[go: up one dir, main page]

WO2024055020A2 - Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs - Google Patents

Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs Download PDF

Info

Publication number
WO2024055020A2
WO2024055020A2 PCT/US2023/073808 US2023073808W WO2024055020A2 WO 2024055020 A2 WO2024055020 A2 WO 2024055020A2 US 2023073808 W US2023073808 W US 2023073808W WO 2024055020 A2 WO2024055020 A2 WO 2024055020A2
Authority
WO
WIPO (PCT)
Prior art keywords
aav
capsid protein
vector
peptide
tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/073808
Other languages
English (en)
Other versions
WO2024055020A3 (fr
WO2024055020A9 (fr
Inventor
Prashant MALI
Andrew PORTELL
Kyle Ford
Amanda SUHARDJO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Priority to JP2025514363A priority Critical patent/JP2025531824A/ja
Priority to AU2023338576A priority patent/AU2023338576A1/en
Priority to EP23864068.4A priority patent/EP4584371A2/fr
Priority to CA3265850A priority patent/CA3265850A1/fr
Priority to CN202380064555.XA priority patent/CN119855901A/zh
Publication of WO2024055020A2 publication Critical patent/WO2024055020A2/fr
Publication of WO2024055020A3 publication Critical patent/WO2024055020A3/fr
Publication of WO2024055020A9 publication Critical patent/WO2024055020A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • 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
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 AAV is AAV5 or AAV9.
  • peptide sequences were generated via pooled oligonucleotide synthesis and inserted into 4 distinct loop regions: AAV5-Loopl (N443) , AAV5-Loop2 (S576) , AAV9-Loopl (Q456) , and AAV9- Loop2 (A587) to generate over 1 million AAV variants.
  • a 20'mer ligand peptide is inserted into one or both of 2 surface-exposed loops of AAV5 (SEQ ID NO: 2) or AAV9 (SEQ ID NO: 4) .
  • 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 heterologous targeting peptide targets the viral vector to neuronal cells or brain tissue. In still another embodiment, the heterologous targeting peptide targets the viral vector to pancreatic cells or pancreas tissue. In yet another embodiment, the heterologous targeting peptide targets the viral vector to cardiac cells or heart tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to lung tissue. In still yet another embodiment, the heterologous targeting peptide targets the viral vector to intestinal tissue. In yet another embodiment, the heterologous targeting peptide targets the viral vector to spleen tissue. In still another embodiment, the heterologous targeting peptide targets the viral vector to renal cells or kidney tissue. In another embodiment, the heterologous targeting peptide targets the viral vector to muscle cells or tissue .
  • the disclosure also provides an adeno-associated virus
  • AAV 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 within or comprises any one of the peptides of SEQ ID NO: 5-820 or 870-911.
  • the capsid protein is a VP1 capsid protein.
  • the capsid protein is a VP2 capsid protein.
  • the capsid protein is a VP3 capsid protein.
  • the heterologous targeting peptide is about 15-25 amino acids in length.
  • the heterologous targeting peptide is about 20 amino acids in length.
  • the heterologous targeting peptide is flanked by a linker peptide at the N-terminal and C-terminal ends of the heterologous targeting peptide.
  • the heterologous targeting peptide targets hepatocytes or liver tissue.
  • the heterologous targeting peptide targets neuronal cells or brain tissue.
  • the heterologous targeting peptide targets pancreatic cells or pancreas tissue.
  • the heterologous targeting peptide targets cardiac cells or heart tissue.
  • the heterologous targeting peptide targets lung tissue.
  • the heterologous targeting peptide targets intestinal tissue.
  • the heterologous targeting peptide targets spleen tissue.
  • the heterologous targeting peptide targets renal cells or kidney tissue. In yet another embodiment, the heterologous targeting peptide targets muscle cells or tissue.
  • the disclosure also provides a recombinant AAV (rAAV) comprising a capsid protein of any of the foregoing embodiments. [0012]
  • the disclosure provides 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 Nos: 5-820 or 870-911.
  • a targeting peptide is present in both Loop 1 and Loop 2.
  • the targeting peptide has the same tropism.
  • the recombinant AAV further comprises a heterologous polynucleotide for gene delivery.
  • the heterologous polynucleotide is a therapeutic gene.
  • the rAAV is present in a pharmaceutical composition.
  • the disclosure also provides a method for delivering a transgene to a subject comprising: administering a recombinant AAV (rAAV) to a subject, wherein the rAAV comprises: (i) a capsid protein of the disclosure, and (ii) at least one transgene, and wherein the rAAV infects cells of a target tissue of the subject.
  • the at least one transgene encodes a protein.
  • the protein is an immunoglobulin heavy chain or light chain or fragment thereof.
  • the at least one transgene encodes a small interfering nucleic acid.
  • the small interfering nucleic acid is a miRNA.
  • 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.
  • the miRNA is expressed in a cell of the target tissue.
  • the target tissue is skeletal muscle, heart, liver, pancreas, brain or lung.
  • the transgene expresses a transcript that comprises at least one binding site for a miRNA, wherein the miRNA inhibits activity of the transgene, in a tissue other than the target tissue, by hybridizing to the binding site.
  • the at least one transgene encodes a gene product that mediates genome editing.
  • the transgene comprises a tissue specific promoter or inducible promoter.
  • the tissue specific promoter is a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PRY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.
  • TSG liver-specific thyroxin binding globulin
  • an insulin promoter a glucagon promoter
  • a somatostatin promoter a pancreatic polypeptide (PRY) promoter
  • a synapsin-1 (Syn) promoter a creatine kinase (MCK) promoter
  • MCK mammalian desmin
  • 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
  • Significantly enriched AAV variants in a particular organ are defined as those with a log2FC > 1 and an FDR-adjusted p-value ⁇ 0.05. Bar plots show the number of significantly enriched variants detected per organ, as well as a comparison of peptide hits for each loop insertion site, for both AAV5 and AAV9.
  • CNN convolutional neural network
  • Model performance was separately evaluated on each organ, via accuracy, area under the receiver operator characteristic curve (AUROC) , Fl score, and Matthews Correlation Coefficient (MCC) . Models were trained on of the data, and the remaining was held out as a validation dataset to evaluate performance.
  • Figure discloses SEQ ID NOs: 867-869, respectively, in order of appearance.
  • FIG. 4A-D In vivo screen identified AAV variants demonstrate reprogrammed tropism,
  • (a) Heatmap showing log 2 FC values for each AAV variant which was significantly enriched in at least one organ. Rows are individual variants, and columns are organs (n 2 per organ) .
  • 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,
  • BLAST Basic local alignment search tool
  • This initial list was then filtered to exclude truncated genomes, redundant samples, human and non-mammalian serotypes, as well as close orthologs.
  • the final resulting list contained 23 AAV capsid sequences for subsequent investigation,
  • 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) .
  • the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.
  • the term "about” meaning within an acceptable error range for the particular value can be assumed.
  • the ranges and/or subranges can include the endpoints of the ranges and/or subranges. In some cases, variations can include an amount or concentration of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.
  • 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.
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • AAV refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus , family Parvoviridae . Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types.
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, e.g. , AAV2, AAV5, and AAV8, or variant serotypes such as AAV-DJ.
  • the AAV structural particle is composed of 60 protein molecules made up of VP1, VP2 and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins ordered into an icosahedral structure.
  • Non-limiting exemplary VP1 sequences useful in the methods disclosed herein are provided below.
  • 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 the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, y-carboxyglutamate, and O- phosphoserine .
  • an amino acid analog refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.
  • 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 refers to a CRISPR-associated, RNA-guided endonuclease such as Streptococcus pyogenes Cas9 (spCas9; see Accession Number Q99ZW2.1, the seguence of which is incorporated herein by reference) and orthologs and biological equivalents thereof.
  • Biological equivalents of Cas9 include, but are not limited to, C2cl from Allcyclobacillus acldeterrestrls and Cpfl (which performs cutting/cleaving functions analogous to Cas9) from various bacterial species including Acidaminococcus spp. and Francisella novicida U112.
  • 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 refers to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) .
  • CRISPR may also refer to a technique or system of sequence-specific genetic manipulation relying on the CRISPR pathway.
  • a CRISPR recombinant expression system can be programmed to cleave a target polynucleotide using a CRISPR endonuclease and a guideRNA.
  • a CRISPR system can be used to cause double stranded or single stranded breaks in a target polynucleotide.
  • a CRISPR system can also be used to recruit proteins or label a target polynucleotide.
  • 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.
  • delivery vehicle refers to a composition useful for delivering a payload to a cell, tissue or subject.
  • the delivery vehicle can deliver various payloads including biological agents and small molecule drugs.
  • exemplary, but non-limiting, delivery vehicles include a liposome, a nanoparticle, a bacteria, a bacteriophage, a virus-like particle (VLP) , a erythrocyte ghost, and an exosome.
  • VLP virus-like particle
  • 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.
  • the term "functional" may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.
  • gRNA or "guide RNA” as used herein refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique.
  • Techniques of 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.
  • 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) .
  • 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 a position in each sequence which can be aligned for purposes of comparison. For example, when a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A 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 shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure.
  • Homology refers to a percent (%) identity of a sequence to a reference sequence.
  • 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 particular peptide, polypeptide or nucleic acid sequence has a particular identity/homology can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) .
  • 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 percent identity can be corrected by calculating the number of residues of the query sequence that are lateral to the N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the island C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence are considered for this manual correction. For example, a 90 residue subject sequence can be aligned with a 100 residue query sequence to determine percent identity.
  • 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.
  • isolated can refer to molecules or biologicals or cellular materials being substantially free from other materials.
  • the term “isolated” can refer to nucleic acid, such as DNA or RNA, or protein or polypeptide (e.gr. , an antibody or derivative thereof) , or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
  • isolated also can refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and may not be found in the natural state.
  • the term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • the term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
  • 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.
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall; L-asparaginase ; anthracenedione substituted urea; methyl hydrazine derivatives; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocar zinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores) , aclacinomysins , actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, car zinophilin, chromomycinis , dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including
  • TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J. ) , ABRAXANE® Cremophor-f ree, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.
  • 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.
  • growth hormone including human growth hormone, bovine growth hormone, and other members of the GH supergene family; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-l-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA) ; bombazine; thrombin; alpha tumor necrosis factor, beta tumor necrosis factor; enkephalinase ; RANTES (regulated on activation normally T-cell expressed and secreted) ; human macrophage inflammatory protein (MIP-l-alpha)
  • 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.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide .
  • the sequence of nucleotides can be interrupted by non-nucleotide components .
  • a polynucleotide can be further modi fied after polymeri zation, such as by conj ugation with a labeling component .
  • the term also can refer to both double and single stranded molecules . Unles s otherwise specified or required, any embodiment of this disclosure that is a polynucleotide can encompass both the double stranded form and each of two complementary s ingle stranded forms known or predicted to make up the double stranded form.
  • polynucleotide sequence can be the alphabetical representation of a polynucleotide 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 genomics and homology searching .
  • 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,
  • treat refers to ameliorating symptoms associated with a disease or disorder (e.g., cancer, Covid-19 etc.) , including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or freguency of symptoms of the disease or disorder .
  • a disease or disorder e.g., cancer, Covid-19 etc.
  • 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.
  • viral vectors examples include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Infectious tobacco mosaic virus (TMV) -based vectors can be used to manufacturer proteins and have 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 Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al.
  • a vector construct can refer to the polynucleotide comprising the retroviral genome or part thereof, and a gene of interest. Further details as to modern methods of vectors for use in 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 that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked 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. ) .
  • 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
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and 'Vector” can be used interchangeably.
  • the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.
  • the vector or plasmid contains sequences directing transcription and translation of a relevant gene or genes, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the species chosen as a production host.
  • the vector or plasmid contains sequences directing transcription and translation of a gene fragment, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the species chosen as a production host. [0075] Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art.
  • 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 .
  • Adeno-associated viruses are common gene therapy vectors, however, their effectiveness is hindered by poor target tissue transduction and off-target delivery. Hypothesizing that naturally occurring receptor-ligand interactions could be repurposed to engineer tropism.
  • the disclosure provides a method wherein all annotated protein ligands known to bind human receptors were fragmented into tiling 20-mer peptides and displayed onto the surface loops of AAV5 and AAV9 capsids at two sites. The resulting capsid libraries, comprising >1 million AAV variants, were screened across 9 tissues in C57BL/6 mice.
  • certain peptides also displayed consistent activity across mice strains, capsid insertion contexts, and capsid serotypes, including novel immune orthogonal serotypes. Further analyses of displayed peptides revealed that biophysical attributes were highly predictive of AAV variant packaging, and there was a machine learnable relationship between peptide sequence and tissue tropism.
  • the disclosed comprehensive ligand peptide tiling and display approach can enable engineering of tropism across diverse viral, viral-like and non-viral delivery platforms, and shed light into basic receptor-ligand biology.
  • the disclosure provides a plurality of peptides useful for targeting a desired tissue.
  • the disclosure also provides peptides that are immunosilent .
  • the disclosure also provides vectors, delivery vehicles and peptide-payload conjugates comprising one or more of the peptides of the disclosure having a desired tropism.
  • Rational screening strategies have immense potential to expand the molecular tools available for clinical gene therapy applications. While AAV engineering efforts have been conducted for over a decade, advances in DNA synthesis have enabled us to create here a data-driven library of AAV variants leveraging existing functional biomolecules from nature. Using natural biomolecules as a defined source of inserted peptides has multiple benefits over random hexamers (and similar methods) . First, natural biomolecules have been pre-filtered for biological functionality by millennia of evolutionary selection pressure. Second, a defined library allows for robust quantification of the fitness of each AAV variant, enabling facile stratification of AAV variants by infectivity across organs of interest.
  • AAVs adeno-associated viruses
  • AAV variants have been engineered to specifically target tis sues such as the brain and muscle .
  • This has predominantly been established using strategies of iteratively screening random peptides inserted into the AAV capsid, or caps id shuffling, or randomly mutageni zing the caps id sequence as a whole , or direct chemical engineering .
  • mutagenizing AAV capsids via random oligomers has yielded functional capsids with novel properties a stochastic mutational screening strategy limits the ability to predict future functional variants , and thus rational and programmable engineering of viral phenotypes remains an elus ive goal .
  • Short peptides grafted into stabili zing molecular scaffolds can recapitulate local protein domain structure , and as protein-protein interface sites are typically 1200 -2000 A 2 , with speci fic peptide hot loops ranging from 4 -8 amino acids (AAs ) contributing maximally to the binding energy of protein-protein interactions , it was hypothes i zed that 20 amino acid peptide insertions into the AAV capsid could drastically alter its binding ability, and therefore , its transduction profile in vivo .
  • ligand tiling enabled robust guantitation of tissue transduction rates for all variants screened. Furthermore, systematic examination of the activity of similar peptides generate predictions of putative receptor interactions driving AAV variant tropism. Quantifying transduction rates across nine organs, extremely specific variants targeting the brain and lung were identified, as well as muscle and heart targeting variants with broader organ transduction. The resulting data linking AAV variant genotype 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.
  • FIG. 3a The general schematic for the AAV screens is outlined in Fig. 3a. Briefly, in the primary screen, a 20 amino acid peptide library consisting of tiled fragments from all known receptorinteracting ligands along with other interesting protein classes flanked by glycine-serine linkers were displayed into the 2 surface- exposed loops of two clinically relevant AAV serotypes, AAV5 and AAV9 with their sequences and specific amino acid site of insertion shown in Table 1.
  • Table 1 Showing both the DNA and amino acid sequences and insertion sites of the two wild-type AAV serotypes utilized in the screens, AAV5 and AAV9. Bolded and underlined are the flanking residues of the of the 'Loop 1' site and shown in bold/ital/underline are the flanking residues of the 'Loop 2' site.
  • a candidate list of "hit peptides" in the AAV9Loopl and AAV9Loop2 scaffolds which were synthesized and subjected to deep mutagenesis to yield AAV libraries with millions of variants that were then profiled in a secondary screen.
  • AAV capsid particles were produced from these libraries and injected into mice where the tropism was quantified.
  • the AAV capsid libraries were pooled from the primary and secondary screens and injected into rhesus macaques as an important preclinical non-human primate (NHP) model.
  • variable peptide region was amplified out and the AAV abundance quantified in the liver, lung, and 4 lobes of the brain, moreover, to apply additional selected pressure, the variable peptide regions from the heart, lung, pancreas, intestine, brain, and muscle were amplified out and cloned back into the AAV9Loopl and AAV9Loop2 scaffolds.
  • This tertiary screen was then subjected to two weeks on in vivo selective pressure in mice before the tropism was quantified by NGS.
  • a "hit" was defined as an engineered AAV variant that exhibited a log2FC relative to the capsid > 1 in both replicates and a p- value ⁇ 0.1 in at least one organ.
  • Table 2 Table for "wild-type" peptides that were present in the primary screen library and showed in vivo activity across all 4 scaffolds in which they were grafted (i.e. AAVsLocpl, AAV5Loop2,
  • AAV9Loopl AAV9Loop2
  • Table 3 Table for "wild-type" peptides that were present as hits in both the primary, secondary, tertiary, and NHP screens.
  • Table 4A-B Shown in Table 4A-B are the top performing peptides across all of these described screens. These include the mutant versions of the peptides that were originally identified in the primary screen.
  • the information includes the amino acid sequence of the peptide, the AAV scaffold in which that peptide was inserted, the organ where that peptide was a hit, the log2FC in that organ, and the screen in which that peptide was identified. While the peptides shown here were highlighted for their ability to transduce a particular organ, many of these also display other interesting properties such as their increased ability to form functional capsids (high titers) , their significant de-targeting away from the liver, and their ability to target unique combinations of organs simultaneously.
  • these peptides and mutants thereof to have the ability to modulate tropism of AAV capsids, such as AAV5, AAV9, and the 23 immune orthogonal capsids (shown in Table 5) and beyond as well as in tropism of other delivery vehicles and peptide-payload conjugates.
  • AAV capsids such as AAV5, AAV9, and the 23 immune orthogonal capsids (shown in Table 5) and beyond as well as in tropism of other delivery vehicles and peptide-payload conjugates.
  • Table 4A Table for mutant peptides that were identified as the top hits in the primary, secondary, tertiary, and NHP screens. Shown are the peptide amino acid sequences, the scaffold where they were grafted, the log2FC in the organ they were a hit, the organ they were a hit in, and the screen in which that log2FC was observed.
  • Table 5 table of the identified 23 immune-orthogonal AAV serotypes with their DNA and amino acid (AA) sequences.
  • 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 .
  • the disclosure presents a massive functional screen of engineered AAV variants, spanning over one million total variants derived from two capsids and multiple sites of insertional mutagenesis.
  • validation of 21 AAV variants identifying AAV capsids with increased organ transduction across multiple organs (heart, muscle, and lung for AAV9.PDGFC, Fig. 4c) , as well as highly specific AAV capsids (AAV5.AP0A1, AAV9.DKK1, Fig. 5a) .
  • Improved broad targeting AAV variants have massive potential for genetic diseases such as hemophilia A, where total factor VIII expression levels are most critical.
  • 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) .
  • AAV5 and AAV9 structure files were downloaded from the Protein Data Bank. These were visualized using the PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC with the ramp new function utilized for coloring the surface capsid representation.
  • Each AAV library consisted of 275,298 peptides, derived from 6,465 proteins. These protein sources were mined from a variety of protein families, including all protein ligands cataloged in the Guide to Pharmacology database, toxins, nuclear localization signals (NLS) , viral receptor binding domains, albumin and Fc binding domains, transmembrane domains, histones, granzymes, and predicted cell penetrating motifs. In addition to peptides coding for functional biomolecules, 444 control peptides coding for FLAG-tags with premature stop codons were included.
  • Oligonucleotide libraries were synthesized by GenScript as three 91,766 element pools. Each oligonucleotide library was amplified using KAPA Hifi Hotstart Readymix, and the manufacturer recommended cycling conditions with an annealing temperature of 60 °C and an extension time of 30 seconds. The number of PCR cycles was optimized to avoid over-amplification of the peptide libraries. After amplifying each oligonucleotide pool and confirming amplicon size on an agarose gel, the amplified sub-libraries were pooled to yield the total 275,298 element peptide library.
  • each AAV capsid library was produced by transfecting HEK293T cells in 40 15 cm dishes with the plasmid library pool (diluted 1:100 with pUC19 filler DNA to prevent capsid cross-packaging) and an adenoviral helper plasmid (pHelper) . Titers were determined via qPCR using the iTaq Universal SYBR green supermix and primers binding to the AAV ITR region.
  • 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.
  • capsid particles As templates for PCR, 2 pL of virus was added to 50 pL of alkaline digestion buffer (25mM NaOH, 0.2 rnM 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. 1 pL of this digested capsid mix was then used as a template for a 50 pL PCR reaction. For each sample, the number of cycles was optimized to avoid overamplification, and a secondary PCR was subsequently performed to add illumina compatible adapters and indices.
  • alkaline digestion buffer 25mM NaOH, 0.2 rnM EDTA
  • neutralization buffer 40mM Tris-HCl, .05% Tween-20, pH 5
  • AAV cap genes from each tissue for the pooled screen as with the plasmid/capsid libraries, a two-step PCR based library prep method was used. For each organ and replicate, a 300 pL PCR reaction was performed with 120 uL of genomic DNA used as a template. For each tissue, the number of cycles was optimized via an initial qPCR to avoid overamplification of the library. All other parameters such as primers, and melting temperatures were identical to the PCRs for the plasmid libraries. Following this initial PCR, a secondary PCR was performed as above to add illumina compatible adapters and indices. The libraries were then sequenced on a NovaSeq 6000 with an S4 flowcell generating lOObp paired end reads.
  • Either saline or the AAV-variant-mCherry, AAV9-mCherry, or AAV5-mCherry capsids were systematically administered to mice in duplicate at a dose of 5E11 vg/mouse.
  • the lungs were inflated with a PBS/OCT solution and the lungs, heart, liver, intestine, spleen, pancreas, kidneys, brain, and gastrocnemius muscle were harvested.
  • Each organ was split with one portion placed in RNAlater and the other embedded in OCT blocks and flash frozen in a dry-ice/ethanol slurry.
  • mCherry transgene expression was normalized to that of an internal GAPDH control, using GAPDH specific primers.
  • OCT frozen blocks were cryosectioned at approximately 10 pm thickness and tissue slides were then imaged on an Olympus SlideScanner S200.
  • Exposure times between 5-1000 ms were used, with identical exposure times used for all samples of a given tissue type.
  • mCherry expression from histological sections was then quantified using the Olympus OlyVIA software to calculate the mean pixel intensity across the entire organ section.
  • the MAGeCK (94) 'count' function was used to generate count matrices describing AAV abundance in each sample (plasmids/capsids/tissues) .
  • the count matrices were normalized (via multiplication with a constant size-factor) for each sample to account for non-identical read depth.
  • the sequencing counts were then transformed by taking the log base 2 of the raw counts, after addition of a pseudocount. Variants with no counts across all of the experimental samples were excluded from analysis.
  • the biophysical characteristics of the inserted peptides was calculated using the "ProteinAnalysis” module within the Biopython Python package. A variant was considered a successful packager if it had higher abundance in the capsid particles compared to the plasmid pool. Support vector machine training and visualization was accomplished via the "svm” module within the sklearn Python package (96) . UMAP projection of peptide biophysical characteristics was accomplished via the "plot” functionality within the UMAP Python package. All default parameters were used for the visualization. Boxplots and hexbin plots were generated using the matplotlib and seaborn Python packages.
  • Heatmaps for visualizing AAV transduction were generated using the 'clustermap' function within the seaborn Python package. Rows and columns were ordered via the scipy 'optimal_leaf_ordering' function to minimize the euclidean distance between adjacent leaves of the dendrogram.
  • UMAP projections visualizing AAV tissue specificity were generated by embedding the tissue level log2 fold change into two dimensions via the "plot" functionality within the UMAP Python package. All default parameters were used for generating the embedding. The variants were colored by the organ in which they had the max log 2 fold change.
  • 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.
  • HEK293T cells were seeded in a 12-well plate at -20% confluency the day before transduction.
  • 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.
  • AAV orthologs were curated by first identifying sequences which exhibited similarity to the AAV2 cap gene using the National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) . Next, incomplete, heavily truncated, and highly homologous sequences to one another were filtered out of the selection criteria. Viruses within the human AAV clade and those from non-mammalian hosts were then filtered out. Finally, sequences with high similarity to previously identified human serotypes were removed, resulting in the 23 potential AAV orthologues assessed. [00142] Immune orthogonal AAV production
  • AAV orthologs To clone computationally identified AAV orthologs, the capsid sequences were codon-optimized and cloned downstream of the AAV2 rep gene using Gibson Assembly. Immune orthogonal AAV capsids were produced as described for the AAV variant validation capsids and AAV production titer was measured via qPCR with primers binding to the AAV ITR region. Any ortholog with a production titer within a power of 10 of AAV5 was considered to have successfully packaged.
  • 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) .
  • 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 'AAV MG2' vector at the appropriate Loop 1 location with an identical protocol as was done for cloning peptides into AAV9.
  • PDGFC' vector capsid was then assayed in vivo identically to the above validation experiments using AAV9.
  • 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) .
  • AAV variants targeting the skeletal muscle and brain were identified, in line with the high therapeutic AAV doses needed to achieve clinical efficacy for muscle targeting gene therapies, and the challenge of delivery across the blood-brain barrier.
  • a substantial number of the AAV variants were comprised of the same peptide inserted across different AAV capsids and insertion sites, giving credence to the hypothesis that tropism reprogramming is, at least partially, peptide-specific (Fig. 3c) .
  • transduction of multiple organs is a near ubiquitous phenotype among the infectious AAV variants identified (Fig. 4a) .
  • Significant transduction of the liver and spleen was observed for the majority of infectious variants regardless of which other organs were cotransduced. This is true even for variants with insertions in surface loops known to be involved in WT capsid receptor binding.
  • liver and spleen targeting were near ubiquitous, allowing for identifying variants which specifically target the liver/spleen plus one other organ, as well as variants which transduced all tissues at high levels (Fig. 4a) .
  • variants were hierarchically clustered based on their tissue detection levels, variants derived from the same sub-library tended to cluster together, suggesting that the tissue specificity of the wild-type scaffold was at least partially a determinant of engineered variant tropism.
  • Hierarchical clustering of the organ samples resulted in replicates clustering together, giving further confidence to the reliability of the screen results.
  • the tissue detection levels for each variant were embedded into two dimensions using UMAP, coloring the variants by the organ they most readily transduce (Fig. 4b) . In this reduced dimensional space, organ specific clusters can be readily identified, with the liver and spleen targeting variants especially prominent.
  • 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
  • the result highlights the fidelity of the ligand tiling display approach and further lends credence to the exciting possibility that displayed peptides identified in the initial screening approach could be utilized to re-engineer a diverse clade of AAV serotypes via peptide transfer.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés de modification génétique de protéines et de virus afin d'améliorer le tropisme, et des protéines et des virus fabriqués à l'aide desdits procédés.
PCT/US2023/073808 2022-09-09 2023-09-08 Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs Ceased WO2024055020A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2025514363A JP2025531824A (ja) 2022-09-09 2023-09-08 受容体リガンドをタイリングするディスプレイされたペプチドを介したトロピズムのリプログラミング
AU2023338576A AU2023338576A1 (en) 2022-09-09 2023-09-08 Reprogramming tropism via displayed peptides tiling receptor-ligands
EP23864068.4A EP4584371A2 (fr) 2022-09-09 2023-09-08 Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs
CA3265850A CA3265850A1 (fr) 2022-09-09 2023-09-08 Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs
CN202380064555.XA CN119855901A (zh) 2022-09-09 2023-09-08 经由展示肽平铺受体-配体重编程趋向性

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263405360P 2022-09-09 2022-09-09
US63/405,360 2022-09-09

Publications (3)

Publication Number Publication Date
WO2024055020A2 true WO2024055020A2 (fr) 2024-03-14
WO2024055020A3 WO2024055020A3 (fr) 2024-05-10
WO2024055020A9 WO2024055020A9 (fr) 2024-06-06

Family

ID=90191985

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/073808 Ceased WO2024055020A2 (fr) 2022-09-09 2023-09-08 Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs

Country Status (6)

Country Link
EP (1) EP4584371A2 (fr)
JP (1) JP2025531824A (fr)
CN (1) CN119855901A (fr)
AU (1) AU2023338576A1 (fr)
CA (1) CA3265850A1 (fr)
WO (1) WO2024055020A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6855314B1 (en) * 2000-03-22 2005-02-15 The United States Of America As Represented By The Department Of Health And Human Services AAV5 vector for transducing brain cells and lung cells
US20200121746A1 (en) * 2017-06-30 2020-04-23 The Regents Of The University Of California Adeno-associated virus virions with variant capsids and methods of use thereof
US20230203102A1 (en) * 2020-05-13 2023-06-29 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids

Also Published As

Publication number Publication date
AU2023338576A1 (en) 2025-02-20
CA3265850A1 (fr) 2024-03-14
WO2024055020A3 (fr) 2024-05-10
WO2024055020A9 (fr) 2024-06-06
JP2025531824A (ja) 2025-09-25
EP4584371A2 (fr) 2025-07-16
CN119855901A (zh) 2025-04-18

Similar Documents

Publication Publication Date Title
US20240173434A1 (en) Compositions and methods for gene editing for hemophilia a
JP2024520534A (ja) 環状ｒｎａを調製するための構築物及び方法並びにその使用
CN112585268A (zh) 通过插入供体多核苷酸用于基因组编辑的组合物和方法
US20210348159A1 (en) Compositions and methods for delivering transgenes
KR20180092989A (ko) 트랜스포존 시스템, 이를 포함한 키트, 및 이들의 용도
WO2022167009A1 (fr) Arnsg ciblant l'arnm de l'aqp1, et vecteur et utilisation associés
WO2022120103A1 (fr) Ingénierie d'aav orthogonal immunitaire et de furtif immunitaire crispr-cas
WO2023231959A2 (fr) Compositions d'arn circulaire synthétiques et leurs procédés d'utilisation
Ghosh et al. Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns
WO2019106522A1 (fr) Criblage de crispr/cas9 regroupé dans des cellules primaires à l'aide d'une technologie de permutation de guide
WO2024055020A2 (fr) Reprogrammation de tropisme par l'intermédiaire de peptides présentés recouvrant des ligands-récepteurs
EP4326290A2 (fr) Usines de protéines à base de lymphocytes b techniques pour traiter des maladies graves
US20230002756A1 (en) High Performance Platform for Combinatorial Genetic Screening
CN116217741B (zh) 一种高效率且低脱靶率的精确基因编辑方法
JP2025503617A (ja) タンパク質の発現のための最適化されたポリヌクレオチド
EP3635098B1 (fr) Lymphocytes t modifiés pour surexprimer lephf19
Xu et al. mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo
WO2025190256A1 (fr) Protéine cas de type ii et utilisations associées
US20220364073A1 (en) Engineering immune orthoganol aav and immune stealth crispr-cas
WO2025036482A1 (fr) Protéine cas de type ii, système crispr-cas et utilisations associées
WO2025245269A1 (fr) Procédé de criblage pour identifier des mécanismes de résistance au cancer et de létalité synthétique dans des cellules cancéreuses résistantes
US12497611B2 (en) Compositions and methods for high-throughput activation screening to boost T cell effector function
Raghavan Engineering minimally immunogenic cargos and delivery modalities for gene therapy
CN119487200A (zh) 用于基因治疗方法的组织特异性基因外安全港的鉴定
Manjunath Analysis of the Role of EIF5A in Mammalian Translation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23864068

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: AU2023338576

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2023338576

Country of ref document: AU

Date of ref document: 20230908

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2025514363

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2025514363

Country of ref document: JP

Ref document number: 202380064555.X

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 202547032987

Country of ref document: IN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23864068

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2023864068

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023864068

Country of ref document: EP

Effective date: 20250409

WWP Wipo information: published in national office

Ref document number: 202380064555.X

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 202547032987

Country of ref document: IN

WWP Wipo information: published in national office

Ref document number: 2023864068

Country of ref document: EP