administration, RNA was isolated from different tissues of each cynomolgus monkey. Specifically, retinal tissue was collected from macaques that received an AAV capsid variant library containing a retina-specific promoter. Similarly, CNS tissue was collected from macaques that received an AAV capsid variant library containing a neuron-specific promoter. In all cases, mRNA was collected and analyzed as described in Example 1 of this document. Furthermore, cDNA from each sample was amplified using forward primers whose sequences were complementary to the barcode of each sample. Figure 4 This allows for the individual amplification of each member library prior to NGS analysis.

[0131] equivalent Those skilled in the art will understand that various changes, modifications, and improvements to this disclosure will readily occur to them. These changes, modifications, and improvements are part of this disclosure and are within the spirit and scope of the invention. Therefore, the foregoing description and drawings are merely exemplary, and any invention described in this disclosure is further described in detail by the appended claims.

[0132] Those skilled in the art will understand that the values ​​obtained by the measurements or other methods described herein are typically subject to standard deviations or errors. All publications, websites, and other references cited herein to describe the background of the invention and to provide additional details about its practice are hereby incorporated by reference in their entirety.

Claims

1. A method for identifying adeno-associated virus (AAV) capsid variants having desired characteristics, comprising: (a) Transducing cell populations or tissues using multiple AAV capsid variant libraries. Each member library within the plurality of AAV capsid variant libraries contains a plurality of AAV capsid variants, and Each AAV capsid variant within the member library contains a nucleic acid sequence comprising: (i) Selected from the following gene regulatory elements: (1) Cell type-specific and / or tissue-specific gene regulatory elements, or (2) Pervasive gene regulatory elements; and (ii) A nucleic acid sequence encoding a variant capsid protein and containing a barcode. 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 identical for each AAV capsid variant within the member library. Within the plurality of AAV capsid variant libraries, each member library has a barcode distinct from all other member libraries, and The barcodes in one or more member libraries contain one or more silent mutations; (b) Recovering the AAV capsid variant from the cell population or tissue; and (c) Identify AAV capsid variants that have the desired characteristics.

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

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

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

5. The method of claim 4, wherein at least two member libraries in the second group are paired because they have the same gene regulatory elements and different barcodes, wherein the gene regulatory elements in the second group are different from the gene regulatory elements in the first group.

6. The method of any of the preceding claims, wherein the barcode of one or more member libraries contains one or more silent mutations in at least one codon encoding the sequence of 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 as described in any of the preceding claims, wherein each AAV capsid variant includes 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 within the plurality of AAV capsid variant libraries, each member library has one or more additional barcodes that are different from each of the other member libraries.

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

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

12. The method as described in any of the preceding claims, wherein the barcode and / or one or more additional barcodes comprise a Hamming distance of one or more nucleic acids.

13. The method as described in any of the preceding claims, wherein the barcode and / or one or more additional barcodes comprise the minimum Hamming distance of nine nucleic acids and five nucleic acids.

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

15. The method of any of the preceding claims, wherein at least one member library within the plurality of AAV capsid variant libraries contains pervasive gene regulatory elements.

16. The method of any of the preceding claims, wherein the ubiquitous gene regulatory element is or comprises a hybrid chicken β-actin (CBh) promoter, a CAG promoter, a simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter, a ubiquitin C (UBC) promoter, an elongation factor-1α (EF1A) promoter, a phosphoglycerate kinase 1 (PGK1) promoter, a phosphoglycerate kinase (PGK) promoter, a human β-actin promoter, a β-actin long (BActL) promoter, a chimeric CMV-chicken β-actin promoter (CBA) promoter, a TRE promoter, a U6 promoter, an H1 promoter, a 7SK promoter, or a GUSb promoter, or any non-naturally occurring ubiquitous gene regulatory element.

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

18. The method of claim 17, wherein the cell type-specific and / or tissue-specific gene regulatory element is the human synaptic protein I (hSyn1) promoter, glial fibrillary acidic protein (GFAP) promoter, MHCK7 promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, cerebellar Purkinje cell-specific L7-6 promoter, synaptic protein I with minimal CMV sequence (SynI-minCMV) promoter, homeobox Dlx5 / 6, glutamate receptor 1 (GluR1) promoter, protakininogen 1 (Tacl) promoter, dopaminergic receptor 1. (Drdla) promoter, tubulin αI promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor β chain promoter, glutamate decarboxylase (GAD67) promoter, photoreceptor-specific rhodopsin kinase (RK) promoter, L-opsin promoter, human GRM6 gene-derived (hGRM6) promoter, cone cell transduction α subunit (TαC) promoter, CK8 promoter, desmin promoter, tMCK promoter, dMCK promoter, CK6 promoter, SPc5-12 promoter, D-site binding protein (DBP) promoter, CCAAT enhancer binding protein α or β (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 of the preceding claims, wherein each member library contains one or more additional gene regulatory elements operatively linked to the sequence encoding the variant capsid protein.

20. The method as claimed in any of the preceding claims, wherein the AAV capsid variants within each member library are distinct from one another based on the presence of one or more of the following modifications within the variant capsid protein: (i) Peptides inserted into the hypervariable region and / or surface-exposed loop of the said variant capsid protein, (ii) point mutations, and (iii) Missing.

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

22. The method of any 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 of the preceding claims, wherein the recovery step comprises separating the desired cell type from the cell population or separating the desired tissue from the tissue.

24. The method as described in any of the preceding claims, wherein the identification step comprises: (i) Isolate mRNA from the cell population or tissue. (ii) Convert the mRNA into cDNA. (iii) Amplify the cDNA to generate a cDNA amplicon, and (iv) Sequencing the cDNA amplicon.

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

26. The method of claim 24 or 25, wherein the transformation step (ii) and / or amplification step (iii) comprises using primers that specifically bind to the barcode or a nucleic acid sequence upstream or downstream of the barcode.

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

28. The method of any one of claims 24 to 27, wherein each of the cDNA amplicon is less than about 500 base pairs in length.

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

30. The method as described in any of the preceding claims, wherein the desired feature is cell type specificity and / or tissue specificity.

31. The method as described in any of the preceding claims, wherein the desired feature is the absence of cell type specificity and / or tissue specificity.

32. The method as claimed in any of the preceding claims, wherein the AAV capsid variant is an AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAVrh.74, or AAV12 capsid variant.

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

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

35. The method as described in any of the preceding claims, wherein the plurality of AAV capsid variant libraries are merged and transduced substantially simultaneously.

36. The method as claimed in any of the preceding claims, wherein the transduction step comprises administration to a mammal.

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

38. A screening system containing multiple AAV capsid variant libraries, Each member library within the plurality of AAV capsid variant libraries contains a plurality of AAV capsid variants, and Each AAV capsid variant within the member library contains a nucleic acid sequence comprising: (i) Selected from the following gene regulatory elements: (1) Cell type-specific and / or tissue-specific gene regulatory elements, or (2) Pervasive gene regulatory elements; and (ii) A sequence encoding a variant capsid protein and containing a barcode. The gene regulatory element is operatively linked to the sequence encoding the variant capsid protein, and the gene regulatory element and the barcode are identical for each AAV capsid variant within the member library. Within the plurality of AAV capsid variant libraries, each member library has a barcode distinct from all other member libraries, and The barcode of one or more member libraries contains one or more silent mutations.

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