Tiling receptor-ligand body weight programming tropism via display peptides

Abstract

Described herein are methods for engineering proteins and viruses to improve tropism, as well as proteins and viruses prepared by using the methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 63 / 405,360, filed on September 9, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under Grants OD032742, CA222826, and GM123313 from the National Institutes of Health and Grant W81XWH-22-1-0401 from the Department of Defense. The government has certain rights in this invention. Technical Field

[0005] Described herein are methods for engineering proteins and viruses for improved tropism, as well as proteins and viruses made by using the methods.

[0006] Incorporation by Reference into the Sequence Listing

[0007] Attached to this application is a sequence listing named "00015-417WO1_SL.xml", which was created on September 8, 2023 and has 872,924 bytes of data, machine formatted on an IBM-PC, MS-Windows operating system. For all purposes, this sequence listing is hereby incorporated herein by reference in its entirety. Background Art

[0008] Nucleic acid- and protein-based therapeutics that modulate health and disease states are poised to become the next frontier in human medicine. However, their successful deployment depends on the ability to effectively deliver them in a safe and targeted manner. To this end, a host of viral and non-viral delivery agents have been developed, but the ability to directly modulate their tropism remains challenging. Summary of the invention

[0009] The present disclosure provides a method for improving the tropism of a virus or other delivery agent, the method comprising identifying a ligand protein sequence derived from all known receptor-interacting ligands; systematically tiling the ligand peptide into a peptide of 5-50 or 10-20 or 20 amino acids, which is inserted into the surface exposed loop of the AAV capsid; assessing the packaging capacity, in vivo tropism and enhanced protein interactions of the engineered capsid. In one embodiment, the virus is an adeno-associated virus (AAV). In another embodiment, AAV is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9. In another embodiment, AAV is AAV5 or AAV9. In another embodiment, a peptide sequence is generated via a merged oligonucleotide synthesis and inserted into 4 different loop regions: AAV5-Loop1 (N443), AAV5-Loop2 (S576), AAV9-Loop1 (Q456) and AAV9-Loop2 (A587) to generate more than 1 million AAV variants. In another embodiment, the 20'mer ligand peptide is inserted into one or both of the two surface exposed loops of AAV5 (SEQ ID NO: 2) or AAV9 (SEQ ID NO: 4).

[0010] The present disclosure provides a recombinant vector comprising a capsid protein, the capsid protein comprising one or two peptides inserted into one or both of the two surface exposed loops in the capsid protein, wherein the vector has a desired tropism or immunological orthogonality, and wherein the peptides are independently selected from the group consisting of SEQ ID NOs: 5 to 820 and 870-911. In one embodiment, the vector is an adeno-associated virus (AAV). In another embodiment, the AAV vector is an AAV5 serotype. In another embodiment, AAV5 comprises a capsid protein having a sequence shown in SEQ ID NO: 2. In another embodiment, the AAV vector is an AAV9 serotype. In another embodiment, AAV9 comprises a capsid protein having a sequence shown in SEQ ID NO: 4. In another embodiment, the vector has a tropism for the pancreas, heart, brain, lung, liver, kidney, muscle, spleen or intestine. In another or additional embodiment, the peptides of SEQ ID NOs: 530-820, 870-910 or 911 are inserted into loop 1 and / or loop 2 of the AAV5 capsid or AAV9 capsid. In another embodiment, the vector is immunoorthogonal. In another embodiment, the vector is an adeno-associated virus (AAV). In another embodiment, the AAV comprises any one of SEQ ID NOs: 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864 or 866, or a capsid protein having a sequence at least 85% to 99% identical to any one of the above sequences.

[0011] The present disclosure also provides a method for preparing a delivery vehicle or vector with a desired tropism, the method comprising selecting a peptide sequence from any one of SEQ ID NO: 5-820 or 870-911, and (i) cloning a nucleic acid sequence encoding a peptide into a coding sequence of a capsid protein at an exposed loop site to obtain a recombinant capsid coding sequence and using the recombinant capsid coding sequence to produce a vector, or (ii) inserting the peptide into the exposed surface of the delivery vehicle. In one embodiment, the vector is an adeno-associated virus (AAV). In another embodiment, AAV is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9. In another embodiment, AAV is AAV5 or AAV9. In another embodiment, the AAV5 capsid coding sequence comprises SEQ ID NO: 1. In another embodiment, the AAV9 capsid coding sequence comprises SEQ ID NO: 2. The present disclosure also provides an AAV vector comprising a capsid protein modified by any of the foregoing embodiments.

[0012] The present disclosure also provides an AAV vector comprising a capsid protein, wherein the capsid protein expresses a peptide of any one of SEQ ID NOs: 5-820 or 870-911 in the exposed surface loop 1 and / or loop 2 of the capsid protein. In one embodiment, the wild-type capsid protein sequence comprises SEQ ID NO: 2 or 4. In another embodiment, the AAV vector has an AAV5 or AAV9 serotype. In yet another embodiment, the vector has a desired tropism. In another embodiment, the AAV vector has a tropism for the pancreas, heart, brain, lung, liver, kidney, muscle, spleen, or intestine.

[0013] The present disclosure also provides a viral vector with a capsid protein, the capsid protein comprising a heterologous targeting peptide of 10-30 amino acids in length inserted into the surface exposed portion of the capsid protein, and wherein the targeting peptide is as described in any one of SEQ ID NO:5-820 or 870-911. In one embodiment, the length of the heterologous targeting peptide is about 15-25 amino acids. In another embodiment, the length of the heterologous targeting peptide is about 20 amino acids. In another embodiment, the viral vector is an adeno-associated virus (AAV). In yet another embodiment, the viral vector is a lentiviral vector. In another embodiment, the capsid protein is a VP1 capsid protein. In yet another embodiment, the capsid protein is a VP2 capsid protein. In another embodiment, the capsid protein is a VP3 capsid protein. In another embodiment, the heterologous targeting peptide is inserted into the AAV capsid protein at loop 1 and / or loop 2. In another embodiment, the viral vector is AAV5. In another embodiment, the viral vector is AAV9. In yet another embodiment, the heterologous targeting peptide is flanked by a linker peptide at the N-terminus and C-terminus of the heterologous targeting peptide. In another embodiment, the heterologous targeting peptide targets the viral vector to hepatocytes or liver tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to neuronal cells or brain tissue. In yet another embodiment, the heterologous targeting peptide targets the viral vector to pancreatic cells or pancreatic tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to cardiomyocytes or heart tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to lung tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to intestinal tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to spleen tissue. In yet another embodiment, the heterologous targeting peptide targets the viral vector to renal cells or renal tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to muscle cells or tissue.

[0014] The present disclosure also provides an adeno-associated virus (AAV) capsid protein, the AAV capsid protein comprising a heterologous targeting peptide cloned into loop 1 and / or loop 2 of the capsid protein, wherein the heterologous targeting peptide is about 10-30 amino acids in length and is contained in or comprises any one of the peptides of SEQ ID NO:5-820 or 870-911. In one embodiment, the capsid protein is VP1 capsid protein. In another embodiment, the capsid protein is VP2 capsid protein. In yet another embodiment, the capsid protein is VP3 capsid protein. In another embodiment, the length of the heterologous targeting peptide is about 15-25 amino acids. In another embodiment, the length of the heterologous targeting peptide is about 20 amino acids. In yet another embodiment, the heterologous targeting peptide is flanked by a linker peptide at the N-terminus and C-terminus of the heterologous targeting peptide. In another embodiment, the heterologous targeting peptide targets hepatocytes or liver tissue. In yet another embodiment, the heterologous targeting peptide targets neuronal cells or brain tissue. In another embodiment, the heterologous targeting peptide targets pancreatic cells or pancreatic tissue. In another embodiment, the heterologous targeting peptide targets cardiomyocytes or cardiac tissue. In another embodiment, the heterologous targeting peptide targets lung tissue. In another embodiment, the heterologous targeting peptide targets intestinal tissue. In another embodiment, the heterologous targeting peptide targets spleen tissue. In another embodiment, the heterologous targeting peptide targets kidney cells or kidney tissue. In another embodiment, the heterologous targeting peptide targets muscle cells or tissue. The present disclosure also provides a recombinant AAV (rAAV) comprising the capsid protein of any of the preceding embodiments.

[0015] The present disclosure provides a recombinant AAV (rAAV), which comprises a capsid protein having a targeting peptide in loop 1 and / or loop 2, wherein the targeting peptide is independently selected from SEQ ID No: 5-820 or 870-911. In one embodiment, the targeting peptide is present in both loop 1 and loop 2. In another embodiment, the targeting peptide has the same tropism. In another embodiment, the recombinant AAV also comprises a heterologous polynucleotide for gene delivery. In another embodiment, the heterologous polynucleotide is a therapeutic gene. In another or additional embodiment of the aforementioned embodiment, the rAAV is present in a pharmaceutical composition.

[0016] The present disclosure also provides a method for delivering a transgene to a subject, the method comprising: administering a recombinant AAV (rAAV) to a subject, wherein the rAAV comprises: (i) a capsid protein of the present disclosure, and (ii) at least one transgene, and wherein the rAAV infects cells of a target tissue of the subject. In one embodiment, at least one transgene encodes a protein. In another embodiment, the protein is an immunoglobulin heavy chain or light chain or a fragment thereof. In another embodiment, at least one transgene encodes a small interfering nucleic acid. In another embodiment, the small interfering nucleic acid is a miRNA. In another embodiment, the small interfering nucleic acid is a miRNA sponge or TuDRNA that inhibits the activity of at least one miRNA in a subject or animal. In another embodiment, the miRNA is expressed in cells of a target tissue. In yet another embodiment, the target tissue is skeletal muscle, heart, liver, pancreas, brain or lung. In another embodiment, the transgene expresses a transcript comprising at least one binding site for a miRNA, wherein the miRNA inhibits the activity of the transgene in tissues other than the target tissue by hybridizing with the binding site. In another embodiment, at least one transgene encodes a gene product that mediates genome editing. In another embodiment, the transgene comprises a tissue-specific promoter or an inducible promoter. In another embodiment, the tissue-specific promoter is a liver-specific thyroxine binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a sarcosine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an α-myosin heavy chain (α-MHC) promoter, or a cardiac troponin T (cTnT) promoter. In another embodiment, the rAAV is administered intravenously, intravascularly, transdermally, intraocularly, intrathecally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation. In yet another embodiment, the subject is selected from mice, rats, rabbits, dogs, cats, sheep, pigs, and non-human primates. In another embodiment, the subject is a human.

[0017] The present disclosure also provides an isolated nucleic acid encoding an AAV capsid protein comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 5-820 and 870-911.

[0018] The present disclosure also provides a delivery vehicle for delivering a small molecule drug or biologic with a desired tropism, wherein the delivery vehicle comprises a peptide or peptide fragment of at least 10-20 amino acids of any one of SEQ ID NO:5-820 or 870-911. In one embodiment, the delivery vehicle is selected from the group consisting of liposomes, nanoparticles, bacteria, bacteriophages, virus-like particles (VLPs), red blood cell ghosts, and exosomes. In another embodiment, biologics include siRNA, antisense molecules, proteins or polypeptides, insulin, vaccines, or antibodies. In yet another embodiment, small molecule drugs include chemotherapeutic agents, anti-inflammatory agents, steroids, and antibiotics.

[0019] The present disclosure also provides a biologic having a desired tropism, the biologic being linked to a peptide or peptide fragment of at least 10-20 amino acids of any one of SEQ ID NOs: 5-820 or 870-911. In one embodiment, the biologic is a nucleic acid, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, a non-immunoglobulin binding agent, or an enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A -B shows the design of an AAV library of peptide-tiled receptor-ligands. (a) Schematic diagram of a method for rationally engineering and characterizing AAV variants. Ligand protein sequences derived from all known receptor-interacting ligands were systematically tiled into 20 amino acid peptides, which were inserted into surface-exposed loops of the AAV capsid. These engineered variants were then evaluated for packaging capacity, in vivo tropism, and enhanced protein interactions. (b) Distribution of protein classes of ligands that were tiled to form the screening library. These include known receptor-interacting ligands (orange), cell membrane permeable proteins (green), and protein domains (blue), as well as negative controls containing stop codons (purple). Peptide sequences were generated via combined oligonucleotide synthesis and inserted into 4 different loop regions: AAV5-Loop1 (N443), AAV5-Loop2 (S576), AAV9-Loop1 (Q456), and AAV9-Loop2 (A587) to generate more than 1 million AAV variants. Capsid surface residues are colored according to their distance from the capsid core, and residues flanking the insertion site are shown in red.

[0021] Figure 2A-F. AAV library packaging analysis reveals biophysical features that contribute to capsid fitness. (a) Schematic diagram of the recombinant generation of a pooled AAV library in HEK293T cells is illustrated. (b) Normalized abundance of each inserted peptide in the plasmid library relative to DNA isolated from a recombinantly generated AAV variant capsid library. The red dashed line shows the position where the plasmid abundance equals the capsid abundance. (c) Normalized abundance of negative control peptides containing stop codons in the plasmid library and the recombinantly generated AAV capsid library (d) Distribution of peptide biophysical parameters associated with packaging. Peptide charge, α-helical content, flexibility, and hydrophobicity distributions of peptides enriched in the capsid pool (“packaging”) and depleted peptides (“non-packaging”) are shown. Statistical significance between groups was calculated via a T test (****p<0.0001). (e) A support vector machine classifier that predicts which AAV variants are successfully packaged into the capsid is trained using the biophysical parameters of the inserted peptides as features. The receiver operating characteristic curve of the resulting model is shown, with an area under the curve of 0.89. (f) UMAP embeddings of each AAV variant, colored by packaging state. Inserted peptide charge, α-helical content, flexibility, and hydrophobicity were used as input features for the embedding.

[0022] Figure 3A -D. In vivo screening analysis enables computational modeling to predict tropism. (a) Overview of the in vivo screening method. Four AAV variant libraries were injected retroorbitally into C57 / BL6 mice in duplicate. Two weeks after injection, nine organs were harvested from each mouse, and the inserted peptide-containing region of the AAV capsid was amplified and subjected to next-generation sequencing. (b) Log of each library and organ 2 Fold change (log 2 (c) Summary of screening results. AAV variants significantly enriched in a specific organ were defined as log 2FC>1 and FDR adjusted p value<0.05. The bar graph shows the number of significantly enriched variants detected for each organ, as well as a comparison of peptide hits for each loop insertion site of AAV5 and AAV9. (d) Overview of the classification model for predicting AAV tissue tropism based on peptide sequence alone. The inserted peptide sequence is converted to a binary one-hot encoding (for each peptide, 20 rows correspond to positions, and 20 columns correspond to the presence of specific amino acids). The one-hot encoding scheme is then used as the input of a convolutional neural network (CNN) to predict organ targeting. The model performance of each organ is evaluated separately via accuracy, area under the receiver operating characteristic curve (AUROC), F1 score, and Matthews correlation coefficient (MCC). The model is trained on 2 / 3 of the data, and the remaining 1 / 3 is retained as a validation data set to evaluate performance. The accompanying drawings disclose SEQ ID NO:867-869 in the order of appearance.

[0023] Figure 4A -D. AAV variants identified by in vivo screening showed reprogramming tropism. (a) The log of each AAV variant significantly enriched in at least one organ is shown. 2 Heatmap of FC values. Rows are individual variants and columns are organs (n=2 per organ). (b) UMAP embedding of significantly enriched AAV variants. Each point represents the region with the largest log 2 Variants of organ coloration of FC. (c) AAVs were selected from a collection of significant hits for validation based on their tissue specificity, broad tropism, and / or internal consistency. Internal consistency was quantified by counting the number of similar (>50% homology) inserted peptides that were also detected as hits for a given organ. AAV variants were structurally characterized via transmission electron microscopy and functionally characterized via in vivo delivery of mCherry transgenes. The heat map depicts all variants selected for validation (n=21), the left heat map shows the Z-normalized log2FC values ​​from the combined in vivo screening (n=2), and the right heat map shows the Z-normalized mCherry expression quantified by RT-qPCR relative to AAV9 (n=2). (d) Comparison between liver RT-qPCR quantification of mCherry delivery and protein level quantification via fluorescence microscopy.

[0024] Figure 5A-D. Characterization and mechanism exploration of AAV variants with displayed ligand peptides. (a) Complete characterization experiment of variant AAV9.DKK1. At the top left, lung screening count values ​​for all AAV9Loop2 variants with inserted DKK1-derived peptides are shown. The x-axis indicates the position where a given peptide starts in the DKK1 structure. Blue shows lung counts, and orange shows capsid counts. The red arrow indicates the position of the peptide inserted into AAV9.DKK1. The bar graph shows RT-qPCR quantification of mCherry transgene expression via individual verification (n=2), normalized to the quantification of AAV9. Electron microscopy images of AAV variant capsids, as well as fluorescence microscopy images of mCherry protein expression levels in the lungs and liver are also shown. (b) Knockout experiment to verify AAV9.DKK1 receptor dependence. Lentivirus containing Cas9 was generated with non-targeting control (NTC) and LRP6 targeting sgRNA (n=2 each). HEK293T cells were then transduced with lentivirus and positively selected with puromycin. Then, the lentiviral-transduced cells were plated in 24-well plates and transfected with wild-type AAV9 (4 × 10 9 viral genomes) or AAV9.DKK1 virus (1×10 9 (a) AAV9.PDGFC was transduced with 100 μg of 1 μg of 5 μg of 1 μg of 1 μg of 2 ... The known receptors of PDGFC ligands were cloned into overexpression plasmids and transfected into HEK293T cells in 24-well plates. After 24 h, AAV9 (4 × 10 9 viral genomes) or AAV9.PDGFC (4×10 9The cells were transduced with AAV9 (1000 viral genomes). 24 hours after transduction, cells were collected and mCherry expression was quantified by flow cytometry. The bar graph shows the MFI normalized to the average MFI of AAV9 transduced cells overexpressing empty vector. Statistical significance between groups was calculated by T test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0025] Fig. 6A -D. Inserted peptides promote efficient liver off-targeting across multiple AAV scaffolds in a mouse strain-independent manner. (a) Significantly enriched AAV variants from all capsids were projected into 2 dimensions via UMAP. Distances between points were explicitly calculated via Levenshtein distance of the amino acid sequence of the inserted peptides and subsequently embedded via UMAP. AAV variants were identified by their log in the brain. 2 (a) Brain and capsid counts of all AAV5Loop2 and AAV9Loop1 variants with inserted APOA1-derived peptides. Brain counts are shown in blue, and capsid counts are shown in orange. Red arrows indicate the locations of peptides inserted into AAV5.APOA1 and AAV9.APOA1, respectively. Electron micrographs of AAV5.APOA1 and AAV9.APOA1 are also shown. (c) RT-qPCR values ​​for individual in vivo validation of AAV5.APOA1 and AAV9.APOA1 in C57 / BL6 mice (relative to transduction with AAV9), confirming liver off-target. (d) Performance of AAV5.APOA1 in BALB / c mice. The bar graph shows RT-qPCR values ​​for AAV5.APOA1 in eight organs relative to transduction with AAV9 in BALB / c, demonstrating that liver off-target is strain independent.

[0026] Fig. 7A-E. Novel immunoorthogonal AAV serotype mining, characterization and tropism reprogramming by displayed ligand peptides. (a) Schematic diagram of the computational pipeline used to identify novel AAV serotypes for testing. The Basic Local Alignment Search Tool (BLAST) was used to identify 687 starting capsids with sequence homology to the AAV2 cap gene. This initial list was then filtered to exclude truncated genomes, redundant samples, human and non-mammalian serotypes, and near-orthologs. The final list contained 23 AAV capsid sequences for subsequent studies. (b) Hierarchical clustering dendrogram of AAV capsid sequences. The previously identified AAV serotypes currently in use are shown in red. The novel AAVs identified and used for downstream testing are shown in black. (c) Two-stage filtering of novel AAVs. AAVs are first evaluated by measuring their packaging capacity and then by their ability to transduce the liver in vivo (using the mCherry transgene). All values ​​shown are relative to the orthologous wild-type AAV5. (d) Four novel AAVs (AAV MM2, AAV MG2, AAV MG1, and AAV CH1) that can infect the liver were tested for immunological cross-reactivity with AAV8. Mice were immunized with the specified AAVs and then tested for antibody cross-reactivity via ELISA 3 weeks after injection. (e) The PDGFC peptide from AAV9.PDGFC was inserted into loop 1 of AAV MG2 to generate AAV MG2.PDGFC. AAV MG2 and AAVMG2.PDGFC were injected into C57BL / 6 mice, and muscle transduction was quantified via RT-qPCR three weeks later. The bar graph shows muscle transduction relative to wild-type AAV MG2. Statistical significance between groups was calculated via T-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). DETAILED DESCRIPTION

[0027] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "a fragment" includes reference to one or more fragments and equivalents thereof known to those skilled in the art, and so forth.

[0028] Furthermore, the use of "or" means "and / or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising," "includes," and "including" are interchangeable and are not intended to be limiting.

[0029] It should also be understood that where the description of various embodiments uses the term “comprising,” those skilled in the art will understand that, in some specific cases, the embodiments may alternatively be described using the language “consisting essentially of” or “consisting of.”

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, exemplary methods and materials are disclosed herein.

[0031] All publications mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing methods that may be used in conjunction with the description herein. In addition, with respect to any terms that appear in one or more publications that are similar or identical to terms explicitly defined in this disclosure, the definitions of the terms as explicitly provided in this disclosure will control in all respects.

[0032] It is to be understood that the present disclosure is not limited to the particular methodology, protocols, reagents, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments or aspects only and is not intended to limit the scope of the present disclosure.

[0033] Except in the operating examples, or when otherwise specified, all the numbers used herein representing the amount of ingredients or reaction conditions should be understood to be modified by the term "about" in all cases. When used to describe the present invention, the term "about" related to percentage means ± 1%. As used herein, the term "about" can mean within the acceptable error range of a specific value as determined by a person of ordinary skill in the art, which may depend in part on how to measure or determine the value, such as the limitation of the measurement system. Alternatively, "about" can mean the range of plus or minus 20%, plus or minus 10%, plus or minus 5% or plus or minus 1% of a given value. Alternatively, particularly with respect to a biological system or method, the term can mean within the order of magnitude of a value, within 5 times or within 2 times. In the case of describing a specific value in the present application and claims, unless otherwise specified, it can be assumed that the term "about" means within the acceptable error range of a specific value. In addition, in the case of providing a range and / or subrange of a value, the range and / or subrange can include the endpoints of the range and / or subrange. In some cases, a change may include an amount or concentration of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of a specified amount.

[0034] For the recitation of numerical ranges herein, each intermediate value with the same degree of precision is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are expressly contemplated.

[0035] As used herein, the term "adeno-associated virus" or "AAV" refers to members of the class of viruses associated with the name and belonging to the genus Dependoviridae of the Parvoviridae family. Multiple serotypes of the virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. Non-limiting exemplary serotypes that can be used in the methods disclosed herein include any of 11 or 12 serotypes, such as AAV2, AAV5, and AAV8, or variant serotypes, such as AAV-DJ. AAV structural particles are composed of 60 protein molecules, which are composed of VP1, VP2, and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins, and 50 VP3 proteins, which are arranged in an icosahedral structure. Non-limiting exemplary VP1 sequences that can be used in the methods disclosed herein are provided below.

[0036] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code and subsequently modified amino acids, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In some embodiments, amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., a carbon a bonded to hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, amino acid mimetics refer to compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. The terms "non-naturally occurring amino acids" and "non-natural amino acids" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature. In some cases, one or more D-amino acids can be used in various peptide compositions disclosed herein. The present disclosure provides a variety of peptides that can be used to treat various diseases and infections. These peptides can include naturally occurring amino acids. In other embodiments, the peptides can include non-natural amino acids. The use of non-natural amino acids can improve the stability of the peptides, reduce degradation and / or improve biological activity. For example, in some embodiments, one or more D-amino acids. In other embodiments, it is contemplated to use retro-inverse peptides of various amino acid configurations.

[0037] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0038] The term "Cas9" refers to a CRISPR-associated, RNA-guided endonuclease, such as Streptococcus pyogenes Cas9 (spCas9; see Accession No. Q99ZW2.1, whose sequence is incorporated herein by reference) and its orthologs and biological equivalents. Bioequivalents of Cas9 include, but are not limited to, C2c1 from Alicyclobacillus acideterrestris and Cpf1 from various bacterial species (including Acidaminococcus spp. and Francisella novicida U112) (which performs a cutting / cleavage function similar to Cas9). Cas9 may refer to an endonuclease that causes double-strand breaks in DNA, a nickase variant that causes single-strand breaks in DNA, such as RuvC or HNH mutants, and other variants that lack endonuclease activity, such as deadCas-9 ("dCas9"). Cas9 may also refer to "split Cas9", in which Cas9 is split into two halves (C-terminal Cas9 (C-Cas9) and N-terminal Cas-9 (N-Cas9)), which can be fused to two intein portions. See, e.g., U.S. Patent 9,074,199B1; Zetsche et al. (2015) Nat Biotechnol. 33(2):139-42; Wright et al. (2015) PNAS 112(10)2984-89. Non-limiting examples of commercially available sources of SpCas9 containing plasmids can be found under the following AddGene reference numbers:

[0039] 42230: PX330; SpCas9 and single guide RNA;

[0040] 48138: PX458; SpCas9-2A-EGFP and single guide RNA;

[0041] 62988: PX459; SpCas9-2A-Puro and single guide RNA;

[0042] 48873: PX460; SpCas9n (D10A nickase) and single guide RNA;

[0043] 48140: PX461; SpCas9n-2A-EGFP (D10A nickase) and single guide RNA;

[0044] 62987: PX462; SpCas9n-2A-Puro (D10A nickase) and single guide RNA; and

[0045] 48137:PX165;SpCas9;

[0046] These examples are incorporated herein by reference.

[0047] As used herein, the term "CRISPR" refers to clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR can also refer to a technique or system for sequence-specific gene manipulation that relies on the CRISPR approach. The CRISPR recombinant expression system can be programmed to cut the target polynucleotide using CRISPR endonucleases and guide RNAs. The CRISPR system can be used to cause double-stranded or single-stranded breaks in the target polynucleotide. The CRISPR system can also be used to recruit proteins or mark target polynucleotides. In some aspects, CRISPR-mediated gene editing utilizes non-homologous end joining (NHEJ) or homologous recombination approaches for editing. These applications of CRISPR technology are known in the art and are widely practiced. See, for example, U.S. Patent 8,697,359 and Hsu et al. (2014) Cell 156 (6): 1262-1278.

[0048] As used herein, the term "delivery vehicle" refers to a composition that can be used to deliver a payload to a cell, tissue, or subject. Delivery vehicles can deliver a variety of payloads, including biologics and small molecule drugs. Exemplary but non-limiting delivery vehicles include liposomes, nanoparticles, bacteria, bacteriophages, virus-like particles (VLPs), red blood cell ghosts, and exosomes.

[0049] As used herein, the term "domain" can refer to a specific region of a larger molecule (e.g., a specific region of a protein or polypeptide) that can be associated with a specific function. For example, a "domain that binds to a homolog" can refer to a domain of a protein that binds to one or more receptors or other protein parts. Similarly, the corresponding coding sequence of a specific polypeptide domain can be referred to as a polynucleotide domain.

[0050] When applied to polynucleotides, the term "encoding" can refer to a polynucleotide that is said to "encode" a polypeptide, which, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or its fragments. In some cases, the antisense strand is the complementary sequence of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0051] The terms "equivalent" or "bioequivalent" are used interchangeably when referring to specific molecular, biological, or cellular materials and refer to those that have minimal homology while still retaining the desired structure or function.

[0052] As used herein, "expression" may refer to the process of polynucleotide transcription into mRNA and / or the process of transcribed mRNA being subsequently translated into peptides, polypeptides or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0053] As used herein, the term "functional" may be applied to the modification of any molecular, biological or cellular material with the intent to cause it to achieve a specific, designated effect.

[0054] As used herein, the term "gRNA" or "guide RNA" refers to a guide RNA sequence used to target a specific gene for correction using CRISPR technology. Techniques for designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J. et al., Nature biotechnology 2014; 32(12): 1262-7, Mohr, S. et al. (2016) FEBS Journal 283: 3232-38 and Graham, D. et al., Genome Biol. 2015; 16: 260. The gRNA comprises or alternatively consists essentially of, or the polynucleotide consists of: a fusion polynucleotide comprising CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA). In some aspects, the gRNA is synthetic (Kelley, M. et al. J of Biotechnology 233 (2016) 74-83).

[0055] "Homology" or "identity" or "similarity" can refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for the purpose of comparison. For example, when a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% identity, or alternatively less than 25% identity, to one of the sequences of the present disclosure.

[0056] Homology refers to the percent identity (%) of a sequence to a reference sequence. In fact, any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein. Whether such a particular peptide, polypeptide or nucleic acid sequence has a particular identity / homology can be routinely determined using known computer programs such as Bestfit programs (Wisconsin Sequence Analysis Package, Version 8 of Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When Bestfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to a reference sequence, parameters can be set so that the percent identity is calculated over the full length of the reference sequence, and homology gaps of up to 5% of the total reference sequence are allowed. A number of sequences are provided herein, and sequences that are at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% and 100% identical to any of the sequences herein are contemplated for use in any of the compositions and methods described herein.

[0057] For example, in a specific embodiment, the identity between a reference sequence (query sequence, i.e., sequence of the present disclosure) and a target sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6: 237-245 (1990)). In some cases, the parameters of the specific embodiments of the identity narrowly interpreted in the FASTDB amino acid alignment may include: scoring scheme = PAM (percentage of mutations accepted) 0, K-tuple = 2, mismatch penalty = 1, connection penalty = 20, random grouping length = 0, cutoff value = 1, window size = sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the length of the target sequence, whichever is shorter. According to this embodiment, if the target sequence is shorter than the query sequence due to N-terminal or C-terminal deletions rather than due to internal deletions, the result can be manually corrected to take into account the fact that the N-terminal and C-terminal truncations of the target sequence are not considered by the FASTDB program when calculating the global identity percentage. For the target sequence truncated at the N-terminus and C-terminus, relative to the query sequence, the percentage of the residue number of the query sequence located at the side of the N-terminus and C-terminus of the target sequence that does not match / align with the corresponding subject residues can be calculated as a percentage of the total bases of the query sequence to correct the identity percentage. Whether the residue is matched / aligned can be determined by the result of the FASTDB sequence comparison. The percentage can then be subtracted from the identity percentage calculated using the specified parameters by the FASTDB program to obtain the final identity percentage score. The final identity percentage score can be used for the purpose of this embodiment. In some cases, for the purpose of manually adjusting the identity percentage score, only the residues at the N-terminus and C-terminus of the target sequence that do not match / align with the query sequence are considered. That is, for this manual correction, only the query residue position outside the farthest N-terminus and C-terminal residues of the target sequence is considered. For example, a target sequence of 90 residues can be compared with a query sequence of 100 residues to determine the identity percentage. The deletion occurs at the N-terminus of the target sequence, so the FASTDB comparison does not show the matching / alignment of the first 10 residues at the N-terminus. 10 unpaired residues represent 10% of the sequence (the number of residues that the N-terminus and C-terminus do not match / the total number of residues in the query sequence), so 10% is subtracted from the identity percentage score calculated by the FASTDB program. If the remaining 90 residues are fully matched, the final identity percentage can be 90%. In another example, the target sequence of 90 residues is compared with the query sequence of 100 residues. This time the deletion is an internal deletion, so there is no residue that does not match / align with the query at the N-terminus or C-terminus of the target sequence. In this case, the identity percentage calculated by FASTDB is not manually corrected.Again, only residue positions outside the N-terminus and C-terminus of the subject sequence that were not matched / aligned with the query sequence as shown in the FASTDB alignment were manually corrected.

[0058] "Hybridization" may refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may include two chains that form a duplex structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. A hybridization reaction may constitute a step in a broader process, such as the initiation of a PC reaction, or enzymatic cleavage of a polynucleotide by a ribozyme.

[0059] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6x SSC to about 10x SSC; a formamide concentration of about 0% to about 25%; and a washing solution of about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9x SSC to about 2x SSC; a formamide concentration of about 30% to about 50%; and a washing solution of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a washing solution of about 1x SSC, 0.1x SSC or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the washing incubation time is about 1 minute, 2 minutes or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It will be appreciated that the equivalent of SSC using other buffer systems may be employed.

[0060] As used herein, the term "immunoorthogonal" refers to the lack of immune cross-reactivity between two or more antigens. In some embodiments, the antigen is a protein (e.g., Cas9). In some embodiments, the antigen is a viral antigen associated with a specific viral vector (e.g., AAV). As recognized in the art, antigens generally include antigenic determinants of a specific sequence with a 3-dimensional structure. In addition, the antigenic determinant may include a domain or subsequence of a larger polypeptide or molecular sequence. In some embodiments, the antigens of the immunoorthogonal do not share an amino acid sequence greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15 or greater than 16 continuous amino acids. In some embodiments, the antigens of the immunoorthogonal do not share any highly immunogenic peptides. In some embodiments, the antigens of the immunoorthogonal do not share affinity for major histocompatibility complexes (e.g., MHC class I or class II). The antigens of the immunoorthogonal are suitable for continuous administration to escape the host immune system.

[0061] The term "immunosilent" refers to an epitope or exogenous peptide, polypeptide or protein that does not elicit an immune response in a host when administered. In some embodiments, the peptide, polypeptide or protein does not elicit an adaptive immune response. In some embodiments, the peptide, polypeptide or protein does not elicit an innate immune response. In some embodiments, the peptide, polypeptide or protein does not elicit an adaptive or innate immune response. In some embodiments, the immunosilent peptide, polypeptide or protein has reduced immunogenicity.

[0062] As used herein, the term "isolated" can refer to molecules or biological products or cell materials that are substantially free of other substances. In one aspect, the term "isolated" can refer to nucleic acids separated from other DNA or RNA, or proteins or polypeptides, or cells or organelles, or tissues or organs present in natural sources, such as DNA or RNA, or proteins or polypeptides (e.g., antibodies or derivatives thereof), or cells or organelles, or tissues or organs. The term "isolated" can also refer to nucleic acids or peptides that are substantially free of cell materials, viral materials or culture media when produced by recombinant DNA technology, or nucleic acids or peptides that are substantially free of chemical precursors or other chemical substances when chemically synthesized. In addition, "isolated nucleic acids" are intended to include nucleic acid fragments that are not naturally present in fragment form and may not exist in natural states. In some cases, the term "isolated" is also used herein to refer to polypeptides separated from other cell proteins, and is intended to include purified and recombinant polypeptides. In some cases, the term "isolated" is also used herein to refer to cells or tissues separated from other cells or tissues, and is intended to include cultured and engineered cells or tissues.

[0063] "Messenger RNA" or "mRNA" is a nucleic acid molecule that is transcribed from DNA and then processed to remove non-coding portions called introns. In some cases, the resulting mRNA is exported from the nucleus (or another locus where the DNA is present) and translated into protein. The term "pre-mRNA" can refer to the chain before processing to remove the non-coding portions. mRNA replaces "T" with "U" in the cDNA coding sequence.

[0064] The term "ortholog" is used to refer to another gene or protein, and means a homolog of the gene or protein that has evolved from the same ancestral source or has been artificially evolved using molecular biology and genetic engineering. Orthologs may or may not retain the same function as their orthologous genes or proteins. Non-limiting examples of Cas9 orthologs include Staphylococcus aureus (S.aureus) Cas9 ("spCas9"), Streptococcus thermophilus (S.thermophiles) Cas9, Legionella pneumophila (L.pneumophilia) Cas9, Neisseria lactamica (N.lactamica) Cas9, Neisseria meningitidis (N.meningitides) Cas9, Bifidobacterium longum (B.longum) Cas9, A.muciniphila (A.muciniphila) Cas9, and O.laneus (O.laneus) Cas9.

[0065] The term "payload" refers to therapeutic and diagnostic agents that can be loaded into or onto a delivery vehicle. Such payloads include biological and small molecule entities. Exemplary payload agents include small molecule drugs, biomolecules, viruses, therapeutic agents, prodrugs, gene silencing agents, chemotherapeutic agents, diagnostic agents, and / or components of gene editing systems. Examples of biomolecules include, but are not limited to, nucleic acids (e.g., DNA, RNA, mRNA, modified mRNA, small RNA, siRNA, miRNA, genes, and transgenes), peptides / proteins (including antibodies, enzymes, transcription factors, etc.), viruses, hormones, carbohydrates, lipids, and vitamins. Examples of gene silencing agents include siRNA, chRNA, miR, ribozymes, morpholinos, and esiRNA. Examples of gene editing systems include, but are not limited to, CRISPR-Cas systems, zinc finger nucleases, and TALENs. Examples of diagnostic agents include, but are not limited to, dyes and stains, radioactive tracers, and contrast agents. Examples of anticancer agents and chemotherapeutic agents that can be used with or loaded into a delivery vehicle include, but are not limited to, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethyleneamine, triethylenephosphoramide, triethylenethiophosphoramide and tiimethylolomelamine; annona lactones (e.g., bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin derivative); ), carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; vinca alkaloids; epipodophyllotoxins; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma ll and calicheamicin omega ll; L-asparaginase; anthracenedione-substituted ureas; methylhydrazine derivatives; enediyne anthracyclines (dynemicins), including enediyne anthracycline A;bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tuberculocidal tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitiaerine; pentostatin; phenamet; pirarubicin; losoxantione; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2 2"-Trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verrucosporin A, baculosporin A and baculosporin); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, for example Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Albumin-engineered paclitaxel nanoparticle formulations without Cremophor oil (American Pharmaceutical Partners, Schaumberg, Ill.) and (docetaxel) (Rhone-Poulenc Rorer, Antony, France); chloranbucil; (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; leucovorin (LV); irinotecan; adrenocortical inhibitors; adrenocortical steroids; progestins; estrogens; androgens; gonadotropin-releasing hormone analogs; and any pharmaceutically acceptable salts, acids or derivatives thereof. Anticancer agents also include antihormonal agents for regulating or inhibiting the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene; aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, for example, 4(5)-imidazoles, aminoglutethimide, Megestrol acetate, exemestane, formestanie, fadrozole, Vorozole, Letrozole and anastrozole; antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation (e.g., PKC-α, Ralf, and H-Ras); ribozymes, such as inhibitors of VEGF-A expression (e.g., ribozymes) and inhibitors of HER2 expression; vaccines, such as gene therapy vaccines, e.g. vaccine, Vaccines and vaccine; rJL-2; Topoisomerase 1 inhibitors; rmRH; antibodies, such as trastuzumab, and any pharmaceutically acceptable salts, acids or derivatives thereof. Examples of protein payloads include mammalian proteins, such as growth hormone (GH), including human growth hormone, bovine growth hormone, and other members of the GH supergene family; growth hormone episomal; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX tissue factor, and von Hippel-Lindau; Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; tumor necrosis factor alpha, tumor necrosis factor beta; enkephalinase; RANTES (normally expressed and secreted by T cells to regulate activation); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; mullerian inhibitory substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-related peptide; DNA enzyme; inhibin; activin; vascular endothelial growth factor (VEGF); receptor for a hormone or growth factor; integrin; protein A or D; rheumatoid factor; neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factor, such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor, such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II); de s(1-3)-IGF-I (brain IGF-D); insulin-like growth factor binding protein; CD proteins, such as CD3, CD4, CD8, CD19 and CD20; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); T-cell receptors; surface membrane proteins; decay accelerating factors (DAFs); viral antigens, such as part of the AIDS envelope; transporters; homing receptors; addressins; regulatory proteins; immunoadhesins; antibodies; and biologically active fragments or variants of any of the above polypeptides.

[0066] Members of the GH supergene family include growth hormone, prolactin, placental lactogen, erythropoietin, thrombopoietin, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-11, interleukin-12 (p35 subunit), interleukin-13, interleukin-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, interferon alpha, interferon beta, interferon gamma, interferon omega, interferon tau, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, cardiotrophin-1, and other proteins identified and classified as members of this family.

[0067] Other payload agents that can be incorporated into the delivery vehicle include gastrointestinal therapeutic agents such as aluminum hydroxide, calcium carbonate, magnesium carbonate, sodium carbonate, etc.; nonsteroidal antifertility agents; parasympathomimetic agents; psychotherapeutic agents; primary sedatives such as chlorpromazine hydrochloride, clozapine, mesoridazine, metiapine, reserpine, and thioridazine; secondary sedatives such as chlordiazepoxide, diazepam, meprobamate, temazepam, etc.; nasal decongestants; sedative-hypnotic drugs such as codeine, phenobarbital, sodium pentobarbital, etc. pentobarbital), sodium secobarbital, etc.; other steroids such as testosterone, testosterone propionate; sulfonamides; sympathomimetics; vaccines; vitamins and nutrients such as essential amino acids, essential fats, etc.; antimalarials such as 4-aminoquinoline, 8-aminoquinoline, pyrimethamine, etc.; antimigraine agents such as mazindol, phentermine, etc.; antiparkinson's agents such as levodopa; antispasmodics such as atropine, methscopolamine bromide, etc.; antispasmodics and anticholinergics such as bile therapy, digestives, enzymes, etc.; antitussives such as dextromethorphan, noscapine, etc.; bronchodilators; cardiovascular agents such as antihypertensive compounds, Rauwolfia alkaloids, etc. alkaloids), coronary vasodilators, nitroglycerin, organic nitrates, pentaerythritol tetranitrate, etc.; electrolyte substitutes, such as potassium chloride; ergot alkaloids, such as ergotamine with and without caffeine, hydrogenated ergot alkaloids, dihydroergocrine mesylate, dihydroergotamine mesylate, dihydroergocryptine mesylate, and combinations thereof; alkaloids, such as atropine sulfate, Belladonna, scopolamine hydrobromide, etc.; analgesics; narcotics, such as codeine, dihydrocodeine, meperidine, morphine, etc.; non-narcotics, such as salicylates, aspirin, acetaminophen, dextropropoxyphene, etc.

[0068] Antibiotic payloads include, for example, cephalosporins, chloramphenicol, gentamicin, kanamycin A, kanamycin B, penicillin, ampicillin, streptomycin A, antimycin A, chloropamtheniol, metronidazole, oxytetracycline, penicillin G, tetracycline, and the like.

[0069] Other payload agents may include vaccines or antigenic agents. For example, payload antigens are derived from microorganisms such as Neisseria gonorrhea, Mycobacterium tuberculosis, herpes viruses (human, type 1 and type 2), Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus, Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.It can carry Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetusintestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovi, equine herpesvirus 1, equine arteritis virus, IBR-IBP virus, BVD-MB virus, Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi), Pseudomonas aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridium botulinum, etc. In other embodiments, the payload may comprise neutralizing antibodies against the above-mentioned microorganisms.

[0070] In other embodiments, the payload can comprise an enzyme such as ribonuclease, neuraminidase, trypsin, glycogen phosphorylase, sperm lactate dehydrogenase, sperm hyaluronidase, adenosine triphosphatase, alkaline phosphatase, alkaline phosphatase esterase, aminopeptidase, trypsin chymotrypsin, amylase, muramidase, acrosomal protease, diesterase, glutamate dehydrogenase, succinate dehydrogenase, β-glycosylphosphatase, lipase, ATP-ase α-peptate γ-glutaminyl transpeptidase, sterol-3-β-alcohol-dehydrogenase, DPN-di-aprorase.

[0071] The present disclosure also encompasses peptide-payload conjugates in which the peptides of the present disclosure are directly linked or fused to payload molecules as described herein, such that the peptide-payload conjugates can be delivered directly (without loading into a delivery vehicle) to target desired tissues based on the tropism of the peptide.

[0072] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (such as a gene). For example, a promoter can be constitutive, inducible, repressible or tissue-specific. A "promoter" is a control sequence that is a region in a polynucleotide sequence that controls transcription initiation and rate. It can contain genetic elements that can bind regulatory proteins and molecules, such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include CMV promoter and U6 promoter.

[0073] The terms "protein", "peptide" and "polypeptide" are used interchangeably and refer to compounds of two or more subunit amino acids, amino acid analogs or peptide mimetics in their broadest sense. The subunits can be connected by peptide bonds. In another embodiment, the subunits can be connected by other bonds (e.g., esters, ethers, etc.). A protein or peptide can contain at least two amino acids, and there is no restriction on the maximum number of amino acids that can contain a protein or peptide sequence. As described above, the term "amino acid" can refer to natural and / or non-natural or synthetic amino acids, including glycine and D and L optical isomers, amino acid analogs and peptide mimetics. As used herein, the term "fusion protein" can refer to a protein composed of domains from more than one naturally occurring or recombinantly produced protein, wherein each domain generally has a different function. In this regard, the term "linker" can refer to a peptide fragment used to connect these domains together-optionally maintaining the conformation of the fusion protein domain and / or preventing adverse interactions between the fusion protein domains, which can impair their respective functions.

[0074] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymer form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs) of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA separated from any sequence, RNA separated from any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after the polynucleotide assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugating with a labeling component. The term can also refer to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the present disclosure, ie, a polynucleotide, may include a double-stranded form and each of two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0075] The term "polynucleotide sequence" may be an alphabetic representation of a polynucleotide molecule. This alphabetic representation may be input into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searches.

[0076] Similarly, the term "polypeptide sequence", "peptide sequence" or "protein sequence" can be an alphabetic representation of a polypeptide molecule. This alphabetic representation can be input into a database in a computer having a central processing unit and used for bioinformatics applications such as functional proteomics and homology searches.

[0077] As used herein, the term "recombinant expression system" refers to one or more genetic constructs used to express certain genetic material formed by recombination.

[0078] As used herein, the term "recombinant protein" may refer to a polypeptide or peptide produced by recombinant DNA technology, wherein typically DNA encoding the polypeptide or peptide is inserted into an appropriate expression vector, which in turn is used to transform host cells to produce the heterologous polypeptide or peptide.

[0079] As used herein, the term "sequencing" may include bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, massively parallel feature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, Enigma sequencing, or any combination thereof.

[0080] As used herein, the term "subject" means any animal. In some embodiments, the subject can be a mammal; in another embodiment, the subject can be a cow, horse, cat, mouse, pig, dog, human or rat.

[0081] As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing exogenous nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents, e.g., (Invitrogen Corp., San Diego, CA), (Invitrogen), (Roche Applied Science, Basel, Switzerland), JETPEI TM (Polyplus-transfection Inc., New York, NY), (Qiagen, Valencia, CA), DREAMFECT TM(OZ Biosciences, France) or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, (1989) and Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, (1984); and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience (1987), each of which is hereby incorporated by reference in its entirety.Additional useful methods are described in handbooks including Advanced Bacterial Genetics (Davis, Roth and Botstein, Cold Spring Harbor Laboratory, 1980), Experiments with Gene Fusions (Silhavy, Berman and Enquist, Cold Spring Harbor Laboratory, 1984), Experiments in Molecular Genetics (Miller, Cold Spring Harbor Laboratory, 1972), Experimental Techniques in Bacterial Genetics (Maloy, in Jones and Bartlett, 1990), and A Short Course in Bacterial Genetics (Miller, Cold Spring Harbor Laboratory 1992), each of which is hereby incorporated by reference in its entirety.

[0082] As used herein, the terms "treat / treating / treatment" refer to improving symptoms associated with a disease or condition (e.g., cancer, Covid-19, etc.), including preventing or delaying the onset of disease or condition symptoms, and / or reducing the severity or frequency of disease or condition symptoms.

[0083] As used herein, the term "vector" may refer to a nucleic acid construct designed to transfer between different hosts, including but not limited to plasmids, viruses, cosmids, phages, BACs, YACs, etc. In some embodiments, "viral vector" is defined as a recombinant virus or viral particle, which comprises a polynucleotide to be delivered to a host cell in vivo, in vitro or in vitro. In some embodiments, a plasmid vector may be prepared by a commercially available vector. In other embodiments, a viral vector may be produced by baculovirus, retrovirus, adenovirus, AAV, etc. according to techniques known in the art. In one embodiment, a viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors, alphavirus vectors, etc. A vector based on infective tobacco mosaic virus (TMV) can be used to manufacture protein, and has been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106 (15): 6099-6104). Alphavirus vectors, such as vectors based on forest encephalitis virus (Semliki Forest virus) and vectors based on Sindbis virus (Sindbis virus), have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al. (1999) Nat. Med. 5 (7): 823-827. In aspects where gene transfer is mediated by retroviral vectors, a vector construct may refer to a polynucleotide comprising a retroviral genome or a portion thereof and a gene of interest. Further details on modern methods for vectors used for gene transfer can be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors containing a promoter and a cloning site that can be operably linked to a polynucleotide are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). In one aspect, the promoter is a pol III promoter.

[0084] Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to as "expression vectors" herein. Generally, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably. However, the disclosure is intended to include such other forms of expression vectors with equivalent functions, such as viral vectors (such as replication-defective retroviruses, adenoviruses and adeno-associated viruses). Generally, vectors or plasmids contain sequences that guide the transcription and translation of one or more related genes, selective markers, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors include 5' regions of genes with transcription initiation control and 3' regions of DNA fragments that control transcription termination. Both control regions can be derived from genes homologous to the transformed host cells, although it should be understood that such control regions can also be derived from genes that are not natural to the species selected as the production host.

[0085] Typically, the vector or plasmid contains sequences that direct the transcription and translation of the gene fragment, a selective marker, and a sequence that allows autonomous replication or chromosomal integration. Suitable vectors contain a 5' region of the gene with a transcription initiation control and a 3' region of the DNA fragment that controls transcription termination. Both control regions can be derived from genes homologous to the transformed host cell, although it should be understood that such control regions can also be derived from genes that are not natural to the species selected as the production host.

[0086] There are many initiation control regions or promoters that can be used to drive expression of the coding regions of the relevant pathways in the desired host cells and are well known to those skilled in the art. In fact, any promoter capable of driving these genetic elements is suitable for use in the present invention, including but not limited to lac, ara, tet, trp, IPL, IPR, T7, tac and trc (can be used for expression in Escherichia coli and Pseudomonas); amy, apr, npr promoters and various phage promoters that can be used for expression in Bacillus subtilis and Bacillus licheniformis; nisA (can be used for expression in Gram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64 (8): 2763-2769 (1998)); and the synthetic Pll promoter (can be used for expression in Lactobacillus plantarum, Rud et al., Microbiology 152: 1011-1019 (2006)). Termination control regions may also be derived from various genes native to the preferred hosts.

[0087] Adeno-associated virus (AAV) is a common gene therapy vector, however, its effectiveness is hampered by poor target tissue transduction and off-target delivery. It is hypothesized that naturally occurring receptor-ligand interactions can be repurposed to engineer tropism. The present disclosure provides a method in which all annotated protein ligands known to bind human receptors are fragmented into tiled 20-mer peptides and displayed at two sites onto surface loops of AAV5 and AAV9 capsids. The resulting capsid library containing >1 million AAV variants was screened in nine tissues of C57BL / 6 mice. Variant abundance was tracked, and >250,000 variants packaged into capsids were identified, as well as >15,000 variants that effectively transduced at least one mouse organ. Twenty-one (21) AAV variants were validated, with 74.3% of organ tropism predictions accurately reproduced, confirming the overall screening efficacy. Systematic ligand tiling enabled prediction of putative AAV-receptor interactions, which were successfully validated by targeted genetic perturbations. Comprehensive peptide tiling also enabled examination of homologous peptide activity. Interestingly, the observed functional peptides often derive from specific domains on the ligand. Notably, certain peptides also showed consistent activity across mouse strains, capsid insertion backgrounds, and capsid serotypes, including novel immunoorthogonal serotypes. Further analysis of the displayed peptides revealed that biophysical properties were highly predictive of AAV variant packaging, and that there was a machine-learnable relationship between peptide sequence and tissue tropism. The disclosed comprehensive ligand peptide tiling and display methods can enable engineering tropism between different viral, virus-like, and non-viral delivery platforms, and elucidate basic receptor-ligand biology.

[0088] The present disclosure provides a variety of peptides that can be used to target desired tissues. The present disclosure also provides immunologically silent peptides. The present disclosure also provides vectors, delivery vehicles, and peptide-payload conjugates containing one or more peptides of the present disclosure having desired tropism.

[0089] Rational screening strategies have great potential to expand the molecular tools available for clinical gene therapy applications. Although AAV engineering work has been underway for more than a decade, advances in DNA synthesis have enabled us to create data-driven libraries of AAV variants that utilize existing functional biomolecules from nature. Compared to random hexamers (and similar methods), using natural biomolecules as a defined source of inserted peptides has multiple benefits. First, under thousands of years of evolutionary selection pressure, the biological functions of natural biomolecules have been pre-filtered. Second, the defined library allows for robust quantification of the fitness of each AAV variant, making it possible to easily stratify AAV variants by infectivity across organs of interest. Although this method has been primarily applied to engineered AAV, mining the properties of functional biomolecules has applications in a wide range of protein engineering challenges, such as engineering orthogonal viral (including lentiviral) and non-viral (including lipid nanoparticles) vectors or identifying biological inhibitors of key protein / protein interactions.

[0090] Additionally, it is believed that the rational ligand tiling approach described herein may lead to important insights into basic AAV-receptor binding and transduction more generally. Previous efforts have utilized large-scale genetic screens, or unique observations of the transduction behavior of specific AAV variants. Since the AAV variants described herein display peptides derived from natural receptor-interacting ligands, it is believed that the AAV variants may retain some of this binding ability, which in part explains their altered transduction profiles.

[0091] The present disclosure focuses on adeno-associated virus (AAV), which has emerged as the main vector for gene delivery in clinical applications. Although a variety of AAV-mediated therapies have been approved by regulatory authorities, it is challenging to effectively direct the treatment to the target tissue by systemic injection. To overcome this, high viral titers are often used in treatment, which is related to potential liver toxicity in clinical trials. Local injections are also problematic, often requiring invasive procedures that may cause organ damage and long recovery times. Due to these delivery challenges, some gene therapy agents have chosen to perform ex vivo treatment designs to overcome targeting issues where feasible, but this can lead to dependence on complex laboratory procedures and high manufacturing costs.

[0092] Therefore, there are many ongoing research efforts to improve in vivo therapeutic targeting, and AAV variants have been engineered to specifically target tissues, such as brain and muscle. This has been primarily established using strategies that repeatedly screen random peptides inserted into the AAV capsid, or capsid shuffling, or random mutagenesis of the capsid sequence as a whole, or direct chemical engineering. Although functional capsids with new properties have been generated via random oligomer mutagenesis of AAV capsids, random mutation screening strategies limit the ability to predict future functional variants, and thus rational and programmable engineering of viral phenotypes remains an elusive goal.

[0093] For the rational engineering of viral function, deep mutation libraries and related functional screening have enabled the systematic mapping of capsid mutation fitness, providing key information that can be used to predict the activity of future variants. In addition, a limited library of pooled oligonucleotides has been used to insert gene fragments derived from proteins with known affinity for synapses into AAV capsids, with the aim of improving reverse axonal transport. Although these methods have provided important insights into AAV engineering, there are still many unknowns about how AAV genotypes affect packaging and tissue transduction. Therefore, there is an urgent need for systematic data sets that map AAV genotypes to clinically relevant properties (such as organ specificity). Given the clinical risk of hepatotoxicity and other efficacy issues associated with off-target transduction, the use of screening techniques to produce highly specific AAV capsids is of great value to the medical and scientific communities.

[0094] With the goal of developing rational and potentially programmable strategies to achieve tissue targeting, it was hypothesized that receptor-ligand interactions that mediate the naturally occurring spectrum of cell-protein and cell-cell interactions could be repurposed to engineer AAV tropism. However, since neither the interaction interfaces nor the cell type-specific profiles of receptor-ligand interactions have been fully mapped, they cannot be comprehensively studied and engineered. To investigate and address this issue, all annotated protein ligands known to bind human receptors were fragmented into tiled short peptides and displayed onto surface loops of the AAV capsid, an approach termed “AAV-PepTile”. Short peptides grafted into stable molecular scaffolds can recapitulate local protein domain architecture, and since protein-protein interface sites are often Specific peptide thermoloops in the 4-8 amino acid (AA) range contribute most to the binding energy of protein-protein interactions, and it was hypothesized that a 20-amino acid peptide inserted into the AAV capsid could significantly alter its binding capacity and, therefore, its transduction profile in vivo. Although insertion of longer peptides could, in principle, more faithfully recapitulate the local ligand structure, AAV generally cannot tolerate insertions larger than 20-25 amino acids without severely compromising capsid packaging efficiency. On this basis, experiments were performed using 20-amino acid peptides + flanking 2-amino acid linker insertions, while a library of >1 million AAV variants was constructed by inserting gene fragments encoding potential receptor-ligands and cell membrane permeabilizing proteins synthesized from corresponding oligonucleotide pools into one of two surface loops on AAV5 and AAV9. Unlike random peptide libraries, ligand tiling enabled robust quantification of tissue transduction rates for all screened variants. Furthermore, systematic examination of the activity of similar peptides yielded predictions of putative receptor interactions that drive the tropism of AAV variants. Quantification of transduction rates in nine organs identified extremely specific variants targeting brain and lung, as well as muscle and heart-targeted variants with broader organ transduction. The resulting data linking AAV variant genotypes to packaging efficacy and tissue specificity expands the understanding of the AAV fitness landscape and provides a unique resource from which further data-driven engineering efforts can be built.

[0095] A general schematic of AAV screening is outlined in Figure 3a. Briefly, in the primary screen, a 20-amino acid peptide library consisting of tiled fragments from all known receptor-interacting ligands and other protein classes of interest flanked by glycine-serine linkers was displayed into two surface-exposed loops of two clinically relevant AAV serotypes (AAV5 and AAV9), with sequences and specific amino acid sites of insertion listed in Table 1.

[0096] Table 1: Shows the DNA and amino acid sequences of the two wild-type AAV serotypes AAV5 and AAV9 used in the screen and the insertion site. In bold and underlined are the flanking residues of the ‘Loop1’ site, and in bold / italic / underlined are the are the flanking residues of the 'Loop2' site.

[0097]

[0098]

[0099]

[0100] These libraries were used to prepare AAV capsid particles, which were injected systemically into mice in duplicate. Two weeks later, the heart, lungs, liver, small intestine, spleen, pancreas, kidneys, brain, and skeletal muscle were removed. Genomic DNA was isolated from these tissues, and the variable peptide regions of interest were amplified and subjected to next generation sequencing (NGS) to measure the log relative to the injected pool in each organ. 2Fold change (log2FC) values ​​were used to quantify AAV abundance in each tissue.

[0101] A candidate list of "hit peptides" in the AAV9Loop1 and AAV9Loop2 scaffolds was synthesized and subjected to deep mutagenesis to generate an AAV library with millions of variants, which was then analyzed in the secondary screening. As performed in the primary screening, AAV capsid particles were produced from these libraries and injected into mice, where tropism was quantified. In addition, to further evaluate the species dependence of the engineered viral particles, AAV capsid libraries were pooled from the primary and secondary screenings and injected into rhesus macaques, which are important preclinical non-human primate (NHP) models. Similar to the experiments performed in mice, various organs were isolated from NHPs 3 weeks after injection, variable peptide regions were amplified, and AAV abundance was quantified in the 4 lobes of the liver, lungs, and brain. In addition, to apply additional selection pressure, variable peptide regions from the heart, lungs, pancreas, intestines, brain, and muscle were amplified and cloned back into the AAV9Loop1 and AAV9Loop2 scaffolds. This tertiary screen was then subjected to two weeks of in vivo selection pressure in mice before tropism was quantified by NGS. For all screens, a “hits” was defined as engineered AAV variants that exhibited a log2FC>1 relative to capsid in two replicates and a p-value<0.1 in at least one organ.

[0102] Shown in Table 2 are peptides present in the starting library of the primary screen that showed significant in vivo activity in all four scaffolds in which they were transplanted. In addition to this list, there is a group of peptides that were consistently identified as hits in the primary, secondary, tertiary and NHP screens. These screen-independent hits for AAV9Loop1 and AAV9Loop2 are shown in Table 3.

[0103] Table 2: All 4 scaffolds present in the primary screening library and in which they were grafted (i.e., AAV5Loop1, Table of "wild-type" peptides that showed in vivo activity in AAV5Loop2, AAV9Loop1, AAV9Loop2). Amino acids are shown sequence .

[0104]

[0105]

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[0107] Table 3: Table of "wild-type" peptides present as hits in primary, secondary, tertiary and NHP screens. is the peptide amino acid sequence

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[0118] Shown in Table 4A-B are the peptides that performed best in all these described screenings. These include mutant forms of peptides initially identified in primary screening. This information includes the amino acid sequence of the peptide, the AAV scaffold into which the peptide is inserted, the organ to which the peptide is hit, the log2FC in the organ, and the screening for identifying the peptide. Although the peptides shown here are highlighted for their ability to transduce specific organs, many of these peptides also show other interesting properties, such as their ability to form functional capsids increases (high titers), their significant de-targeting away from the liver, and their ability to simultaneously target a unique combination of organs. It is expected that these peptides and their mutants have the ability to regulate the tropism of AAV capsids (such as AAV5, AAV9, and 23 kinds of immune orthogonal capsids (as shown in Table 5), etc.), as well as the tropism of other delivery vehicles and peptide-payload conjugates.

[0119] Table 4A: Table of mutant peptides identified as top hits in primary, secondary, tertiary and NHP screening. Shown are Peptide amino acid sequences, the scaffolds into which they were implanted, the log2FC in the organs into which they were hit, and A screen of log2FC was observed. (P = pancreas; H = heart; B = brain; L = lung; Lv = liver; K = kidney; M = muscle; S = spleen; I =Intestine) :

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[0136] Table 4B: Identification of peptides derived from domains showing consistent organ transfection across many variants The following table shows the amino acid sequence, the AAV scaffold in which the peptide was transplanted, the log2 fold change value and its transfection efficiency. The corresponding organs induced by the peptides were screened (P = pancreas; H = heart; B = brain; L = lung; Lv = liver; K = kidney; M = muscle; S = spleen; I = intestine) ：

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[0140] Table 5: Table of the 23 immunoorthogonal AAV serotypes identified and their DNA and amino acid (AA) sequences 。

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[0171] The disclosure shows that one of the variants discovered (AAV9.DKK1) exhibited disrupted transduction when the cognate receptor for the ligand from which the peptide was derived was knocked out (Fig. 5a). Furthermore, due to the systematic tiling nature of the peptide library, it was possible to generate full-length transduction maps across the entire residue space of the ligands used in this study (Fig. 5a, c). This not only increases the confidence of screening hits, as cognate peptides behave similarly, but also provides insight into key domains mediating virus-receptor interactions and, more broadly, provides a platform for further expanding the known protein interactome, particularly when the dataset is integrated with the cell surface group.

[0172] Additionally, in recent years, the development of predictive models for AAV infectivity has attracted great interest from multiple research groups. The application of machine learning in AAV engineering parallels the significant advances in machine learning in multiple areas of protein science, such as structure prediction, enzyme activity prediction, and antibody binding optimization. Although deep learning and similar black-box approaches have rapidly become mature technologies, the application of these methods to AAV engineering is still severely limited by the lack of available training data. The AAV screening data are an ideal training dataset for several reasons: (1) the experimental design features a large, defined library of variants (Figure 1), meaning that each variant has reliable quantification of packaging infectivity, (2) each variant was screened across a panel of nine major organs to map infectivity across different tissue types, (3) the library was inserted into two AAV serotypes and two surface loops, providing important functional information about the capsid context in which the peptides are present, and (4) a large number of variants were validated to demonstrate that the screening data are trustworthy (Figure 4), and (5) illustrating the utility of the dataset as training data, the packaging efficiency of AAV variants can be accurately predicted based on the biophysical properties of the inserted peptides (Figure 2e), and the peptide amino acid sequence also directly predicts tissue tropism across multiple capsids and insertion sites (Figure 3d). Therefore, the screening data will have great utility for the machine learning and computational biology communities.

[0173] Although the variants identified by combined screening have tissue transduction exceeding AAV9 in many organs (Fig. 5a, c), and several variants show severe liver off-target (Fig. 4d, Fig. 6c), further engineering can be performed to enhance efficacy and specificity. Therefore, peptides with at least 85% identity to any of the peptide sequences provided herein are also considered. This may be particularly relevant to certain peptides derived from ligands that bind to receptors expressed on a variety of cell types or have promiscuous binding activity. In the validation experiment, a standard promoter (CMV) was used to drive the expression of the mCherry transgene. Alternatively, tissue-specific promoters can be used to increase the specificity and quantity of transgenic expression in organs of interest. In addition, the hit capsid identified herein can be further engineered to increase activity. Existing hits can be used as scaffolds for additional rounds of targeted mutagenesis and screening, or peptides can be inserted into loops 1 and 2 of the AAV capsid to increase the valence of the displayed ligand. In addition, recently developed direct chemical engineering or peptide display strategies and / or alternative peptide linkers can be used to enhance peptide-mediated transduction. In addition, scRNAseq can be used to screen for hit variants that target more specific cell types within an organ of interest.

[0174] While screening strategies address barriers to organ-targeted gene therapy, pre-existing AAV immune issues may also limit clinical translation. Therefore, experiments computationally identified and fully characterized a panel of four immunoorthogonal AAV capsids (Fig. 7a-d), and showed that insertion of a retargeting peptide identified by the screen could alter the in vivo tropism of one of these identified AAVs (Fig. 7e). This not only increases the number of known serotypes with interesting immunological properties studied with the current AAV panel, but also provides evidence that peptide insertions identified by the screening approach can be transplanted onto different biological scaffolds to achieve unique targeting in a wide range of delivery settings.

[0175] In summary, the present disclosure provides a large number of functional screenings of engineered AAV variants, which span more than one million total variants derived from two capsids and multiple insertion mutagenesis sites. Using this screening data, 21 AAV variants were verified, and AAV capsids with increased organ transduction in multiple organs (heart, muscle and lung of AAV9.PDGFC, Figure 4c) were identified, as well as highly specific AAV capsids (AAV5.APOA1, AAV9.DKK1, Figure 5a). Improved broadly targeted AAV variants have great potential for genetic diseases such as hemophilia A, in which total factor VIII expression levels are the most critical. At the same time, highly specific AAV capsids such as AAV5.APOA1 (which has less than 1% of the liver infectivity of WT AAV9) will have great utility for neurodegenerative disorders, in which maximizing transgenic expression in the brain is essential. In addition to the new variants identified herein, a large amount of screening data itself is also of high value. Considering the scale, reliability and translation relevance of the screening data set, this data set can be used as the basis for future computational engineering of designing AAV capsids.

[0176] The following examples are intended to illustrate rather than limit the present disclosure. Although they are typical procedures that may be used, other procedures known to those skilled in the art may alternatively be used.

[0177] Example

[0178] Mouse

[0179] All animal care and experimental procedures were performed in accordance with the University of California Institutional Animal Care and Use Committee. Male C57Bl / 6J (JAX, #000664) and Balb / cJ (JAX, #000651) mice aged 6-8 weeks were purchased from Jackson Laboratories and injected retro-orbitally with AAV or PBS.

[0180] Cell lines and culture conditions

[0181] All cells were cultured in a humidified incubator at 37° C. with 5% CO 2 . HEK293T cells were cultured in DMEM medium supplemented with 10% FBS, GlutaMAX (1×) (GIBCO) and penicillin-streptomycin (100 U / mL) (GIBCO).

[0182] Visualizing AAV capsid structure

[0183] To obtain the structural representation of AAV capsid, AAV5 and AAV9 structure files were downloaded from the Protein Data Bank. The PyMOL Molecular Graphics System, Version 2.0 These are visualized using LLC and the ramp_new function for coloring the surface capsid representation.

[0184] Design of display peptide libraries

[0185] Each AAV library consists of 275,298 peptides derived from 6,465 proteins. These protein sources were mined from various protein families, including all protein ligands, toxins, nuclear localization signals (NLS), viral receptor binding domains, albumin and Fc binding domains, transmembrane domains, histones, granzymes, and predicted cell penetration motifs cataloged in the Pharmacology Guidance Database. In addition to peptides encoding functional biomolecules, 444 control peptides encoding FLAG tags with premature stop codons are also included. For all human proteins, the cDNA encoding each protein is fragmented in silico to generate DNA encoding all possible 20mer peptides. For viral proteins, cell penetration motifs, and FLAG stop codon controls, the protein sequence is reverse-translated into DNA using the most abundant human codon for each amino acid.

[0186] Oligonucleotide array synthesis, amplification and cloning

[0187] The oligonucleotide library was synthesized by GenScript as three pools of 91,766 elements. Each oligonucleotide library was amplified using KAPA HifiHotstart Readymix, and the manufacturer's recommended cycling conditions were annealing temperature 60°C and extension time 30 seconds. The number of PCR cycles was optimized to avoid over-amplification of the peptide library. After amplification of each oligonucleotide pool and confirmation of the amplicon size on an agarose gel, the amplified sub-libraries were combined to generate a total peptide library of 275,298 elements.

[0188] These pools were cloned into either loop 1 or loop 2 sites to generate pAAV5L1 screen, pAAV5L2 screen, pAAV9L1 screen, and pAAV9L2 screen. AAV rep and cap are flanked by AAV inverted terminal repeat (ITR) sequences to facilitate packaging of the cap gene into recombinant AAV particles.

[0189] Recombinant AAV production

[0190] Using the above library plasmid pools (AAV5-Loop1, AAV5-Loop2, AAV9-Loop1, and AAV9-Loop2), each AAV capsid library was generated by transfecting HEK293T cells with a plasmid library pool (filled with pUC19 DNA at a dilution of 1:100 to prevent capsid cross-packaging) and an adenovirus helper plasmid (pHelper) in 40 15 cm dishes. Titers were determined via qPCR using iTaqUniversal SYBR Green Supermix and primers that bind to the AAV ITR region. To prepare capsid particles as qPCR templates, 2 μL of virus was added to 50 μL of alkaline digestion buffer (25 mM NaOH, 0.2 mM EDTA) and boiled for 8 minutes. Thereafter, 50 μL of neutralization buffer (40 mM Tris-HCl, .05% Tween-20, pH 5) was added to each sample.

[0191] In vivo evaluation of AAV display libraries

[0192] Each AAV capsid library was administered to mice retro-orbitally in duplicate at a dose of 2E12 vg / mouse (for AAV9-based libraries) or 1E12 vg / mouse (for AAV5-based libraries). Two weeks after injection, the heart, lung, liver, intestine, spleen, pancreas, kidney, brain, and gastrocnemius muscle were collected and placed in RNAlater storage solution. Total DNA was extracted from all mouse tissues using TRIzol reagent and TNES-6U reverse extraction method. The resulting precipitated DNA was centrifuged at 18,000G for 15 minutes, and the supernatant was discarded. The DNA pellet was then washed three times with 70% ethanol and finally resuspended in 300 μL EB after the pellet was air-dried.

[0193] Preparation of plasmid and capsid DNA for next-generation sequencing

[0194] In order to sequence the plasmid library (AAV5 / 9 and loop 1 / 2 peptide insertion), 50ng plasmid was used as a template for 50μL KAPAHifi Hotstart Readymix PCR reactions, with an annealing temperature of 60°C and an extension time of 30 seconds. Primers were designed to amplify the peptide coding region from each sub-library. The number of cycles was optimized to avoid over-amplification. According to the manufacturer's protocol, the PCR reaction was purified using a QIAquick PCR purification kit. Thereafter, 50ng PCR amplicon was used as a template for the second 50μL KAPAHifi Hotstart Readymix PCR reaction to add an illumina-compatible adapter and index (NEBNext catalog number E7600S). The PCR reaction was performed with an annealing temperature of 60°C and an extension time of 30 seconds. In order to sequence the capsid library, a similar protocol was performed, using a modified template amount in step-1PCR. To prepare the capsid particles for the PCR template, 2 μL of virus was added to 50 μL of alkaline digestion buffer (25 mM NaOH, 0.2 mM EDTA) and boiled for 8 minutes. Thereafter, 50 μL of neutralization buffer (40 mM Tris-HCl, .05% Tween-20, pH 5) was added to each sample. 1 μL of the digested capsid mixture was then used as a template for a 50 μL PCR reaction. For each sample, the number of cycles was optimized to avoid over-amplification, followed by a secondary PCR to add an illumina-compatible adapter and index. After generating an illumina-compatible library, the plasmid and capsid samples were sequenced on a NovaSeq6000 with an S4 flow cell to generate 100 bp paired-end reads.

[0195] Preparation of tissue DNA for next-generation sequencing

[0196] As with the plasmid / capsid library, a two-step PCR-based library preparation method was used to sequence the AAV cap gene from each tissue for combined screening. For each organ and parallel determination, 300 μL PCR reactions were performed with 120 μl of genomic DNA as a template. For each tissue, the number of cycles was optimized via initial qPCR to avoid over-amplification of the library. All other parameters (such as primers and melting temperatures) were the same as for the PCR of the plasmid library. After this initial PCR, secondary PCR was performed as described above to add illumina-compatible adapters and indexes. The library was then sequenced with an S4 flow cell on a NovaSeq6000 to generate 100 bp paired-end reads.

[0197] In vivo validation of AAV variants

[0198] Physiological saline or AAV- variant-mCherry, AAV9-mCherry or AAV5-mCherry capsids were systematically administered to mice at a dose of 5E11vg / mouse in duplicate. Three weeks after injection, the lungs were inflated with PBS / OCT solution, and lungs, heart, liver, intestines, spleen, pancreas, kidney, brain and gastrocnemius were collected. Each organ was separated, a portion was placed in RNAlater, and the other portion was embedded in an OCT block and rapidly frozen in dry ice / ethanol slurry. Total RNA was then isolated from all mouse tissues using TRIzol reagent and RNA isolation kit by DNA enzyme treatment on column (Zymo catalog number R2072). cDNA synthesis was performed using random primers from Protoscript cDNA synthesis kit (NEB catalog number E6560S). iTaq Universal SYBR green supermix and primers combined with mCherry transcripts were then used to quantify transgenic expression via qPCR. Using GAPDH-specific primers, mCherry transgene expression was normalized to the expression of an internal GAPDH control. For histological examination, OCT frozen blocks were cryosectioned at approximately 10 μm thickness, and tissue slides were imaged on an Olympus SlideScanner S200. An exposure time of 5ms-1000ms was used, with the same exposure time used for all samples of a given tissue type. mCherry expression from tissue sections was then quantified using Olympus OlyVIA software to calculate the average pixel density of the entire organ section.

[0199] Quantification of AAV variant abundance from NGS data

[0200] Starting from the FASTQ sequencing files, a count matrix describing the abundance of AAV in each sample (plasmid / capsid / tissue) was generated using the MAGeCK (94) 'count' function. Following this, the count matrix for each sample was normalized (by multiplying with a constant size factor) to account for unequal read depths. Sequencing counts were then transformed by taking the base-2 logarithm of the raw counts after adding pseudocounts. Variants with no counts in all experimental samples were excluded from the analysis.

[0201] Biophysical analysis of AAV capsids

[0202] The biophysical properties of the inserted peptides were calculated using the “ProteinAnalysis” module within the Biopython Python package. A variant was considered a successful packager if it had a higher abundance in the capsid particles compared to the plasmid pool. Support vector machine training and visualization were performed using the “svm” module within the sklearn Python package (96). UMAP projections of the peptide biophysical properties were performed using the “plot” function within the UMAP Python package. All default parameters were used for visualization. Boxplots and hexagonal boxplots were generated using matplotlib and seaborn Python packages.

[0203] Identification of significantly enriched variants in each tissue

[0204] To identify variants that successfully transduced each tissue, a one-sample T-test was applied to each variant, comparing abundance in capsid particles to abundance in tissues. The resulting p-values ​​were adjusted for multiple hypothesis testing via the Benjamini-Hochberg procedure. Variants were considered positive if their FDR-adjusted p-values ​​in both replicates were < .05 and Log 2 If FC>1, the variant was considered a significant transducer of the organ. When selecting variants for validation experiments, priority was given to variants with insertion peptides identified as hits in multiple capsid / loop contexts, as well as variants that identified similar insertion peptides infecting the same organ.

[0205] Visualizing tissue transduction from pooled screening

[0206] Heatmaps for visualization of AAV transduction were generated using the ‘clustermap’ function in the seaborn Python package. Rows and columns were ordered to minimize the Euclidean distance between adjacent leaves of the dendrogram via the scipy ‘optimal_leaf_ordering’ function. Tissue-level log-squared plots were plotted using the ‘plot’ function in the UMAP Python package. 2 Fold changes were embedded in 2D to generate UMAP projections to visualize AAV tissue specificity. All default parameters were used to generate embeddings. Variants were identified by the variant with the largest log 2 Fold changes in organ coloration.

[0207] Assessing the accuracy of predicted AAV variant tropism

[0208] For each variant that was individually validated, the accuracy of positive and negative predictions for tissue infectivity was assessed. For variants predicted to target a specific organ, a prediction was considered accurate if the individual validation showed greater than 50% wild-type AAV9 infectivity in that organ. For variants predicted not to target a specific organ, a prediction was considered accurate if the individual validation showed less than 50% wild-type AAV9 activity.

[0209] Peptide distance projection

[0210] To calculate the Levenshtein distance between the inserted peptides, the "levenshtein" function from the Python package "rapidfuzz" was used with default parameters (100). After constructing a pairwise distance matrix between all significantly enriched peptides, the matrix was projected into two dimensions via UMAP with metric = "precomputed", n_neighbors = 1500, and min_dist = .1. Clusters of peptides with similar functions were then manually annotated onto the resulting map.

[0211] Convolutional Neural Networks

[0212] To train a convolutional neural network (CNN) to predict tissue specificity of AAV, the AA sequences of the inserted peptides were converted to one-hot encoding via the “get_dummies” function from the pandas Python package. Among the significantly enriched variants, the log relative to the capsid in both replicates was 2If FC is greater than 0, the variant is considered to be a transducer for a given organ. The data was then randomly divided into a training dataset (2 / 3) and a validation dataset (1 / 3). For each variant, the one-hot encoding was reorganized into a 20×20 matrix, where the rows represent the residue positions and the columns represent the presence or absence of a specific amino acid. The model architecture was instantiated via the Keras sequential model (102). In brief, a convolutional layer (Conv1D) with 32 filters, a kernel size of 3, and a "relu" activation was fed to a maximum pooling layer (MaxPool1D) with a pool size of 2. These layers were followed by another set of convolutional and maximum pooling layers, this time with 64 filters in the convolutional layer. These layers were followed by a dense layer with units = 20. Finally, a dropout layer was added with a dropout rate = .5. A flattening layer and a final dense layer (with a sigmoid activation) were then used to output the resulting class probabilities. A separate independent model was trained for each organ. When training the model, the categories (infectious vs. non-infectious variants) were weighted proportionally to the inverse of the number of class examples. When calling 'model.fit()' to train the CNN, a dictionary describing the class weights is passed via the 'class_weight' parameter. Model performance was evaluated via accuracy, area under the receiver operating characteristic curve (AUROC), F1 score, and Matthews correlation coefficient (MCC). Metrics were calculated via built-in Keras functions and plotted via matplotlib. To evaluate how model accuracy changes with changes in the edit distance of the training data, the levenshtein distance between peptides in the test and training datasets was calculated using the 'rapidfuzz' python package described above.

[0213] Transmission Electron Microscopy (TEM)

[0214] To obtain transmission electron microscopy images of selected AAV variants, 20 μL of AAV solution was applied to polyvinyl formal / carbon-coated EM grids. After washing 3 times with water, the sample-containing grids were then negatively stained using a 2% aqueous solution of uranyl acetate for 1 min and blot dried. The EM grids with each AAV variant were then imaged at 68,000x magnification using a FEI Tecnai SpiritG2 BioTWIN transmission electron microscope operating at 80 keV.

[0215] Mechanistic knockout experiments

[0216] sgRNA sequences targeting LRP6 or non-targeting controls were identified using CRISPick and cloned into the lentiCRISPR v2 plasmid backbone. Lentivirus was then produced as described in (33). Briefly, HEK293T cells were seeded at ~40% confluence the day before transfection. On the day of transfection, Optimem low serum medium was mixed with Lipofectamine2000 (Thermo Fisher), 3 μg pMD2.G plasmid, 12 μg pCMV deltaR8.2 plasmid, and 9 μg corresponding lentiCRISPRv2 plasmid and added dropwise to HEK293T cells after a 30-minute incubation period. 48 hours after transfection, the culture medium was collected and replaced with fresh DMEM containing 10% FBS. 72 hours after transfection, the supernatant containing viral particles was collected again, combined, and concentrated to 1 mL using an Amicon-15 centrifugal filter (EMD Millipore) with a 100,000 NMWL cutoff. For lentiviral transduction, HEK293T cells were seeded in 12-well plates at ~20% confluence the day before transduction. On the day of transduction, lentivirus was added to the cells in DMEM (containing 8 μg / mL polybrene). The medium was then changed after 24 hours and then to DMEM containing puromycin (2 μg / mL) 28 hours after transfection. After selection, once the cells reached confluence, they were passaged into 24-well plates at ~40% confluence. After 24 hours, lentivirus was added to the cells in DMEM containing AAV9 (4×10 9 viral genomes) or AAV9.DKK1 (1×10 9 The cells were then collected 24 hours later and subjected to flow cytometry to quantify mCherry transgene expression.

[0217] Identification of immunoorthogonal AAV capsids

[0218] Potential AAV orthologs were first selected by identifying sequences with similarity to the AAV2 cap gene using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST). Next, incomplete, severely truncated, and highly homologous sequences to each other were filtered out from the selection criteria. Viruses within the human AAV clade and viruses from non-mammalian hosts were then filtered out. Finally, sequences with high similarity to previously identified human serotypes were removed, resulting in an evaluation of 23 potential AAV orthologs.

[0219] Immunoorthogonal AAV production

[0220] To clone the computationally identified AAV orthologs, the capsid sequences were codon optimized and cloned downstream of the AAV2 rep gene using Gibson Assembly. Immunoorthogonal AAV capsids were produced as described for validation of capsids for AAV variants, and AAV production titers were measured via qPCR with primers that bind to the AAV ITR region. Any ortholog with a production titer within 10 of AAV5 was considered to have been successfully packaged.

[0221] Immunoorthogonal AAV transduction in vivo

[0222] Then, immunoorthogonal AAV capsids with sufficient packaging titer were added at 1 × 10 12 The dose of 10 viral genomes / mouse was injected retro-orbitally into C57BL / 6 mice. Liver was harvested 3 weeks after injection and total RNA was isolated as described above. cDNA was then generated and transgene expression was quantified via qPCR using iTaq Universal SYBR Green Supermix and primers that bind to mCherry transcripts. mCherry transgene expression was then normalized to GAPDH and relative expression was compared to AAV5.

[0223] Assessment of immune cross-reactivity

[0224] Assays for assessing cross-reactivity of the identified immunoorthogonal immune antibodies were performed as described previously. Prior to injection, sera were collected by tail-snipping procedures and mice were then injected in triplicate with 1×10 12 After 3 weeks, serum was collected from each mouse and subjected to antibody cross-reactivity ELISA. To this end, 1×10 9 AAV8, AAV MM2, AAV MG1, AAV MG2, or AAV CH1 containing 1× viral genomes were diluted in 1× coating buffer and incubated overnight in each well of a 96-well Nunc MaxiSorp plate. The plate was washed three times with 1× wash buffer (Bethyl) for 5 minutes and blocked with 1× BSA blocking buffer (Bethyl) for 2 hours at room temperature. The wells were then washed again and serum samples were added at a dilution of 1:40. The plate was incubated at 4°C with shaking for 5 hours. The wells were washed 3 times and 100 μL of HRP-labeled goat anti-mouse IgG1 (Bethyl; diluted 1:100,000 in 1% BSA) was added to each well. The secondary antibody was incubated for 1 hour at room temperature, the wells were washed 3 times, and 100 μL of TMB substrate was added to each well. The optical density at 450 nm was measured using a microplate absorbance reader (BioRad iMark).

[0225] Engineering immunoorthogonal AAV variants

[0226] To engineer the immunoorthogonal 'AAV MG2' capsid, the AAV9 capsid amino acid sequence was aligned to the MG2 sequence using Clustal Omega (108). The PDGFC peptide coding sequence was then ligated into the appropriate loop 1 position of the 'AAV MG2' vector using the same protocol as that performed for cloning the peptide into AAV9. The engineered 'AAV MG2.PDGFC' vector capsid was then assayed in vivo using AAV9 in the same manner as the validation experiments described above.

[0227] Ethical Compliance

[0228] All experiments involving live vertebrate animals performed at UCSD were performed in accordance with ethical regulations approved by the UCSD IACUC committee.

[0229] Statistics

[0230] Unless otherwise stated, differences in means were calculated by unpaired t-test. P values ​​were adjusted for multiple hypothesis testing by the Benjamini-Hochberg procedure (99) when necessary. For all figures: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 unless otherwise stated.

[0231] Systematic library of AAV variants displaying fragmented proteins

[0232] To create the library, AAV5 and AAV9 were chosen as starting serotypes. This was due to their established clinical utility as well as two key properties: first, AAV5 is evolutionarily more distant from other AAV serotypes in clinical use and has been previously shown to be immunologically orthogonal (to other popular AAV serotypes, thus enabling their serial re-administration); second, AAV9 has been extensively used in clinical trials and has been shown to outperform other AAV serotypes in crossing the blood-brain barrier in most tissues. To generate the library of different AAV variants, a pool of DNA oligonucleotides of 275,298 gene fragments was generated (Figure 1a-b). Each gene fragment encodes a 20-amino acid peptide derived from the coding sequence of a ligand with a known extracellular receptor, or a gene predicted to have cell penetration or internalization properties (Figure 1a-b). Protein ligands were derived from the Pharmacology Database Guide, a carefully curated list of pharmacological targets and their associated ligands, and cell penetration / internalization functions were inferred by text mining of UniProt entries. Examples of protein classes identified as having potential internalization functions include toxins, histones, granzymes, viral receptor binding domains, and nuclear localization signal domains (NLS). The mouse and human genomes share 80% of protein-coding genes, with 85% amino acid sequence identity between orthologs. These genomic similarities extend to the receptors of the ligands in the library (e.g., 86% of mouse GPCR proteins have human orthologs), which means that many human ligands have similar functions in human and mouse backgrounds. After synthesizing a single-stranded oligonucleotide pool encoding these gene fragments, it is amplified into double-stranded DNA via PCR, digested and connected to two different positions on the AAV5 and AAV9 cap genes. Seamless cloning is achieved by cutting outside its recognition sequence by IIS type restriction enzymes. The PaqCI site engineered to produce a compatible overhang (encoding glycine-serine 2 amino acid linker) is inserted into the end of the DNA library encoding the peptide and the site encoding two different surface loops (hereinafter referred to as loop 1 and loop 2) on the AAV5 / AAV9 cap plasmid DNA. Surface loop 1 (AA 443 and 456 on AAV5 and AAV9, respectively) and loop 2 (AA 576 and 587 on AAV5 and AAV9, respectively) were selected as peptide insertion sites due to their distance from the core of the viral particle promoting potential receptor engagement (Figure 1b). Although many previous AAV engineering efforts have focused on inserting peptides at surface loop 2, the loop 1 site has been shown to accommodate large insertions, even including full-length fluorescent proteins. A total of four variant libraries were generated, spanning two AAV capsids, each with two loop insertion sites. In addition to protein-coding gene fragments, 444 gene fragments containing stop codons were included as negative controls. This defined library synthesis method was used to enable quantitative inference of variant packaging and transduction efficiencies.The starting plasmid library was sequenced to determine the initial relative variant abundance, and packaging efficiency was quantified via comparison to this initial baseline.

[0233] Biophysical drivers of AAV capsid formation

[0234] To quantify the extent to which different AAV cap variants are packaged into functional capsids, recombinant AAV particles were produced with engineered AAV5 and AAV9 cap plasmid libraries via transient triple transfection of HEK293T cells (Figure 2a). These viral particles were treated with a universal nuclease to degrade residual plasmid DNA and then subjected to next-generation sequencing (NGS) to quantify relative variant abundance. The logarithm of the relative capsid abundance of the AAV variants was calculated by 2 Fold change (log 2 Packaging efficiency was quantified by ranking the AAV variants by comparing them to the counts in the plasmid pool (Fig. 2b). Using this approach, more than 250,000 AAV variants were identified that were efficiently packaged to log 2 To validate this packaging metric quantified from screening data, 25 AAV capsids were produced, including 23 identified as successful packagers (log 2 FC>0) and 2 identified as non-packers (log 2 FC<0). In these separate validations, 2 non-packagers produced titers >10-fold lower than those identified as packagers, thus providing confidence in the AAV packaging metric. Consistent with their disruption of AAV capsid structure, there was also a depletion of non-functional stop codon-controlled AAV variants in the capsid pool and, importantly, confirmed the lack of library cross-packaging during AAV production ( Figure 2C ).

[0235] To better understand which features contribute to successful capsid formation, the biophysical characteristics of the inserted peptides that produced successfully packaged AAV variants were analyzed. It was found that peptide charge, flexibility, alpha helical content, and hydrophobicity were significantly different in the packaged AAV variants and the AAV variants that could not be packaged (Figure 2d). The successfully packaged variant group had a narrower charge distribution than the variants that could not be packaged, indicating that peptides with extreme charge density had a negative impact on capsid formation. The successfully packaged AAV variants also had inserted peptides with higher flexibility, lower alpha-helical content, and lower hydrophobicity compared to the variants that could not be packaged. The observed depletion of hydrophobic peptide display variants is consistent with the solvent exposure properties of the AAV surface loops.

[0236] In order to establish an integrated model for predicting whether an AAV variant will be packaged based on the biophysical features of the inserted peptides, the charge, flexibility, alpha helical content, and hydrophobicity of the peptides in the data set were used to train a support vector machine classifier (Figure 2e). Although all of these biophysical features were significantly different when comparing packaged and non-packaged AAV variants, the size of this difference was relatively modest for each individual feature (Figure 2d). However, these features were collectively sufficient to train a model that could distinguish between packaged and non-packaged AAV variants (area under the receiver operating characteristic curve = 0.89). For each AAV variant, the charge, flexibility, alpha helical content, and hydrophobicity of the inserted peptides were embedded in two dimensions using uniform manifold approximation and projection (UMAP), allowing such separability to be visualized (Figure 2f). The resulting embedding arranged the AAV variants into distinct clusters, indicating that although each potential biophysical feature is continuous, there are separable groups of AAV variants with similar biophysical features. In this unsupervised embedding, packaged AAV variants tend to cluster with other packaged variants, further supporting the predictive power of these four biophysical blood features. This comprehensive quantitative and analytical framework for assessing AAV packaging, enabled by the diversity and length of the inserted peptide library, is of crucial translational importance for the identified AAV variants, as AAV production costs are directly related to their packaging capacity.

[0237] High-throughput mapping of engineered AAV tissue tropism

[0238] After generating a library of recombinant AAV particles packaging their own cap genes, the next four viral pools were injected into C57BL / 6 mice in duplicate (Figure 3a). Two weeks later, the mice were sacrificed and the peptide-containing region of the AAV cap gene was amplified from DNA isolated from mouse liver, kidney, spleen, brain, lung, heart, skeletal muscle, intestine, and pancreas. The relative abundance of each AAV variant was quantified via NGS, and the log of each variant was calculated relative to its abundance in the capsid pool. 2 enrichment, and good correlation between parallel assays was observed (Fig. 3b). Using this data, more than 15,000 variants that effectively infected at least one mouse tissue were identified (Fig. 3c). Spleen and liver are the most common tissue targets of infectious variants, consistent with the established wild-type (WT) tropism of AAV5 and AAV9 for the liver, and consistent with recent studies showing that AAV5 and AAV9 easily transduce the spleen. The least AAV variants targeting skeletal muscle and brain were identified, which is consistent with the high therapeutic AAV doses required for clinical efficacy of muscle-targeted gene therapy and the challenges of delivery across the blood-brain barrier. A large number of AAV variants consist of the same peptide inserted across different AAV capsids and insertion sites, which confirms the hypothesis that tropism reprogramming is at least partially peptide-specific (Fig. 3c).

[0239] In order to map and understand the tissue transduction patterns mediated by the inserted peptides, the feasibility of training predictive models that link the inserted peptide sequences to tissue tropism was analyzed. Inspired by contemporary work using convolutional neural networks (CNNs) to predict antibody specificity, a CNN multi-label classifier was trained to predict AAV tissue tropism using one-hot encoded inserted peptide sequences as input features (Figure 3d). To evaluate performance, the model was trained using 2 / 3 of the significantly enriched AAV variants randomly selected and evaluated on the 1 / 3 test dataset. The CNN model architecture had good performance in all organs, with the lowest accuracy of 72% in the kidney (Figure 3d). The highest F1 scores and Matthews correlation coefficients (MCCs) were observed in the liver and spleen, which may be due to the large number of liver and spleen targeting variants identified in the combined screening (Figure 3c). It was also observed that for the liver and spleen, the F1 score of the CNN was only moderately reduced when evaluating peptides that were very different from any training examples (greater than 10 edits), meaning that the model has learned generalizable features associated with AAV transduction. For other organs, the model was much less accurate when predicting tissue tropism for peptides that were very different from the training data, likely due to the reduced amount of positive training examples. In summary, the ability to predict AAV tropism supports the conclusion that the inserted peptide mediates retargeting of tissue tropism and that there is a learnable relationship between peptide sequence and tissue tropism.

[0240] Engineered AAV variants with clinically relevant tissue tropism

[0241] Like the WT scaffolds from which they are derived, transduction of multiple organs is an almost universal phenotype in the identified infectious AAV variants (Figure 4a). For most infectious variants, significant transduction of the liver and spleen was observed, regardless of which other organs were co-transduced. This is true even for variants with insertions in surface loops known to be involved in WT capsid receptor binding. Although liver and spleen targeting are almost universal, variants that specifically target the liver / spleen plus one other organ, as well as variants that transduce all tissues at high levels, are allowed to be identified (Figure 4a). When variants are hierarchically clustered based on their tissue detection levels, variants from the same sublibrary tend to cluster together, indicating that the tissue specificity of the wild-type scaffold is at least in part a determinant of the tropism of engineered variants. Hierarchical clustering of organ samples results in parallel determinations clustering together, further improving the reliability of the screening results. In order to visualize the overall screening results, the tissue detection level of each variant was embedded in two dimensions using UMAP, and the variants were colored by the organ they were most likely to transduce (Figure 4b). In this reduced dimensional space, organ-specific clusters can be readily identified, with liver and spleen-targeted variants particularly prominent.

[0242] To confirm the tissue tropism of the new AAV variants identified via the combined screening, 21 variants were generated individually and validated individually by quantifying their ability to package and deliver the mCherry transgene in vivo (Figure 4c). All 21 variants were significantly enriched in at least one organ, and variants that were internally consistent in the screening data were prioritized for validation: consistent AAV capsids were defined as hits, where other variants with similar inserted peptides enriched in the same organ were identified. The variants were initially characterized by in vivo quantification of tissue tropism at the mRNA level (quantified via RT-qPCR of mCherry transgene expression) (Figure 4c). The tissue tropism of the variants largely recapitulated the screening predictions (Figure 4c), with 74.3% of tissue tropism predictions matching expectations. AAV variants that specifically target difficult-to-infect organs such as muscle, lung, and brain, while off-targeting from the liver, were identified (Figure 4c). Notably, quantification of protein levels of mCherry delivered to the liver via fluorescence microscopy confirmed the excellent consistency between mRNA and protein measurements of tissue transduction (R 2 =0.97) (Figure 4d). In summary, of the variants tested individually, 9 / 21 variants were found to exceed AAV9 infectivity in at least one organ. Variants that exceeded AAV9 infectivity in all organs except the liver and pancreas were identified, with the liver and pancreas having maximum relative transduction of 98.8% and 82.2% of WT AAV9, respectively. In addition, liver transduction of 18 / 21 variants was less than half of that of WT AAV9, with three variants falling below 5% AAV9 liver transduction levels (Figure 4d), indicating clinically important liver off-targets. To increase confidence in the fidelity of the individually validated variants in C67BL / 6 mice, three of the above 21 variants were individually validated in BALB / c mice. Notably, their relative tropism was consistent in both mouse strains.

[0243] Mechanistic understanding of AAV reprogramming

[0244] After confirming the efficacy of AAV variants, experiments were conducted to explore the potential mechanism of its reprogramming tropism, particularly to study whether the hypothesis that AAV variants display peptides derived from different ligand domains can indeed drive its transduction pattern. First, experiments were conducted to explore how peptides derived from specific ligand protein regions change AAV tropism. This is driven by observing that the peptide enrichment pattern tiled in screening can produce an in-depth understanding of the functional domains of the protein from which they are derived. How peptides are derived is analyzed. For this reason, experiments were conducted to explore the tissue-specific AAV variants identified in screening, first focusing on the lung-specific variants (called AAV9.DKK1) derived via peptide fragments of the DKK gene displayed on the AAV9-Loop2 scaffold. Specifically, as shown in Figure 5a (upper left), it was observed that the lung transduction of the peptide derived from the specific domain in the N-terminus of the protein was significantly enhanced. After confirming that the variant was packaged into a functional capsid via an electron microscope (Figure 5a, upper right), the following experiment quantified its in vivo transduction profile. This confirmed that AAV9.DKK1 was indeed an efficient lung transducing variant, with 2- to 3-fold higher expression than AAV9 and consistent off-targeting compared to AAV9 in all other organs when quantified at both RNA and protein levels (Fig. 5a, bottom left and right).

[0245] Next, experiments were performed on DKK1 display peptides to determine insights into the transduction mechanism. Interestingly, the lung-enriched N-terminal region (Figure 5a, top left) is concentrated on an evolutionarily conserved linear peptide motif that mediates binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 / 6). Since HEK293T cells robustly express LRP6, lentiviral-mediated CRISPR-Cas9 was used to disrupt LRP6 expression in these cells to study the relative transformability of AAV9.DKK1 variants in this system (Figure 5b). Specifically, cells transduced with a non-targeting control (NTC) guide or an LRP6-targeted guide were subsequently transduced with AAV9.DKK1 or wild-type AAV9. Using flow cytometry to quantify AAV delivery of the mCherry transgene, it was observed that the infectivity of AAV9.DKK1 in the LRP6 knockout population was significantly reduced, while the infectivity of wild-type AAV9 was not correspondingly reduced (Figure 5b). The LRP6-dependent in vitro infectivity of AAV9.DKK1, combined with the known interaction between LRP6 and the N-terminus of DKK1 (see crystal structure, Figure 5b) and its known robust expression in alveolar cells, together strongly support the hypothesis that LRP6 at least partially mediates the reprogramming tropism of AAV9.DKK1.

[0246] On the basis of these observations, and using the brain as a paradigm, experiments were performed to more broadly examine the extent to which inserted peptides mediate retargeting. First, a distance matrix was generated that quantified the similarities between all peptide hits identified as significantly enriched in at least one organ (Figure 6a). By projecting this distance matrix into two dimensions, different clusters of homologous inserted peptides that infect the brain were allowed to be visualized. It was found that similar families of inserted peptides produced similar transduction rates (Figure 6a). This effect was not limited to a specific capsid or insertion site, as the inserted peptides were generally functional in all tested capsids and insertion sites (Figure 6a). The observation that the inserted peptides produced consistent phenotypes in multiple capsids suggests that retargeting is directly attributed to a peptide-mediated mechanism. This result also highlights the power of peptide tiling library design, as multiple overlapping peptides with similar sequences can act as internal controls, giving confidence in identifying specific AAV variant hits.

[0247] Experiments were conducted to specifically study two brain-targeted variants (AAV5.APOA1 and AAV9.APOA1), both of which contained the same inserted apolipoprotein-A 1 (APOA1)-derived peptide. Both variants were packaged into functional capsids as confirmed by electron microscopy (Fig. 6b). Throughout the APOA1 protein, functional APOA1-derived peptides were primarily derived from the tandem repeat region at the C-terminus of the protein (Fig. 6b). The peptide-specific effects of altered AAV tropism were highlighted, with both variants observed to be strongly off-target from the liver at less than 2% AAV9 levels, with uninterrupted brain transduction relative to wild-type AAV9 (Fig. 6c). This is particularly significant for AAV5.APOA1, as the engineered variants were able to overcome the limited transduction of its parental AAV serotype across the blood-brain barrier. The improved specificity of AAV5.APOA1 was subsequently shown to be strain-independent, as it retained its brain-targeting and liver off-target functions in BALB / c mice (Fig. 6d).

[0248] Engineering retargeted immunoorthogonal AAV vectors

[0249] In order to generate additional variants with clinically relevant characteristics, AAV engineering approaches were extended to include new capsids in addition to AAV5 and AAV9. In particular, the potential for engineered tropism of highly divergent AAV orthologs was explored. The motivation lies in the fact that one of the biggest challenges of AAV therapy is the associated immune response: either pre-existing immunity, which currently excludes a large part of the population from receiving any AAV treatment; or immunity induced at the time of AAV injection, which prevents subsequent AAV re-administration. This makes it extremely difficult to create effective therapies because it only allows one chance to treat a patient, thus often requiring extremely high titers for one-time therapy, which in turn can lead to dangerous levels of toxicity. Recently, the concept of immune orthogonality has been implemented to address this challenge. Specifically, it has been proposed that, given sufficient sequence divergence, orthologs will not cross-react with the immune response generated by exposure to other orthologs, thereby allowing re-administration, which avoids neutralization by existing antibodies or elimination of treated cells by activated cytotoxic T cells. Although in this study, experiments focused on commonly used AAV orthologs, experiments were conducted to explore highly divergent natural orthologs from the full spectrum of mammalian species, including those that have not been thoroughly tested for use as in vivo vectors. To this end, a computational experimental pipeline was established to identify new immunologically orthogonal AAV serotypes. First, the Basic Local Alignment Search Tool (BLAST) was used to identify 687 capsids with sequence homology to the AAV2 cap gene. This initial list was then filtered to exclude truncated genomes, redundant samples, human and non-mammalian serotypes, and near-orthologs, resulting in 23 AAV capsid sequences for experimental studies (Figure 7a). Analysis of the evolutionary distances of these potential AAV orthologs confirmed that they exhibited a high degree of divergence from all wild-type AAV capsids (except AAV5, which had previously been shown to be immunologically orthogonal), many of which had less than 60% sequence similarity to AAV2 (Figure 7b). To investigate whether these computationally predicted AAV orthologs are functional, their ability to be packaged into capsids was examined. The titers were compared with AAV5, and 11 of the 23 AAV capsids were identified to have packaging titers within 10 times of AAV5 (Figure 7c). Next, to assess the in vivo transduction potential, these 11 AAV capsids carrying the mCherry transgene were administered to C57BL / 6 mice. Given that most AAV capsids have at least some degree of liver transduction potential, livers from these mice were harvested 3 weeks after infection. Of the 11 AAV capsids analyzed, 4 had detectable levels of mCherry in the liver, and 2 of them generated 3-fold higher mCherry expression than AAV5 (Figure 7c).

[0250] After identifying 4 functional AAV capsids, their immunoorthogonality was characterized by injecting C57BL / 6 mice with each of these and collecting sera at day 0 and day 21 time points (Figure 7d). AAV8 was selected for comparison in this study due to its propensity to transduce the liver and its serological profile similar to other wild-type serotypes. ELISA at the 3-week time point confirmed that antibodies raised against the 4 immunoorthogonal AAVs studied did not show immunological cross-reactivity with AAV8 (Figure 7d). This result highlights the utility of combining computational and experimental methods to identify and characterize new AAV serotypes that can be repurposed to enable gene therapy to be re-administered.

[0251] Next, experiments were conducted to assess whether the tropism of the AAV immune orthogonal excavated can be reengineered by inserting one of the hit peptides identified by the screening. Muscle-targeted AAV variants have a wider range of tissue tropism than variants targeting the brain and lungs, because they are also prone to infect the heart, lungs, intestines and spleen at levels comparable to or exceeding AAV9, assuming that transplanting a peptide from one of these variants to an AAV straight homolog will result in enhanced muscle targeting, while minimal effect on parental AAV infectivity. In order to assess this situation, AAV9.PDGFC was selected as a candidate variant, and its ability to form a functional capsid (Fig. 6d) and its ability to effectively target muscle tissue (as quantified by RT-qPCR, with expression >10 times higher than AAV9) (Fig. 6d) were confirmed. Enhanced transduction of skeletal muscle and myocardium (heart) relative to AAV9 was also confirmed by protein level visualization (Fig. 6d). The 20-mer PDGFC-derived peptide was then grafted onto one of the surface loops of the immunoorthogonal AAV MGS (Fig. 7e), and similar enhanced muscle transduction was significantly observed for the engineered variant over its parental AAV (Fig. 7e). Taken together, the results highlight the fidelity of the ligand tiled display approach and further demonstrate the exciting possibility that the displayed peptides identified in the initial screening approach can be used to reengineer different clades of AAV serotypes via peptide transfer.

[0252] It will be appreciated that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, other implementations are within the scope of the following claims.

Claims

1. A method for improving the tropism of a virus or other delivery agent, the method comprising identifying ligand protein sequences derived from all known receptor-interacting ligands; systematically tiling the ligand peptides into peptides of 5-50 or 10-20 or 20 amino acids, which are inserted into surface-exposed loops of an AAV capsid; and assessing the packaging capacity, in vivo tropism, and enhanced protein interactions of the engineered capsid.

2. The method of claim 1, wherein the virus is an adeno-associated virus (AAV).

3. The method of claim 2, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9.

4. The method of claim 3, wherein the AAV is AAV5 or AAV9.

5. The method of claim 3, wherein the peptide sequence is generated via pooled oligonucleotide synthesis and inserted into four different loop regions: AAV5-Loop1 (N443), AAV5-Loop2 (S576), AAV9-Loop1 (Q456) and AAV9-Loop2 (A587) to generate more than 1 million AAV variants.

6. The method of claim 3, wherein the 20'mer ligand peptide is inserted into one or both of the two surface exposed loops of AAV5 (SEQ ID NO: 2) or AAV9 (SEQ ID NO: 4).

7. A recombinant vector comprising a capsid protein, wherein the capsid protein comprises one or two peptides inserted into one or both of the two surface exposed loops in the capsid protein, wherein the vector has a desired tropism or immunological orthogonality, and wherein the peptides are independently selected from the group consisting of SEQ ID NOs: 5 to 820 and 870-911.

8. The recombinant vector according to claim 7, wherein the vector is an adeno-associated virus (AAV).

9. The recombinant vector according to claim 8, wherein the AAV vector is of AAV5 serotype.

10. The recombinant vector according to claim 9, wherein AAV5 comprises a capsid protein having the sequence shown in SEQ ID NO:

2.

11. The recombinant vector of claim 8, wherein the AAV vector is of AAV9 serotype.

12. The recombinant vector according to claim 11, wherein AAV9 comprises a capsid protein having the sequence shown in SEQ ID NO:

4.

13. The recombinant vector according to claim 7, wherein the vector has a tropism for the pancreas, heart, brain, lung, liver, kidney, muscle, spleen or intestine.

14. The recombinant vector according to claim 10 or 12, wherein the peptide of SEQ ID NO: 530-820, 870-910 or 911 is inserted into loop 1 and / or loop 2 of AAV5 capsid or AAV9 capsid.

15. The recombinant vector of claim 7, wherein the vector is immunoorthogonal.

16. The recombinant vector of claim 15, wherein the vector is an adeno-associated virus (AAV).

17. The recombinant vector of claim 16, wherein the AAV comprises any one of SEQ ID NOs: 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864 or 866, or a capsid protein having a sequence that is at least 85% to 99% identical to any one of the above sequences.

18. A method for preparing a delivery vehicle or vector having a desired tropism, the method comprising selecting a peptide sequence from any one of SEQ ID NOs: 5-820 or 870-911, and (i) cloning a nucleic acid sequence encoding the peptide into a coding sequence of a capsid protein at an exposed loop site to obtain a recombinant capsid coding sequence and using the recombinant capsid coding sequence to produce the vector, or (ii) inserting the peptide into the exposed surface of the delivery vehicle.

19. The method of claim 18, wherein the vector is an adeno-associated virus (AAV).

20. The method of claim 19, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9.

21. The method of claim 20, wherein the AAV is AAV5 or AAV9.

22. The method of claim 21, wherein the AAV5 capsid coding sequence comprises SEQ ID NO:

1.

23. The method of claim 21, wherein the AAV9 capsid coding sequence comprises SEQ ID NO:

2.

24. An AAV vector, wherein the capsid protein has been modified according to the method of claim 18.

25. An AAV vector comprising a capsid protein, wherein the capsid protein expresses a peptide of any one of SEQ ID NOs: 5-820 or 870-911 in the exposed surface loop 1 and / or loop 2 of the capsid protein.

26. The AAV vector of claim 25, wherein the wild-type capsid protein sequence comprises SEQ ID NO: 2 or 4.

27. The AAV vector of claim 25, wherein the AAV vector has an AAV5 or AAV9 serotype.

28. The AAV vector of claim 25, wherein the vector has a desired tropism.

29. The AAV vector of claim 28, wherein the AAV vector has a tropism for the pancreas, heart, brain, lung, liver, kidney, muscle, spleen, or intestine.

30. A viral vector having a capsid protein, wherein the capsid protein comprises a heterologous targeting peptide with a length ranging from 10 to 30 amino acids inserted into a surface-exposed portion of the capsid protein, and wherein the targeting peptide is as described in any one of SEQ ID NOs: 5-820 or 870-911.

31. The viral vector of claim 30, wherein the heterologous targeting peptide is about 15-25 amino acids in length.

32. The viral vector of claim 31, wherein the heterologous targeting peptide is about 20 amino acids in length.

33. The viral vector of claim 30, wherein the viral vector is an adeno-associated virus (AAV).

34. The viral vector of claim 30, wherein the viral vector is a lentiviral vector.

35. The viral vector of claim 30, wherein the capsid protein is VP1 capsid protein.

36. The viral vector of claim 30, wherein the capsid protein is VP2 capsid protein.

37. The viral vector of claim 30, wherein the capsid protein is VP3 capsid protein.

38. The viral vector of claim 30, wherein the heterologous targeting peptide is inserted into the AAV capsid protein at loop 1 and / or loop 2.

39. The viral vector of claim 30, wherein the viral vector is AAV5.

40. The viral vector of claim 30, wherein the viral vector is AAV9.

41. The viral vector of claim 39, wherein the heterologous targeting peptide is flanked by linker peptides at the N-terminus and the C-terminus of the heterologous targeting peptide.

42. The viral vector of claim 40, wherein the heterologous targeting peptide is flanked by linker peptides at the N-terminus and the C-terminus of the heterologous targeting peptide.

43. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to hepatocytes or liver tissue.

44. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to neuronal cells or brain tissue.

45. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to pancreatic cells or pancreatic tissue.

46. ​​The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to cardiomyocytes or cardiac tissue.

47. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to lung tissue.

48. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to intestinal tissue.

49. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to spleen tissue.

50. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to renal cells or renal tissue.

51. The viral vector of claim 30, wherein the heterologous targeting peptide targets the viral vector to muscle cells or tissues.

52. An adeno-associated virus (AAV) capsid protein, comprising a heterologous targeting peptide cloned into loop 1 and / or loop 2 of the capsid protein, wherein the heterologous targeting peptide is about 10-30 amino acids in length and contains or comprises any one of the peptides of SEQ ID NO: 5-820 or 870-911.

53. The AAV capsid protein of claim 52, wherein the capsid protein is a VP1 capsid protein.

54. The AAV capsid protein of claim 52, wherein the capsid protein is a VP2 capsid protein.

55. The AAV capsid protein of claim 52, wherein the capsid protein is a VP3 capsid protein.

56. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide is about 15-25 amino acids in length.

57. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide is about 20 amino acids in length.

58. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide is flanked by linker peptides at the N-terminus and the C-terminus of the heterologous targeting peptide.

59. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets hepatocytes or liver tissue.

60. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets neuronal cells or brain tissue.

61. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets pancreatic cells or pancreatic tissue.

62. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets cardiomyocytes or cardiac tissue.

63. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets lung tissue.

64. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets intestinal tissue.

65. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets spleen tissue.

66. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets renal cells or renal tissue.

67. The AAV capsid protein of claim 52, wherein the heterologous targeting peptide targets muscle cells or tissues.

68. A recombinant AAV (rAAV) comprising the capsid protein of any one of claims 52 to 67.

69. A recombinant AAV (rAAV), comprising a capsid protein having a targeting peptide in loop 1 and / or loop 2, wherein the targeting peptide is independently selected from SEQ ID No: 5-820 or 870-911.

70. The recombinant AAV of claim 69, wherein the recombinant AAV further comprises a heterologous polynucleotide for gene delivery.

71. A recombinant AAV according to claim 70, wherein the heterologous polynucleotide is a therapeutic gene.

72. A composition comprising the recombinant rAAV according to any one of claims 69 to 71.

73. The composition of claim 72, further comprising a pharmaceutically acceptable carrier.

74. A method for delivering a transgene to a subject, the method comprising: administering to a subject a recombinant AAV (rAAV), wherein the rAAV comprises: (i) a capsid protein according to any one of claims 52 to 67, and (ii) at least one transgene, and wherein the rAAV infects cells of a target tissue of the subject.

75. The method of claim 73, wherein the at least one transgene encodes a protein.

76. The method of claim 74, wherein the protein is an immunoglobulin heavy or light chain or a fragment thereof.

77. The method of claim 73, wherein the at least one transgene encodes a small interfering nucleic acid.

78. The method of claim 76, wherein the small interfering nucleic acid is miRNA.

79. The method of claim 76, wherein the small interfering nucleic acid is a miRNA sponge or TuD RNA that inhibits the activity of at least one miRNA in the subject or animal.

80. The method of claim 77, wherein the miRNA is expressed in cells of the target tissue.

81. The method of claim 73, wherein the target tissue is skeletal muscle, heart, liver, pancreas, brain, or lung.

82. The method of claim 73, wherein the transgene expresses a transcript comprising at least one binding site for a miRNA, wherein the miRNA inhibits activity of the transgene in tissues other than the target tissue by hybridizing to the binding site.

83. The method of claim 73, wherein the at least one transgene encodes a gene product that mediates genome editing.

84. The method of claim 73, wherein the transgene comprises a tissue-specific promoter or an inducible promoter.

85. The method of claim 83, wherein the tissue-specific promoter is a liver-specific thyroxine binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a sarcosine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an alpha-myosin heavy chain (a-MHC) promoter, or a cardiac troponin T (cTnT) promoter.

86. The method of claim 73, wherein the rAAV is administered intravenously, intravascularly, transdermally, intraocularly, intrathecally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation.

87. The method of claim 73, wherein the subject is selected from the group consisting of mice, rats, rabbits, dogs, cats, sheep, pigs, and non-human primates.

88. The method of claim 73, wherein the subject is a human.

89. An isolated nucleic acid encoding an AAV capsid protein comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 5-820 and 870-911.

90. A delivery vehicle for delivering a small molecule drug or biologic with a desired tropism, wherein the delivery vehicle comprises a peptide or peptide fragment of at least 10-20 amino acids of any one of SEQ ID NOs: 5-820 or 870-911.

91. The delivery vehicle of claim 89, wherein the delivery vehicle is selected from the group consisting of liposomes, nanoparticles, bacteria, bacteriophages, virus-like particles (VLPs), erythrocyte ghosts, and exosomes.

92. The delivery vehicle of claim 89, wherein the biologic comprises siRNA, an antisense molecule, a protein or polypeptide, insulin, a vaccine, or an antibody.

93. The delivery vehicle of claim 89, wherein the small molecule drugs include chemotherapeutic agents, anti-inflammatory agents, steroids, and antibiotics.

94. A biologic having a desired tropism linked to a peptide or peptide fragment of at least 10-20 amino acids of any one of SEQ ID NOs: 5-820 or 870-911.

95. The biologic of claim 93, wherein the biologic is a nucleic acid, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, a non-immunoglobulin binding agent, or an enzyme.

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