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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C07K2319/00—Fusion polypeptide
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14145—Special targeting system for viral vectors
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source

Definitions

• the disclosure provides a method of improving tropism of a virus or other delivery agent , the method comprising identi fying ligand protein sequences derived from all known receptor-interacting ligands ; systematically tile the ligand peptides into 5 -50 or 10 -20 or 20 amino acid peptides which are inserted into surface-exposed loops of AAV capsids; assessing the engineered capsids for their packaging capacity, in vivo tropism, and enhanced protein interactions.
• the virus is an adeno-associated virus (AAV) .
• the AAV is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7 , AAV8, and AAV9.
• the disclosure provides a recombinant vector comprising capsid proteins containing one or two peptides inserted into one or both of two surface-exposed loops in the capsid protein, wherein the vector has a desired tropism or immune-orthogonality and wherein the peptides are independently selected from the group consisting of SEQ ID NOs : 5 to 820 and 870-911.
• the vector is an adeno-associated virus (AAV) .
• the AAV vector is an AAV5 serotype.
• the AAV5 comprises a capsid protein having a sequence as set forth in SEQ ID NO: 2.
• the AAV vector is an AAV9 serotype.
• the AAV9 comprises a capsid protein having a sequence as set forth in SEQ ID NO: 4.
• the vector has tropism to pancreas, heart, brain, lung, liver, kidney, muscle, spleen or intestine.
• a peptide of SEQ ID NOs: 530-820, 870-910 or 911 is inserted into loop 1 and/or loop 2 of an AAV5 capsid or an AAV9 capsid.
• the vector is immune orthogonal.
• the vector is an adeno-associated virus (AAV) .
• the AAV comprises a capsid protein of 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 sequence that it at least 85% to 99% identical to any of the foregoing sequences.
• the disclosure also provides a method of making a delivery vehicle or vector with 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 for a capsid protein at an exposed loop site to obtain a recombinant capsid coding sequence and producing the vector using the recombinant capsid coding sequence or (ii) inserting the peptide into an exposed surface of the delivery vehicle.
• the vector is an adeno-associated virus (AAV) .
• the AAV is selected from the group consisting of AAV1, AAV2 , AAV5 , AAV6, AAV7, AAV8, and AAV9.
• the AAV is AAV5 or AAV9.
• the AAV5 capsid coding sequence comprises SEQ ID NO:1.
• the AAV9 capsid coding sequence comprises SEQ ID NO : 2.
• the disclosure also provide an AAV vector comprising a capsid protein modified by any of the foregoing embodiments.
• the disclosure also provides an AAV vector comprising a capsid protein wherein the capsid protein expresses a peptide of any one of SEQ ID NO: 5-820 or 870-911 in surface exposed loop 1 and/or loop 2 of the capsid protein.
• the wild-type capsid protein sequence comprises SEQ ID NO: 2 or 4.
• the AAV vector has a AAV5 or AAV9 serotype.
• the vector has a desired tropism.
• the AAV vector has a tropism to pancreas, heart, brain, lung, liver, kidney, muscle, spleen or intestine.
• the disclosure also provides a viral vector having a capsid protein comprising a heterologous targeting peptide of 10-30 amino acids in length inserted into a surface exposed portion of the capsid protein and wherein the targeting peptide is set forth in any one of SEQ ID NOs: 5-820 or 870-911.
• the heterologous targeting peptide is about 15-25 amino acids in length.
• the heterologous targeting peptide is about 20 amino acids in length.
• the viral vector is an adeno-associated virus (AAV) .
• the viral vector is a lentiviral vector.
• the capsid protein is a VP1 capsid protein.
• the capsid protein is a VP2 capsid protein. In yet another embodiment, the capsid protein is a VP3 capsid protein.
• the heterologous targeting peptide is inserted into an AAV capsid protein at loop 1 and/or loop 2.
• the viral vector is an AAV5. In another embodiment, the viral vector is an AAV9.
• the heterologous targeting peptide is flanked by a linker peptide at the N-terminal and C-terminal ends of the heterologous targeting peptide. In another embodiment, the heterologous targeting peptide targets the viral vector to hepatocytes or liver tissue.
• the rAAV is administered intravenously, intravascularly, transdermally, intraocularly, intrathecally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation.
• the subject is selected from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig, and a non-human primate.
• the subject is a human.
• the disclosure also provides an isolated nucleic acid encoding an AAV capsid protein containing an amino acid sequence selected from the group consisting of SEQ ID No: 5-820 and 870-911.
• the disclosure also provides a delivery vehicle for delivery of a small molecule drug or biological agent having 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.
• the delivery vehicle is selected from the group consisting of a liposome, a nanoparticle, a bacteria, a bacteriophage, a virus-like particle (VLP) , a erythrocyte ghost, and an exosome.
• VLP virus-like particle
• the biological agent comprises an siRNA, an antisense molecule, a protein or polypeptide, insulin, a vaccine, or an antibody.
• the small molecule drug comprises a chemotherapeutic agent, an anti-inflammatory, a steroid, and an antibiotic .
• the disclosure also provides a biological agent having a desired tropism, the biological agent 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.
• the biological agent is a nucleic acid, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, a non-immunoglobulin binding agent, or an enzyme.
• FIG. 1A-B Design of AAV libraries displaying peptides tiling receptor- 1 igands .
• AAV5-Loopl N443
• AAV5-Loop2 S576
• AAV9-Loopl Q456
• AAV9-Loop2 A587
• FIG. 2A-F AAV library packaging analyses reveal biophysical features contributing to capsid fitness
• Figure 3A-D In vivo screen analyses enable predictive computational models of tropism
• log2FC log2 fold change
• AAV variants were characterized structurally via transmission electron microscopy, and functionally via delivery of the mCherry transgene in vivo.
• FIG. 5A-D Characterization and mechanistic exploration of AAV variants with displayed ligand peptides
• lung screen count values for all AAV9Loop2 variants with inserted DKK1 derived peptides are shown.
• the x-axis indicates the position in the DKK1 structure a given peptide starts on. Shown in blue are the lung counts, and shown in orange are the capsid counts.
• Red arrow indicates the location of the peptide inserted in AAV9.DKK1.
• MFI mean fluorescence intensity
• Shown to the right is the crystal structure of a 7-mer peptide DKK1 peptide (contained within AAV9.DKK1) in complex with LRP6 (58) .
• (c) Full characterization experiments for the variant AAV9.PDGFC.
• muscle screen count values for all AAV9Loopl variants with inserted PDGFC derived peptides are shown.
• the x-axis indicates the position in the PDGFC structure a given peptide starts on. Shown in blue are the muscle counts, and shown in orange are the capsid counts. Red line indicates the location of the peptide inserted in AAV9. PDGFC.
• AAV9. PDGFC Known receptors for the PDGFC ligand were cloned into an overexpression plasmid and transfected into HEK293T cells in a 24- well plate. After 24 hours, either AAV9 (4x10 s viral genomes) or AAV9. PDGFC (4x10 s viral genomes) were used to transduce the cells. Following 24 hours of transduction, the cells were collected and mCherry expression was quantified via flow cytometry. Bar plots show the MFI normalized to the average MFI of the AAV9 transduced cells with an empty vector overexpressed. Statistical significance between groups was calculated via a T-test (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001) .
• FIG. 6A-D Inserted peptide facilitates efficient liver de-targeting across multiple AAV scaffolds in a mouse strainindependent manner
• AAV variants are colored by their log 2 fold change in the brain. Select variant clusters which are highly enriched in the brain are highlighted.
• AAV variants are also colored by capsid insertion site in the embedding on the right,
• AAVs were first assessed by measuring their ability to package, and then by their ability to transduce the liver (using an mCherry transgene) in vivo. All values shown relative to the orthologous wild-type AAV5.
• the four novel AAVs which could infect the liver (AAV MM2, AAV MG2 , AAV MG1 and AAV CHI) , were tested for immune cross-reactivity with AAV8. Mice were immunized with the indicated AAV, and then 3 weeks post-injection tested for antibody cross reactivity via an ELISA,
• the PDGFC peptide from AAV9.PDGFC was inserted onto loopl of AAV MG2 to yield AAV MG2.PDGFC.
• PDGFC were injected into C57BL/6 mice, quantifying muscle transduction via RT-qPCR after three weeks. Bar plots show muscle transduction relative to wild-type AAV MG2. Statistical significance between groups was calculated via a T-test (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001) .
• each intervening number there between with the same degree of precision is explicitly contemplated.
• the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
• an amino acid mimetic refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
• non-naturally occurring amino acid and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
• one or more D-amino acids can be used in various peptide compositions of the disclosure.
• the disclosure provides various peptides that are useful for treating various diseases and infections. These peptides can comprise naturally occurring amino acid. In other embodiments, the peptides can comprise non-natural amino acids. The use of non-natural amino acids can improve the peptides stability, decrease degradation and/or improve biological activity. For example, in some embodiments, one or more D-amino acids. In other embodiments, retroinverso peptides are contemplated using various amino acid configurations.
• 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. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
• Cas9 may refer to an endonuclease that causes double stranded breaks in DNA, a nickase variant such as a RuvC or HNH mutant that causes a single stranded break in DNA, as well as other variations such as deadCas-9 (“dCas9”) , which lack endonuclease activity.
• Cas9 may also refer to "split-Cas9" in which Cas9 is split into two halves - C-terminal Cas9 (C-Cas9) and an N- terminal Cas-9 (N-Cas9) - which can be fused with two intein moieties. See, e.g. , U.S. Pat. No. 9,074,199 Bl; Zetsche et al.
• Non-limiting examples of commercially available sources of SpCas9 comprising plasmids can be found under the following AddGene reference numbers:
• 48138 PX458; SpCas 9-2A-EGFP and single guide RNA
• 62988 PX459; SpCas 9-2A-Puro and single guide RNA
• 48873 PX460; SpCas9n (D10A nickase) and single guide RNA
• 48140 PX461; SpCas 9n-2A-EGFP (D10A nickase) and single guide RNA;
• CRISPR-mediated gene editing utilizes the pathways of nonhomologous end- joining (NHEJ) or homologous recombination to perform the edits.
• NHEJ nonhomologous end- joining
• homologous recombination to perform the edits.
• domain can refer to a particular region of a larger molecule (e.g. , a particular region of a protein or polypeptide) , which can be associated with a particular function.
• a domain which binds to a cognate can refer to the domain of a protein that binds one or more receptors or other protein moieties.
• a corresponding coding sequence for a particular polypeptide domain can be referred to as a polynucleotide domain.
• encode as it is applied to polynucleotides can refer to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. In some cases, the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
• the terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular molecule, biological, or cellular material and intend those having minimal homology while still maintaining desired structure or functionality.
• expression can refer to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.
• gRNA comprises or alternatively consists essentially of, or yet further consists of a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA) ; or a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA) .
• a gRNA is synthetic (Kelley, M. et al. J of Biotechnology 233 (2016) 74-83) .
• the parameters can be set such that the percentage of identity is calculated over the full length of the reference sequence and that gaps in homology of up to 5% of the total reference sequence are allowed.
• a number of sequences are provided herein, it is contemplated that sequences having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% and 100% to any one of the sequences herein find use in any of the compositions and methods described herein .
• the identity between a reference sequence (query sequence, i.e. , a sequence of the disclosure) and a subject 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) ) .
• the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
• the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity can be 90%.
• a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
• Hybridization can refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
• the hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
• the complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
• a hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
• Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
• Examples of moderate hybridization conditions include: incubation temperatures of about 40 °C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
• Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.
• lx SSC formamide concentrations of about 55% to about 75%
• wash solutions of about lx SSC, O. lx SSC, or deionized water.
• hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
• SSC is 0.15 M NaCl and 15 mN citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
• the term "immune orthogonal” refers to a lack of immune cross-reactivity between two or more antigens.
• the antigens are proteins (e.g., Cas9) .
• the antigens are viral antigens associated with a particular viral vector (e.g. , AAV) .
• antigens typically include antigenic determinants having a particular sequence of 3 dimensional structure.
• an antigenic determinant can comprise a domain or subsequence of a larger polypeptide or molecular sequence.
• antigens that are immune orthogonal do not share an amino acid sequence of 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 consecutive amino acids. In some embodiments, antigens that are immune orthogonal do not share any highly immunogenic peptides. In some embodiments, antigens that are immune orthogonal do not share affinity for a major histocompatibility complex (e.g. , MHC class I or class II) . Antigens that are immune orthogonal are amenable for sequential dosing to evade a host immune system.
• MHC class I or class II major histocompatibility complex
• immunosilent refers to an epitope or foreign peptide, polypeptide or protein that does not elicit an immune response from a host upon administration.
• the peptide, polypeptide or protein does not elicit an adaptive immune response.
• the peptide, polypeptide or protein does not elicit an innate immune response.
• the peptide, polypeptide or protein does not elicit either an adaptive or an innate immune response.
• an immunosilent peptide, polypeptide or protein has reduced immunogenicity.
• RNA essential RNA
• mRNA is a nucleic acid molecule that is transcribed from DNA and then processed to remove non-coding sections known as introns. In some cases, the resulting mRNA is exported from the nucleus (or another locus where the DNA is present) and translated into a protein.
• pre-mRNA can refer to the strand prior to processing to remove non-coding sections. mRNA has "U” in place of "T” in cDNA coding sequences.
• ortholog is used in reference of another gene or protein and intends a homolog of said gene or protein that evolved from the same ancestral source or which are evolved artificially using molecular biology and genetic engineering.
• Orthologs may or may not retain the same function as the gene or protein to which they are orthologous .
• Non-limiting examples of Cas9 orthologs include 5. aureus Cas9 ("spCas9”) , S. thermophiles Cas9, L . pneumophilia Cas9, N. lactamica Cas9, N. meningitides Cas9, B. longum Cas9, A. inuciniphila Cas9, and O. laneus Cas9.
• payload refers to a therapeutic and diagnostic agents that can be loaded into or onto a delivery vehicle.
• payload include biological and small molecule entities.
• exemplary payload agents include small molecule drugs, biological molecules, viruses, therapeutic agents, prodrugs, gene silencing agents, chemotherapeutics, diagnostic agents, and/or components of gene editing systems.
• biological molecules include, but are not limited to, nucleic acids (e.g., DNA, RNA, mRNA, modified mRNA, small RNAs, siRNA, miRNA, genes, and transgenes) , peptides /proteins (including antibodies, enzymes, transcription factors, etc.) , viruses, hormones, carbohydrates, lipids, and vitamins.
• gene silencing agents include siRNA, chRNAs, miRs, ribozymes, morpholines, and esiRNAs.
• gene editing systems include, but are not limited to, CRISPR-Cas systems, zinc finger nucleases, and TALENs.
• diagnostic agents include but are not limited to, dyes and stains, radioactive tracers, and contrast agents.
• anticancer agents and chemotherapeutics that can be used with or loaded into a delivery vehicle include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide ; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide , triethiylenethiophosphoramide and tiimethylolomelamine ; acetogenins (e.g.
• TAXOTERE® docetaxel
• chloranbucil GEMZAR® (gemcitabine)
• 6-thioguanine mercaptopurine
• methotrexate platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g.
• topoisomerase inhibitor RFS 2000 difluoromethylornithine (DFMO) ; retinoids such as retinoic acid; capecitabine ; leucovorin (LV) ; irenotecan; adrenocortical suppressant; adrenocorticosteroids; progestins; estrogens; androgens; gonadotropin-releasing hormone analogs; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• DFMO difluoromethylornithine
• retinoids such as retinoic acid
• capecitabine a leucovorin (LV)
• irenotecan adrenocortical suppressant
• progestins estrogens
• androgens gonadotropin-releasing hormone analogs
• pharmaceutically acceptable salts, acids or derivatives of any of the above and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• anticancer agents are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (including NOLVADEX® tamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene ; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) - imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASL® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARTMIDEX® anastrozole; and anti-androgens
• HER2 expression inhibitor examples include mammalian proteins, such as, e.g.
• the 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, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, cardiotrophin-1 and other proteins identified and classified as members of the family.
• Other payload agents that can be incorporated in the delivery vehicle include gastrointestinal therapeutic agents such as aluminum hydroxide, calcium carbonate, magnesium carbonate, sodium carbonate and the like; non-steroidal antifertility agents; parasympathomimetic agents; psychotherapeutic agents; major tranquilizers such as chloropromazine HC1, clozapine, mesoridazine, metiapine, reserpine, thioridazine and the like; minor tranquilizers such as chlordiazepoxide, diazepam, meprobamate, temazepam and the like; rhinological decongestants; sedative-hypnotics such as codeine, phenobarbital, sodium pentobarbital, sodium secobarbital and the like; other steroids such as testosterone and testosterone propionate; sulfonamides; sympathomimetic agents; vaccines; vitamins and nutrients such as the essential amino acids, essential fats and the like; antimalarials such as 4-
• Antibiotic payloads include, for example, the cephalosporins, chlorarnphenical, gentamicin, kanamycin A, kanamycin B, the penicillins, ampicillin, streptomycin A, antimycin A, chloropamtheniol , metronidazole, oxytetracycline penicillin G, the tetracyclines, and the like.
• Payload agents can include vaccines or antigenic agents.
• payload antigens derived from microorganisms such as Neisseria gonorrhea, Mycobacterium tuberculosis, Herpes virus (humonis, types 1 and 2) , Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp. , Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.
• microorganisms such as Neisseria gonorrhea, Mycobacterium tuberculosis, Herpes virus (humonis, types 1 and 2) , Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp.
• the payload can comprise enzymes such as ribonuclease, neuramidinase, trypsin, glycogen phosphorylase, sperm lactic dehydrogenase, sperm hyaluronidase, adenossinetriphosphatase, alkaline phosphatase, alkaline phosphatase esterase, amino peptidase, trypsin chymotrypsin, amylase, muramidase, acrosomal proteinase, diesterase, glutamic acid dehydrogenase, succinic acid dehydrogenase, beta-glycophosphatase , lipase, ATP-ase alpha-peptate gamma-glutamylotranspeptidase, sterol- 3-beta-ol-dehydrogenase , DPN-di-aprorase .
• enzymes such as ribonuclease, neuramidina
• Peptide-payload conjugates are also encompassed by the disclosure, wherein a peptide of the disclosure is linked or fused directly to a payload molecule as set forth herein such that the peptide-payload conjugate can be directly delivery (without loading into a delivery vehicle) to target a desired tissue based upon the peptide's tropism.
• promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissuespecific, for example.
• a "promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
• Non-limiting exemplary promoters include CMV promoter and U6 promoter.
• protein protein
• peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics .
• the subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc.
• a protein or peptide can contain at least two amino acids and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence.
• amino acid can refer to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
• fusion protein can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function.
• linker can refer to a peptide fragment that is used to link these domains together - optionally to preserve the conformation of the fused protein domains and/or prevent unfavorable interactions between the fused protein domains which can compromise their respective functions.
• polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
• polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag) , exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
• a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
• polypeptide sequence can be the alphabetical representation of a polypeptide molecule .
• This alphabetical representation can be input into databases in a computer having a central process ing unit and used for bioinformatics applications such as functional proteomics and homology searching .
• recombinant expres sion system refers to a genetic construct or constructs for the expres sion of certain genetic material formed by recombination .
• recombinant protein can refer to a polypeptide or peptide which is produced by recombinant DNA techniques , wherein generally, DNA encoding the polypeptide or peptide is inserted into a suitable expression vector which is in turn used to trans form a host cell to produce the heterologous polypeptide or peptide .
• the term "sequencing” as used herein, can comprise bisul fite- free sequencing, bisulfite sequencing, TET-assisted bisul fite (TAB) sequencing, ACE-sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, mas sively parallel s ignature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, I llumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope s ingle molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, Enigma sequencing, or any combination thereof.
• TET-assisted bisul fite (TAB) sequencing ACE-sequencing
• high-throughput sequencing Maxam-Gilbert sequencing
• mas sively parallel s ignature sequencing Polony sequencing
• 454 pyrosequencing Sanger sequencing
• I llumina sequencing SOLiD sequencing
• the term "subject" is intended to mean any animal.
• the subject may be a mammal; in further embodiments, the subject may be a bovine, equine, feline, murine, porcine, canine, human, or rat.
• transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign 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 such as, for example, LIPOFECTIN® (Invitrogen Corp. , San Diego, CA) , LIPOFECTAMINE® ( Invitrogen) , EUGENE® (Roche Applied Science, Basel, Switzerland) , JETPEITM (Polyplus-transfection Inc. , New York, NY) , EFFECTENE® (Qiagen,
• vector can refer to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAG, a YAC, etc.
• a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
• plasmid vectors can be prepared from commercially available vectors.
• viral vectors can be produced from baculoviruses , retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art.
• the viral vector is a lentiviral vector.
• 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. ) .
• the promoter is a pol III promoter.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
• Other vectors e.g. , non-episomal mammalian vectors
• any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli and Pseudomonas) ; the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis , and Bacillus lichen! formis ; nisA (useful for expression in gram positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol.
• Termination control regions may also be derived from various genes native to the preferred hosts .
• the disclosure shows that one of the discovered variants (AAV9 . DKK1 ) exhibit deprecated transduction when the cognate receptor for the ligand from which the peptide was derived is knocked out (Fig . 5a) . Furthermore, due to the systematic tiling nature of the peptide library, it was pos sible to generate full- length transduction maps acros s the entire res idue space of ligands utili zed in this study (Fig. 5a , c) .
• peptides with at least 85% identity to any one of the peptide sequences provided herein are also contemplated. This may be relevant in particular for certain peptides derived from ligands that engage receptors expressed on multiple cell types or which have promiscuous binding activity.
• a standard promoter CMV was used to drive expression of the mCherry transgene.
• tissue-specific promoters could be used to increase the specificity and magnitude of transgene expression in the organ of interest.
• the hit capsids identified here could be further engineered for increased activity.
• Existing hits could serve as a scaffold for further rounds of targeted mutagenesis and screening, or peptides could be inserted on both loopl and loop2 of the AAV capsid to increase the valency of the displayed ligands.
• recently developed direct chemical engineering or peptide display strategies and/or alternative peptide linkers could be utilized to enhance peptide- mediated transduction.
• scRNAseq could be used to screen hit variants towards more specific cell-types within the organ of interest .
• AAV5.APOA1 which has less than 1% the liver infectivity of WT AAV9
• WT AAV9 highly specific AAV capsids
• the bulk screening data itself is high value. Given the scale, reliability, and translational relevance of the screening dataset, the data set can serve as a foundation for future computational engineering of designer AAV capsids.
• mice All animal care and experimental methods were performed in accordance with the University of California Institutional Animal Care and Use Committee. 6-8 week old male C57B1/6J (JAX, #000664) and Balb/cJ (JAX, #000651) mice were purchased from the Jackson Laboratories and systemic injections were administered retro- orbitally with either AAV or PBS.
• HEK293T cells were cultured in DMEM medium supplemented with 10% FBS, GlutaMAX (lx) (GIBCO) , and Penicillin-Streptomycin (100 U/mL) (GIBCO) .
• capsid particles as templates for qPCR, 2 pL of virus was added to 50 pL of alkaline digestion buffer (25mM NaOH, 0.2 mM EDTA) and boiled for 8 minutes. Following this, 50 pL of neutralization buffer (40mM Tris-HCl, .05% Tween-20, pH 5) was added to each sample.
• alkaline digestion buffer 25mM NaOH, 0.2 mM EDTA
• neutralization buffer 40mM Tris-HCl, .05% Tween-20, pH 5
• Each AAV capsid library was retro-orbitally administered to mice in duplicate at a dose of 2E12 vg/mouse for the AAV9-based libraries or 1E12 vg/mouse for the AAV5-based libraries.
• Two weeks after injection the heart, lung, liver, intestine, spleen, pancreas, kidneys, brain, and gastrocnemius muscle were harvested and placed in RNAlater storage solution.
• Total DNA was extracted from all mouse tissues using TRIzol reagent and the TNES-6U back extraction method. The resulting precipitated DNA was centrifuged for 15 minutes at 18,000G, and the supernatant discarded.
• PCR reactions were purified using a QIAquick PCR Purification Kit according to the manufacturer's protocol. Following this, 50 ng of the PCR amplicon was used as template for a secondary 50 pL KAPA Hifi Hotstart Readymix PCR reaction to add illumina compatible adapters and indices (NEBNext Cat# E7600S) . The PCR reaction was performed with an annealing temperature of 60 °C, an extension time of 30 seconds. To sequence the capsid libraries, a similar protocol was performed, with a modified template amount in the step-1 PCR.
• the model architecture was instantiated via a Keras sequential model (102) .
• a convolutional layer (ConvlD) with 32 filters, a kernel size of 3 and "relu" activation was fed into a max pooling layer (MaxPoollD) with pool size of 2.
• MaxPoollD max pooling layer
• These layers were followed with another set of convolutional and max pooling layers, this time with 64 filters in the convolutional layer.
• a flattening layer and final dense layer (with sigmoid activation) was then used to output resulting class probabilities.
• a separate independent model was trained for each organ.
• the classes (infective versus non-inf ective variants) were weighted proportionally to the inverse of the number of class examples.
• a dictionary describing the class weights was passed via the 'class weight' parameter.
• Model performance was evaluated via accuracy, area under the receiver operator characteristic curve (AUROC) , Fl-score, and Matthews Correlation Coefficient (MCC) . Metrics were calculated via builtin Keras functions, and plotted via matplotlib.
• AUROC receiver operator characteristic curve
• MCC Matthews Correlation Coefficient
• 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% confluency the day before transfection. The day of transfection, Optimem serum reduced media was mixed with Lipof ectamine 2000 (Thermo Fisher) , 3 pg of pMD2.
• lentiviral containing DMEM with 8pg/mL polybrene was added to the cells.
• the media was then replaced 24 hours later and then changed into puromycin (2]jg/mL) containing DMEM 28 hours post-transfection.
• Post selection once the cells reached confluency, they were passaged into a 24-well plate at -40% confluency.
• Immune orthogonal AAV capsids with sufficient packaging titer were then injected retro-orbitally into C57BL/6 mice at a dose of IxlO 12 viral genomes/mouse . Livers were harvested 3 weeks postinjection and total RNA was isolated as described above. cDNA was then generated and transgene expression was quantified via qPCR using the iTaq Universal SYBR green supermix and primers binding to the mCherry transcript. mCherry transgene expression was then normalized to GAPDH and the relative expression was compared to AAV5.
• the assay to assess immune antibody cross-reactivity of the identified immune orthogonal was performed as previously described. Prior to injection, serum was collected via tail snip procedure and then the mice were injected with IxlO 12 viral genomes/mouse of AAV or PBS in triplicate. 3 weeks later, serum was collected from each of the mice and the antibody cross-reactivity ELISA was performed. For this, IxlO 9 viral genomes of AAV8, AAV MM2, AAV MG1, AAV MG2 , or AAV CHI were diluted in a lx coating buffer and incubated overnight in each well of 96-well Nunc MaxiSorp plates.
• Plates were washed three times for 5 mins with lx wash buffer (Bethyl) and blocked with lx BSA blocking buffer (Bethyl) for 2 hours at room temperature. The wells were then washed again and serum samples were added at a 1:40 dilutions. Plates were incubated for 5 hours at 4°C with shaking. Wells were 3x washed and 100 pL of HRP-labeled goat anti-mouse IgGl (Bethyl; diluted 1:100,000 in 1% BSA) was added to each well. Secondary antibody was incubated for 1 hour at room temperature, wells were washed 3 times, and 100 pL of TMB substrate was added to each well. Optical density at 450 nm was measured using a microplate absorbance reader (BioRad iMark) .
• AAV5 and AAV9 were chosen as the starting serotypes. This was due to their established clinical utility as well as two key characteristics: one, AAV5 is more evolutionarily distant to other AAV serotypes in clinical use, and has previously been shown to be immune orthogonal (to other prevalent AAV serotypes thereby enabling their sequential redosing) ; and two, AAV9 has been used extensively for clinical trials and has been shown to cross the blood-brain barrier outperforming other AAV serotypes in most tissues. To generate the library of diverse AAV variants, a DNA oligonucleotide pool of 275,298 gene fragments was generated (Fig. la-b) .
• Each gene fragment coded for a 20 amino acid peptide derived from the coding sequence of ligands with known extracellular receptors, or a gene predicted to have cellpenetrating or internalizing properties (Fig. la-b) .
• Protein ligands were sourced from the Guide to Pharmacology database, an expertly curated list of pharmacological targets and their associated ligands, and cell-penetrating/internalizing functionality was inferred through text mining of UniProt entries. Examples of protein classes identified as having potential internalizing function included toxins, histones, granzymes, viral receptor binding domains, and nuclear localization signal domains (NLS) .
• Mouse and human genomes share 80% of their protein coding genes, with 85% amino acid sequence identity between orthologs.
• PaqCI sites engineered to yield compatible overhangs were inserted at the ends of the peptide coding DNA library and on the AAV5/AAV9 cap plasmid DNA at sites coding for two distinct surface loops, hereon referred to as loop 1 and loop 2.
• 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 chosen as peptide insertion locations due to their distance from the viral particle core facilitating potential receptor engagement (Fig. lb) .
• AAV capsids To quantify how well different AAV cap variants package into functional capsids, recombinant AAV particles were generated with the engineered AAV5 and AAV9 cap plasmid libraries via transient triple transfection of HEK293T cells (Fig. 2a) . These viral particles were treated with benzonase to degrade residual plasmid DNA, and then subjected to next generation sequencing (NGS) to quantify relative variant abundance. Packaging efficiency was quantified by ranking AAV variants by the log2 fold change (log2FC) of their relative capsid abundance compared to their count in the plasmid pool (Fig. 2b) . Utilizing this method, over 250,000 AAV variants were identified which package efficiently into functional
• 25 AAV capsids were produced including 23 identified as successful packagers (log 2 FC >0) and 2 identified as non-packagers (log 2 FC ⁇ 0) .
• the 2 non-packagers yielded >10-fold lower titer than those identified as packagers, thus providing confidence in the AAV packaging metric.
• Consistent with their disruption of the AAV capsid structure there was also a depletion of non-functional stop codon control AAV variants in the capsid pool, and importantly this confirmed lack of library cross-packaging during AAV production (Fig. 2C) .
• tissue tropism of the variants largely recapitulated the screen predictions (Fig. 4c) , with 74.3% of the tissue tropism predictions matching expectations.
• AAV variants which specifically targeted hard to infect organs such as the muscle, lung, and brain, while simultaneously de-targeting away from the liver were identified (Fig. 4c) .
• protein level quantification of mCherry delivery to the liver, quantified via fluorescent microscopy, confirmed excellent concordance between mRNA and protein measures of tissue transduction (R 2 0.97) (Fig. 4d) .
• 9/21 variants were found to exceed AAV9 infectivity in at least one organ.
• Variants were identified which exceed AAV9 infectivity in all organs except the liver and pancreas, which had max relative transduction of 98.8% of WT AAV9 and 82.2% of WT AAV9 , respectively. Additionally, 18/21 variants had less than half the liver transduction of WT AAV9, with three variants below 5% AAV9 liver transduction levels (Fig. 4d) indicating clinically important liver de-targeting . To increase confidence in the fidelity of the individually validated variants in C67BL/6 mice, 3 of the above 21 variants were individually validated in BALB/c mice. Notably, their relative tropism was consistent across the two mouse strains.
• DKK1 was indeed an efficient lung transducing variant with greater than 2-3 fold higher expression than AAV9, and with consistent de-targeting across all other organs compared to AAV9 when quantified at both the RNA and protein level (Fig. 5a, bottom left and right) .
• BLAST basic local alignment search tool

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