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WO2024196669A2 - Procédés d'édition in vivo de cellules b - Google Patents

Procédés d'édition in vivo de cellules b Download PDF

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Publication number
WO2024196669A2
WO2024196669A2 PCT/US2024/019736 US2024019736W WO2024196669A2 WO 2024196669 A2 WO2024196669 A2 WO 2024196669A2 US 2024019736 W US2024019736 W US 2024019736W WO 2024196669 A2 WO2024196669 A2 WO 2024196669A2
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WIPO (PCT)
Prior art keywords
cell
protein
adenovirus
recombinant protein
peptide tag
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.)
Pending
Application number
PCT/US2024/019736
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English (en)
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WO2024196669A3 (fr
Inventor
Mark Selby
Hangil Park
Rosa Romano
David T. Curiel
Paul Boucher
Zhi Hong LU
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.)
Washington University in St Louis WUSTL
Walking Fish Therapeutics Inc
Original Assignee
Washington University in St Louis WUSTL
Walking Fish Therapeutics Inc
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Publication date
Application filed by Washington University in St Louis WUSTL, Walking Fish Therapeutics Inc filed Critical Washington University in St Louis WUSTL
Publication of WO2024196669A2 publication Critical patent/WO2024196669A2/fr
Publication of WO2024196669A3 publication Critical patent/WO2024196669A3/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
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    • C12N2510/00—Genetically modified cells
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • B cells are naturally hardwired to present antigens and secrete immunoglobulins.
  • B cells should have great potential as a cellular therapy for targeting certain diseased cell types and expressing therapeutic proteins.
  • alternative treatments such as genetically engineered B cells, for the treatment of a variety of diseases and disorders, including cancer, heart disease, inflammatory disease, muscle wasting disease, neurological disease, and the like.
  • B cells Modifying B cells for the treatment of various diseases, however, is a technique that has not been extensively studied, despite the critical role of B cells in immune responses.
  • Human B cells are easily isolated and can be expanded, making them viable candidates for engineering.
  • B cells can be matured to long-lived cells that are ideal for provision of therapeutic proteins that are required for extended periods.
  • injected B cells can traffic to their normal tissue niches including spleen, lymph nodes and the bone marrow where they can persist.
  • ex vivo modification has been contemplated to avoid in vivo use of recombinant viruses to which immune responses can inactivate.
  • Ads Adenoviral vectors
  • Ads are uniquely suited to achieve this task due to their large packaging capacity, lack of genomic integration, and unparalleled gene transfer efficiency.
  • off-target gene expression and viral particle sequestration may lead to safety concerns in vivo.
  • targeting to specific cell markers remains a challenge, and vectors capable of using existing antibodies for targeting would be of strong utility.
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a recombinant protein capable of recognizing and forming a high affinity interaction with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a recombinant protein capable
  • said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said RNA-guide nuclease is CAS9. In various embodiments, said wherein said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an RNA-guided nuclease a gRNA targeting a locus on the human genome; and an adenovirus, wherein said adenovirus comprises: a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a first and second peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said second peptide tag; and further comprising an RNA binding protein.
  • said second recombinant peptide comprises a Cas protein.
  • said method for editing said B cell is ex vivo.
  • said method for editing said B cell is in vivo.
  • said B cell is selected from a plasma cell or a plasmablast.
  • said adenovirus is a human or primate adenovirus.
  • said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36.
  • the native tropism of said adenovirus has been ablated.
  • said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the method of claim 48, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said second peptide tag; and recognizing and interacting with a molecule capable of binding with a shielding protein.
  • said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct
  • said second recombinant peptide is capable of recognizing and interacting with albumin. In various embodiments, said second recombinant peptide is capable of recognizing and interacting with serum protein. In various embodiments, said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36.
  • the native tropism of said adenovirus has been ablated.
  • said wherein said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM.
  • said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the present invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a second recombinant protein capable of capable of recognizing and forming a high affinity interaction with said second peptide tag; and further comprising an RNA binding protein.
  • said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid
  • said second recombinant peptide comprises a Cas protein.
  • said method for editing said B cell is ex vivo.
  • said method for editing said B cell is in vivo.
  • said B cell is selected from a plasma cell or a plasmablast.
  • said adenovirus is a human or primate adenovirus.
  • said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36.
  • the native tropism of said adenovirus has been ablated.
  • said wherein said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a first protein expressed on the surface of a B cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said second peptide tag; and recognizing and interacting with a second protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a
  • said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said wherein said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to an engineered B cell, wherein said engineered B cell has been edited to express a therapeutic protein of interest, by contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA- guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA- guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat
  • said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said wherein said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to a method of treating a patient in need thereof with an engineered B cell, wherein said engineered B cell has been edited to express a therapeutic protein of interest, by contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further
  • said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM.
  • said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to a composition for editing a B cell, comprising an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a
  • said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • the invention relates to a method of treating a patient in need thereof by administering to said patient a composition comprising an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a pharmaceutical acceptable carrier.
  • said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein;
  • said method for editing said B cell is ex vivo. In various embodiments, said method for editing said B cell is in vivo. In various embodiments, said B cell is selected from a plasma cell or a plasmablast. In various embodiments, said adenovirus is a human or primate adenovirus. In various embodiments, said adenovirus is selected from the group consisting of Ad5, HAdV-C5, SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said therapeutic protein is a recombinant antibody.
  • said therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • said peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • said peptide tag is expressed in the hexon protein of said adenovirus.
  • said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38.
  • the recombinant protein comprises an antigen binding domain.
  • the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.
  • said B cell is contacted with an activating agent, prior to contacting sad B cell with said adenovirus and said recombinant protein.
  • said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.
  • said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.
  • said preconditioning is an immunization or vaccination.
  • the invention relates to a method for editing at least one cell comprising contacting said cell with an adenovirus, wherein said adenovirus comprises: a construct comprising a nucleic acid sequence encoding a therapeutic protein; and wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a first cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a second cell.
  • said adenovirus comprises: a construct comprising a nucleic acid sequence encoding a therapeutic protein; and wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing
  • the method for editing said cell is ex vivo. In various embodiments, the method for editing said cell is in vivo.
  • the adenovirus is a human or primate adenovirus. In various embodiments, the adenovirus is selected from the group consisting of Ad5, HAdV-C5, and SAdV-36. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, the therapeutic protein is a recombinant antibody.
  • the therapeutic protein is for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • the peptide tag is expressed in the Hl loop domain of the Ad fiber.
  • the peptide tag is expressed in the hexon protein of said adenovirus.
  • the peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.
  • the high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.
  • said covalent bond is an isopeptide bond.
  • the first or second recombinant protein comprises a ligand.
  • the ligand is selected from the group consisting of CD20, CD19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD138, PD273, or an Ig receptor.
  • the recombinant protein comprises an antigen binding domain.
  • the first or second recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.
  • the adenovirus is contacted with said recombinant protein, prior to contacting with said one or more cells.
  • the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said one or more cells.
  • the therapeutic protein is an anti-HIV antibody.
  • the anti-HIV antibody comprises SEQ ID No. 23.
  • the anti-HIV antibody comprises SEQ ID NO. 24.
  • FIG. 1 shows a strategy for vector targeting via adenovirus.
  • Transcriptional targeting is achieved utilizing a tissue specific promoter (TSP) to control expression of the transgene.
  • TSP tissue specific promoter
  • Transductional targeting is achieved via replacement of fiber knob with target cell binding domain and/or incorporation of binding domain within fiber knob. Modification of the major capsid protein hexon achieves liver un-targeting. The combination of these approaches constitutes the basis of our novel triple targeting technology
  • FIG. 2 shows tropism modified Ad with in vivo selectivity for endothelial cells of the bone marrow. Comparison was made between GFP reporter-encoding adenoviruses - untargeted human adenovirus serotype 5 with CMV transcriptional control of GFP (Ad5.CMV) and targeted Ad5 with ROBO4 TSP-based transcriptional control of GFP (Ad5.ROBO4). C57BL/6 mice of ten weeks of age were tail vein-injected with l.OxlO 11 viral particles of unmodified Ad5.CMV-eGFP or Ad5.ROBO4-eGFP vector. Femur was harvested from the mice 3 days post virus injection and subject to immunofluorescence analysis of eGFP reporter detection.
  • FIGs. 3A-3B show adenoviral vector-mediated infection of murine primary B cells.
  • (3 A) Ex vivo infection.
  • Murine B cells were isolated and activated for 30h with 50pg/mL LPS, then infected with the indicated vectors encoding GFP. 48h post infection cells were scored for GFP expression using flow cytometry.
  • B cells were gated as live CD 19+ cells.
  • (3B) In vivo infection. 3 x C57BL/6J mice per group were injected with PBS or 5E10 vp of the indicated vectors retro-orbitally.
  • Ad5.CMVeGFP is control adenoviral vector based on human adenovirus serotype 5 (huAd5). It is El A/B-deleted and replication-incompetent with the eGFP reporter expressed from the CMV promoter.
  • AdPK4.CMVeGFP contains the fiber knob domain of porcine adenovirus serotype 4 in place of the corresponding domain from huAd5
  • FIGs. 4A-4B show gene transfer to human B cells.
  • Human B cells were isolated from peripheral mononuclear cells (PBMC) and expanded using standard medium. Cultured cells were infected with various adenoviral vectors expressing the eGFP and reporter expression analyzed by flow cytometry 3 days post-infection.
  • Ad5 RGD central adenovirus vector based on human adenovirus serotype 5 (Ad), Ad5 with fiber knob derived from porcine serotype 4 (Ad5 PK4), Ad5 with polylysine at the fiber knob COOH terminus (Ad5 PK7), and Ad5 with the peptide RGD4C in the fiber knob HI loop (Ad5 RGD).
  • 4B Analysis of adenoviral vectors with various candidate B cell tissue specific promoters. Comparison was made between Ad5RGD vectors with various tissue specific promoters driving the GFP reporter.
  • CMV cytomegalovirus intermediate/early promoter
  • SFFV spleen focus forming virus promoter
  • FEEK synthetic promoter based on human IgK core and enhancers
  • MH synthetic promoter based on human IgH core and enhancers
  • EB VW Epstein-Barr virus W promoter
  • FIGs. 5 A-5B shows somatic integration of hAAT maintains stable long term gene expression in mice.
  • 5 A Non-integrative mouse groups were injected with either PBS (green), 7.5E10 VP of hAAT donor vector (Ad5.EFla-hAAT) and 2.5E10 sham vector (Ad5.CMV-EGFP, solid red) or 5E10 VP of each (dashed-red).
  • Integrative mouse groups were injected with 7.5E10 VP of hAAT donor vector (Ad5.EFla-hAAT) and 2.5E10 CRISPR-containing vector (Ad5.CMV-Cas.U6-gRNA, solid-blue), 5E10 VP of each (dashed- blue), or 2.5E10 VP of hAAT donor vector (Ad5.EFla-hAAT) and 7.5E10 CRISPR- containing vector (dotted-blue).
  • Plasma levels of hAAT were determined intermittently via ELISA. Error bars are standard deviations.
  • 5B One microliter of plasma samples were run on western blot to visualize the temporal change of gene expression between the first week and the sixteenth week in integrative versus episomal groups. Each lane represents one sample from one mouse. Image is representative of two blots.
  • FIG. 6 shows B cell editing strategies. Each diagram shows the heavy-chain locus with a native VDJ-recombined variable-chain gene. In panels B and C, the native VJ- recombined kappa-chain locus is also shown. Beneath each diagram is a homology-directed repair template (HDRT) bounded by its 5’ and 3’ homology arms. These HDRT can be provided as single-stranded DNA or as an AAV episome. (A) A strategy used by most investigators in the field.
  • HDRT homology-directed repair template
  • a single expression cassette consisting of a splice acceptor (SA), poly-adenylation sequence (PA), full light chain (VJCK), a P2A self-cleaving sequence, and full variable heavy sequence (VDJ) is inserted into the intron 5’ to the IgM constant heavy chain (Cp).
  • SA splice acceptor
  • PA poly-adenylation sequence
  • VJCK full light chain
  • VDJ full variable heavy sequence
  • Cp IgM constant heavy chain
  • FIG. 7 shows physiologic BCR editing enables robust in vivo CAR B cell responses.
  • Murine B cells were edited by intron editing (represented in FIG. 6, view A) or our double editing approach (FIG. 6, view C).
  • mice After receiving CAR B cells, mice were vaccinated twice with a 60-meric I3-B505 gpl20 construct.
  • serial dilutions of mouse plasma were tested for neutralizing activity against a CRF250 pseudovirus using TZM-bl assays.
  • Double-edited cells (blue) outperformed intron-edited cells (green) after both vaccine challenges but the contrast was especially clear following the second dose (square symbols).
  • FIG. 8 shows native locus editing of murine heavy- and light-chain genes to express the variable chains of the bNAb VRC26.25.
  • a timeline for a typical edited B cell experiment is represented. Naive B cells are isolated from donor animal spleens, reprogrammed to express a human antibody of choice, and engrafted into recipient animals. If desired, mice can be repeatedly vaccinated over their natural lifetime. In this experiment, mice were engrafted with edited B cells expressing both variable heavy and variable light chains of apex bNAb VRC26.25 using the editing strategy shown in FIG. 6, view C.
  • the murine B cell receptor loci were edited so that the VRC25.25 heavy and light chain genes here expressed from murine heavy and kappa loci, preserving the location and regulatory apparatus of the cell. Mice were then vaccinated three times with a soluble Env construct (chimeric ConM SOSIP.v7/ CRF250) at the time-points indicated and serum samples were collected after each immunization as indicated.
  • a soluble Env construct chimeric ConM SOSIP.v7/ CRF250
  • FIG. 9 shows edited B cells undergo affinity maturation in vivo to further improve bNAbs.
  • the HCDR3-only editing approach (FIG. 6, view D) was used to create tgB cells expressing the HCDR3 of VRC26.25 in the context of otherwise diverse, native murine heavy and light chains.
  • VRC26.25-HCDR3 edited B cells were engrafted into 3 mice, and after 3 challenges with soluble HIV trimer, germinal center cells (CD19+, GL7+, CD38-, CD95+) were isolated for deep sequencing.
  • the bar graphs in FIG 9, views A through C depict the mutational landscape of the HCDR3 of edited B cells recovered from three individual mice (FIG. 9, views A-C).
  • FIGs. 10A-10B shows vector retargeting.
  • FIG. 11 shows assessment of promoter modified vectors in B cells ex vivo.
  • A). Vector schematic. All vectors were based on first-generation E1ZE3 deleted Ads and expressed eGFP from the El region.
  • Ad5.CMV is HAdV-C5 with the ubiquitous cytomegalovirus (CMV) promoter.
  • AdRGD contains the RGD4C peptide inserted in the virus fiber knob HI loop region to expand vector tropism towards integrins.
  • a molecular model of this modification was generated using AlphaFold 2.0 and is shown in the inset. Model visualization was carried out using UCSF ChimeraX.
  • B). Assessment of promoter modified vectors in murine B cells ex vivo.
  • Vectors were first screened at 1000 MOI (left), and standout promoters were then assessed at increasing MOIs (middle). Vector toxicity was assessed using flow cytometry viability dyes (right). C). Assessment of vectors in human B cells ex vivo. Panels are as described in B. In all cases eGFP expression is normalized to a PBS mock control. All data are expressed as means ⁇ SD.
  • FIG. 12 shows in vivo assessment of promoter modified vectors.
  • FIG. 13 shows analysis of the mechanism of gene transfer to B cells via Ad vectors.
  • 5xl0 5 activated B cells were resuspended in 400pL infection media and incubated with the indicated amounts of recombinant Ad5 fiber for 15 minutes at room temperature prior to infection with 1000 MOI of the indicated vectors.
  • Ad5E3gfpFFc3.1uc contains a premature stop codon prior to fiber knob, resulting in a vector unable to bind CAR.
  • Murine B cells were infected via our standard protocol described in the methods. 5xl0 4 A549 cells were seeded in a 24- well plate and infected 24 hours later. D) Blocking of murine B cells with anti-integrin peptide.
  • GRGDSP Sigma SCP0157, Saint Louis, MO
  • GRADSP Sigma SCP0156, Saint Louis, MO
  • 5xl0 5 activated murine B cells were resuspended in 25pL infection media plus 25 pL of the indicated treatment for 30 minutes at room temperature prior to infection.
  • flow cytometry was used to score for eGFP+ cells approximately 48 hours after infection.
  • FIG. 14 shows immunophenotyping of murine B cells after in vivo vector administration.
  • GC germinal center
  • FO follicular
  • MZ marginal zone
  • CD71hi proliferating
  • PBs plasmablasts
  • FIG. 15 shows a preliminary murine B cell gene transfer experiments.
  • A Infection of LPS activated B cells.
  • AdH5/H3.CMV is a standard E1ZE3 deleted HAdV-C5 with our previously described H5/H3 liver de-targeting hexon modification with the cytomegalovirus (CMV) promoter.
  • AdH5/H3RGD.EFla is similar but contains the RGD4C fiber modification described in the text and uses the Elongation Factor 1 alpha promoter.
  • AdPK4.CMV is described in the text.
  • B Infection of resting B cells. Isolated B cells were infected on the day of isolation with the indicated vectors. Flow cytometry was used to score for GFP+ cells approximately 48 hours later.
  • FIG. 16 shows biodistribution of Ad5.CMV in selected tissues. Green, eGFP, blue, DAPI. 1x1011 viral particles were injected via the tail-vein. 48 hours later tissues were harvested and an approximately 1mm mid-sagittal slice of each was fixed in 10% phosphate- buffered formalin. Imaging was then conducted according to our previously established protocols.
  • FIG. 17 shows a schematic diagram depicting an embodiment of a molecular glue strategy for Adenovirus retargeting.
  • DogTag molecular glue peptide is inserted into the virus fiber protein at the HI loop domain to form Ad5FDgT, while antibodies are expressed as fusions with the DogCatcher partner protein. Mixing at room temperature allows the antibody to form a covalent bond with the chimeric fiber, resulting in permanent tethering of the antibody to the virus. These reactions enable cell specific targeting of the vector through the antibody.
  • FIGs. 18A-18B show infection of primary murine B and T lymphocytes with antibody conjugated Ad5FDgT.
  • Primary cells were isolated from C57BL/6J mice and cultured with LPS (B cells) or IL2 + anti-CD3/CD28 beads (T cells).
  • Ad5FDgT was incubated for 2 hours at room temperature with either anti-mCD40 F8 (FIG. 18 A, SEQ ID NO. 14) or anti-mCD8 YTS169 (FIG. 18B, SEQ ID NO. 25) DogCatcher fusion antibodies.
  • FIGs. 19A-19F show development and characterization of Ad- Ab targeting.
  • A Schematic overview of system design. DogTag is genetically inserted into the Ad fiber knob (Ad5FDgT), while DogCatcher is fused to antibody species. Mixing of these reagents results in permanent linkage of the virus and antibody at the fiber knob locale. Fiber model generated with AlphaFold2 and visualized with ChimeraX
  • B Ad5FDgT genome overview. Ad5FDgT is based on an E1ZE3 deleted Ad5 with the CMV promoter driving eGFP expression from the El region. DogTag is inserted with minimal flex linkers at the HI loop domain of the fiber knob.
  • C Antibody-DogCatcher fusion designs.
  • D Antibody-DogCatcher fusion designs.
  • FIGs. 20A-20B show in vitro characterization of Ad- Ab targeting.
  • A Conceptual workflow. Primary lymphocytes are magnetically isolated from mouse splenocytes or human PBMCs then cultured with activating agents. On the day of infection Ad5FDgT is conjugated with the appropriate antibody at the indicated DogTag:DogCatcher molar ratios, then used to infect cells.
  • B Ad-Ab infectivity enhancement in primary murine B cells (top right), murine T cells (bottom left), and human B cells (bottom right). For each antibody, the group with the maximum mean infectivity was compared to the PBS group using a standard t-test. Welch’s correction was used in cases where the F-test revealed significant differences in variances.
  • DgC DogCatcher. Icons generated by BioRender.
  • FIGs. 21 A-21C show in vivo characterization of Ad-Ab targeting.
  • FIGs. 22A-22E show development of purified Ad-Ab complexes.
  • A Purification workflow. Virus particles are purified from cell lysates via cesium chloride (CsCl) ultracentrifugation, then briefly dialyzed against IX PBS to remove excess salts. Conjugation is then carried out, followed by a second CsCl purification, dialysis and final storage.
  • B Viral band images in CsCl gradients after second ultracentrifugation.
  • C SDS-PAGE analysis of purified Ab-Ab complexes.
  • D Western blot against the HAdV-C5 fiber tail of purified Ad- Ab complexes.
  • E In vivo analysis of purified Ad-Ab complexes.
  • C57BL/6J mice aged 6-9 weeks were injected on three separate occasions with 5x1010 of the indicated vectors. Three days later splenocytes were assessed for eGFP expression using flow cytometry. Livers were assessed for tissue eGFP expression as well. Data from all experiments were combined. Icons generated by BioRender.
  • FIGs. 23A-23C show Initial proof-of-concept Ad-Ab targeting using SAd36 reagents.
  • FF-SpT fiber-fibritin configuration
  • SpC-H7 is a control sdAb which was found to bind murine CD40 extremely weakly.
  • SpC-18B12 and SpC-18Bv2 are both scFvs targeting murine CD20.
  • C Analysis of targeting reliance on the conjugated antibody.
  • SAd36-H-SpT was conjugated with SpC-F8, and murine B cells were incubated with either PBS or increasing amounts of Trx-F8 (1/100, 1/10 or IX the amount of SpC-F8 used) for 5 minutes prior to infection.
  • FIGs. 24A-24B show in vivo pilot analyses of Ad-Ab targeting with SAd36 reagents.
  • A Schematic overview of experiment. 2 mice per group were injected with either PBS, SAd36, SAd36-H-SpT, or SAd36-H-SpT conjugated with F8-SpC. Vectors were injected retro-orbitally at 1x1011 vp/mouse. Approximately 72h later spleens were harvested and B and T cells were assessed for eGFP expression using flow cytometry.
  • B Flow cytometry results of SAd36 reagents in vivo infection in B (CD 19+ CD3-) and T (CD 19- CD3+ cells).
  • FIG. 28 shows a strategy for vector targeting via adenovirus.
  • Transductional targeting is achieved via replacement of fiber knob with Ad5.FiberDogTag.
  • Modified adenoparticles are incubated with a DogCatcher-antibody Fc binding domain (AbBD) and an unmodified antibody and photo-crosslinked by incorporating photocrosslinking amino acids (BP A) into the AbBD.
  • the unmodified antibody may be custom or off-the-shelf.
  • Two AbBDs with photocrosslinking amino acids are shown: pZQ32BPA and pGA24BPA.
  • FIG. 29 shows a western blot of adenovirus particles functionalized with an off- the-shelf whole antibody using a pGA24BPA PG fusion.
  • a heavy band (Heavy+binder) is observed on the Western Blot indicating the ability of the DogCatcher fusion protein to bind the native antibody.
  • FIG. 30 shows the ability of the functionalized native off the shelf antibodies to target GFP expressing adenoviral particles using the DogTag / DogCatcher targeting strategy described in Figure 28 to transduce live B cells using a variety of different antibodies known to target cell surface markers on B cells.
  • the invention relates a system comprising an adenoviral particle modified to incorporate one or more peptide tags into one or more components of the capsid protein, and a recombinant protein that comprises a first portion capable of forming a high affinity bond with said peptide tag, and a second portion comprising a ligand or antigen binding protein capable of targeting a specific cell type (for example, a B cell).
  • adenoviral particles carry the genetic machinery to edit the target cells to express a therapeutic protein.
  • the adenoviral particle may express more than one distinct peptide tag and more than one distinct recombinant protein.
  • at least one recombinant protein comprises a protecting component (for example, albumin), that protects the adenovirus particle for immune attack before reaching the target cell.
  • at least one recombinant protein comprises an RNA binding protein.
  • the invention relates to methods for editing a B cell comprising, contacting with said B cell an adenovirus, wherein said adenovirus comprises an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a recombinant protein capable of recognizing and forming a high affinity interaction with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • the invention relates to methods for editing B cells comprising contacting with said B cell with an RNA-guided nuclease a gRNA targeting a locus on the human genome; and an adenovirus, wherein said adenovirus comprises: a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a first and second peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said second peptide tag; and further comprising an RNA binding protein.
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said second peptide tag; and recognizing and interacting with a molecule capable of binding with a shielding protein.
  • said adenovirus comprises an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a second recombinant protein capable of capable of recognizing and forming a high affinity interaction with said second peptide tag; and further comprising an RNA binding protein.
  • said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence
  • the invention relates to a method for editing a B cell comprising contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a capsid protein; and a first recombinant protein capable of recognizing and forming a high affinity interaction with said first peptide tag; and recognizing and interacting with a first protein expressed on the surface of a B cell; and a second recombinant protein capable of recognizing and forming a high affinity interaction with said second peptide tag; and recognizing and interacting with a second protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease a gRNA targeting a locus on the human genome; and a
  • the invention relates to an engineered B cell, wherein said engineered B cell has been edited to express a therapeutic protein of interest, by contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA- guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA- guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat
  • the method of treating a patient in need thereof with an engineered B cell, wherein said engineered B cell has been edited to express a therapeutic protein of interest by contacting with said B cell with an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag
  • the invention relates to a composition for editing a B cell, comprising an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell.
  • said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a
  • the invention relates to a method of treating a patient in need thereof by administering to said patient a composition comprising an adenovirus, wherein said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein; a recombinant protein capable of recognizing and forming a covalent bond with said peptide tag; and recognizing and interacting with a protein expressed on the surface of a B cell; and a pharmaceutical acceptable carrier.
  • said adenovirus comprises: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein; wherein said adenovirus further comprises a peptide tag, expressed on a viral coat protein;
  • polynucleotide includes both singlestranded and double-stranded nucleotide polymers.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2’, 3 ’-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromouridine and inosine derivatives
  • ribose modifications such as 2’, 3 ’-dideoxyribose
  • internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
  • oligonucleotide refers to a polynucle
  • Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
  • control sequence refers to a polynucleotide sequence that affect the expression and processing of coding sequences to which it is ligated.
  • control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence.
  • control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence.
  • Control sequences can include leader sequences (signal peptides) and/or fusion partner sequences.
  • operably linked means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
  • vector means any molecule or entity e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.
  • expression vector or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto.
  • An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
  • the term “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest.
  • the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.
  • transformation refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA.
  • a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques.
  • the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid.
  • a cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell.
  • transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology, 1973, 52:456; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001, supra; Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier; Chu c/ a/., 1981, Gene, 13: 197.
  • transduction refers to the process whereby foreign DNA is introduced into a cell via viral vector. See, e.g., Jones et al., Genetics: Principles and Analysis, 1998, Boston: Jones & Bartlett Publ.
  • polypeptide or “protein” refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
  • polypeptide and protein specifically encompass antigen-binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigenbinding protein.
  • polypeptide fragment refers to a polypeptide that has an aminoterminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein.
  • Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.
  • a “variant” of a polypeptide e.g., an antigen-binding molecule
  • Variants include fusion proteins.
  • isolated means (i) free of at least some other proteins with which it would normally be found, (ii) is essentially free of other proteins from the same source, e.g., from the same species, (iii) separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (iv) operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (v) does not occur in nature.
  • the term “isolated” means a cell that is separated from the molecular and/or cellular components that naturally accompany the cell, including, without limitation, a primary cell that is separated from a subject sample and progeny cells derived therefrom, and modified or engineered cells derived from a primary or progeny isolated cell, each case, whether or not passaged in culture or immortalized.
  • the sample may be blood, bone marrow, or a tissue sample.
  • B cells may be isolated from peripheral blood mononuclear cells (PBMCs), bone marrow, or the spleen. Cells are isolated by any methods known in the art.
  • B cells are isolated by flow cytometry, magnetic cell isolation and cell separation (MACS), RosetteSep, or antibody panning.
  • MCS magnetic cell isolation and cell separation
  • RosetteSep or antibody panning.
  • One or more isolation techniques may be utilized in order to provide an isolated B cell population with sufficient purity, viability, and yield.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (z.e., an “algorithm”).
  • the sequences being compared are typically aligned in a way that gives the largest match between the sequences.
  • One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al. , Nucl. Acid Res., 1984, 12, 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • the sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm).
  • a standard comparison matrix (see, e.g., Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352 for the PAM 250 comparison matrix; Henikoff et aL, 1992, Proc. Natl. Acad. Set. U.S.A., 89, 10915-10919 for the BLO-SUM 62 comparison matrix) is also used by the algorithm.
  • the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Green, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose.
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as alpha-, alpha-di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypeptides of the present invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxy-glutamate, epsilon- N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma. -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • Naturally occurring residues can be divided into classes based on common side chain properties: a) hydrophobic: norleucine, Met, Ala, Vai, Leu, He; b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c) acidic: Asp, Glu; d) basic: His, Lys, Arg; e) residues that influence chain orientation: Gly, Pro; and f) aromatic: Trp, Tyr, Phe.
  • non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
  • derivative refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids).
  • derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties.
  • a chemically modified antigen-binding molecule can have a greater circulating half-life than an antigen-binding molecule that is not chemically modified.
  • a derivative antigen-binding molecule is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J. L., 1986, Adv. Drug Res., 1986, 15, 29; Veber, D. F. & Freidinger, R. M., 1985, Trends in Neuroscience, 8, 392-396; and Evans, B. E., et al., 1987, J. Med. Chem., 30, 1229-1239, which are incorporated herein by reference for any purpose.
  • therapeutically effective amount refers to the amount of immune cells or other therapeutic agent determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
  • patient and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
  • treat and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.
  • prevent does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
  • Enzymatic reactions and purification techniques can be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook el al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “essentially the same” or “substantially the same” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “substantially free of’ and “essentially free of’ are used interchangeably, and when used to describe a composition, such as a cell population or culture media, refer to a composition that is free of a specified substance, such as, 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance, or is undetectable as measured by conventional means. Similar meaning can be applied to the term “absence of,” where referring to the absence of a particular substance or component of a composition.
  • the term “appreciable” refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is readily detectable by one or more standard methods.
  • the terms “not-appreciable” and “not appreciable” and equivalents refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is not readily detectable or undetectable by standard methods.
  • an event is not appreciable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, or less of the time.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5% or 1%, or any intervening ranges thereof.
  • introducing refers to a process that comprises contacting a cell with a polynucleotide, polypeptide, or small molecule.
  • An introducing step may also comprise microinjection of polynucleotides or polypeptides into the cell, use of liposomes to deliver polynucleotides or polypeptides into the cell, or fusion of polynucleotides or polypeptides to cell permeable moi eties to introduce them into a cell.
  • B cell refers to an immune cell (e.g., a white blood cell or leukocyte) that is capable of expressing a B cell receptor (BCR) and/or producing antibodies.
  • B cell may include any cell type that is derived from a B cell including plasma cells (PCs) and plasmablasts (PBs). B cells are known to undergo a “differentiation phase,” which encompasses the antigen-activation of the B cell through to the development of antibody-secreting plasma cells and the development of memory B cells.
  • the invention is based, in part, on the discovery that using ex vivo partially differentiated B cells results in a cell composition that results in in vivo cell survival and differentiation, leading to durable expression of an engineered protein.
  • the B cell compositions described herein are useful for in vivo delivery and expression of therapeutic agents, including, e.g., antigen-specific antibodies.
  • the B cell compositions described herein are useful for long term in vivo delivery and expression of therapeutic recombinant antibodies.
  • the present disclosure relates generally to in vitro culture and production methods for B cells under conditions so as to partially differentiate B cells into plasmablasts and plasma cells prior to in vivo administration, to elicit production of therapeutic recombinant antibodies from these cells. Such cells can continue to express the protein(s) of interest and may become long-lived cells, such as memory, plasmablast or plasma cells.
  • the invention comprises a method for editing a B cell comprising contacting said B cell with an adenovirus.
  • the adenovirus is a human adenovirus.
  • the adenovirus is a primate adenovirus.
  • the adenovirus is a derivative adenovirus.
  • “human adenovirus”, “serotype” or “human adenovirus serotype” means any of the 51 human adenovirus serotypes currently known or isolated in the future. To do.
  • the native tropism of said adenovirus has been ablated.
  • chimeric adenovirus means an adenovirus whose nucleic acid sequence is composed of nucleic acid sequences of at least two serotypes of the above adenovirus serotypes.
  • parent adenovirus serotype means an adenovirus serotype that represents a serotype from which the majority of the genome of the chimeric adenovirus is derived.
  • adenovirus derivative means an adenovirus of the invention that has been modified such that additions, deletions or substitutions are made to or in the genome of the virus.
  • the resulting adenovirus derivatives exhibit their ability and / or therapeutic index higher than that of the parent adenovirus or are more therapeutically useful in other ways (i.e., low immunogens). Improved clearance profile).
  • the derivatives of adenovirus of the present invention can have a deletion in one of the early genes of the viral genome, such as but not limited to the El A or E2B region of the viral genome.
  • the term "ablate” or “ablated” is used to refer to an adenovirus, adenoviral vector or adenoviral particle, in which the ability to bind to a particular cellular receptor is reduced or eliminated, generally substantially eliminated (i.e., reduced more than 10-fold, 100-fold or more) when compared to a corresponding wild-type adenovirus.
  • An ablated adenovirus, adenoviral vector or adenoviral particle also is said to be detargeted, i.e., the modified adenovirus, adenoviral vector or adenoviral particle does not possess the native tropism of the wild-type adenovirus.
  • the reduction or elimination of the ability of the mutated adenovirus fiber protein to bind a cellular receptor as compared to the corresponding wild-type fiber protein can be measured or assessed by comparing the transduction efficiency (gene transfer and expression of a marker gene) of an adenovirus particle containing the mutated fiber protein compared to an adenovirus particle containing the wild-type fiber protein for cells having the cellular receptor.
  • tropism refers to the selective infectivity or binding that is conferred on the particle by a capsid protein, such as the fiber protein and/or penton.
  • adenoviral genome is intended to include any adenoviral vector or any nucleic acid sequence comprising a modified fiber protein. All adenovirus serotypes are contemplated for use in the vectors and methods herein.
  • a packaging cell line is a cell line that is able to package adenoviral genomes or modified genomes to produce viral particles. It can provide a missing gene product or its equivalent.
  • packaging cells can provide complementing functions for the genes deleted in an adenoviral genome (e.g. , the nucleic acids encoding modified fiber proteins) and are able to package the adenoviral genomes into the adenovirus particle.
  • detargeted adenoviral particles have ablated (reduced or eliminated) interaction with receptors with which native particles. It is understood that in vivo no particles are fully ablated such that they do not interact with any cells. Detargeted particles have reduced, typically substantially reduced, or eliminated interaction with native receptors. For purposes herein, detargeted particles have reduced (2 -fold, 5-fold, 1 0-fold, 100-fold or more) binding or virtually no binding to CAR or another native receptor. The particles still bind to cells, but the types of cells and interactions are reduced.
  • pseudotyping describes the production of adenoviral vectors having modified capsid protein or capsid proteins from a different serotype from the serotype of the vector itself.
  • adenovirus 5 vector particle containing an Ad37 or Ad35 fiber protein is produced by producing the adenoviral vector in packaging cell lines expressing different fiber proteins.
  • the adenovirus further comprises a peptide tag, expressed on a capsid protein.
  • Said peptide tag can be expressed on any protein subunit expressed as a part of the capsid.
  • MCPs major capsid proteins
  • Illa, VI, VIII, and IX minor/cement proteins
  • one or more peptide tags are expressed on the capsid shell of said adenovirus.
  • one or more peptide tags are expressed as a part of the hexon protein. In various embodiments, one or more peptide tags are expressed as a part of the penton base. In various embodiments, one or more peptide tags are expressed as a part of the fiber protein. In various embodiments, one or more peptide tags are expressed as part of one or more of the minor/cement proteins. In various embodiments, one or more peptide tags are expressed in the Hl loop domain of the Ad fiber. In various embodiments, one or more peptide tags are expressed in the hexon protein of said adenovirus. In various embodiments, one or more peptide tags are expressed in the pIX protein, the penton base protein, a fiber- fibritin recombinant protein.
  • the invention further comprises a recombinant protein capable of recognizing and forming a high affinity interaction with one or more peptide tags.
  • said interaction is a covalent bond.
  • said interaction is an isopeptide bond.
  • [OHl] Several technologies, for example, enable covalent conjugation of polypeptides at specific pre determined sites.
  • One example is the sortase system (Schmohl et al., 2014), whereby a short peptide (the sorting motif) is genetically fused to the C-terminus of one polypeptide and two glycine residues are genetically fused to the N-terminus of a second peptide (or vice versa). In the presence of the sortase enzyme, the two modified polypeptides are fused together.
  • Other enzymatic protein ligase systems are butelase (Nguyen et al., 2014) or peptiligase (Toplak et al., 2016).
  • Another example is the in-frame addition of nucleotides encoding one or more cysteines to the C- or N- termini of polypeptides.
  • cysteine containing polypeptides When such free cysteine containing polypeptides are mixed under oxidizing conditions, they will form disulfide bridges.
  • Such systems suffer from the synthesis of many side-products and from instability of the disulfide bridge under reducing conditions.
  • Spy Tag/Spy Catcher Reddington et al., 2015
  • FbaB Streptococcus pyogenes protein FbaB
  • the other part, the SpyCatcher is a 116 amino acid protein domain containing the other part (e.g., a lysine) of the center, promoted by a nearby glutamate or aspartate.
  • SpyLigase is a fragment of the FbaB domain comprising a glutamic acid residue that induces or catalyzes the formation of the isopeptide bond between the aspartate and lysine residues in SpyTag and K-tag, respectively.
  • SnoopTag/SnoopCatcher (Veggiani et al, 2016), with a later development of a SnoopLigase system (Bui dun et al, 2018).
  • SnoopTag/SnoopCatcher technologies are hereby incorporated by reference in their entirety.
  • Another system using Streptcoccus pyogenes pilin subunit Spy0128 has also been developed and is called Isopeptag/Split Spy0128 (Abe et al, 2013).
  • SdyTag/SdyCatcherDANG short Yet another system derived from the Streptococcus dysgalactiae fibronectin-binding protein has also been developed and is called SdyTag/SdyCatcherDANG short (Tan et al, 2016).
  • the recombinant protein comprises a SpyCatcher Protein. In various embodiments, the recombinant protein comprises SEQ ID NO: 12. In various embodiments, the recombinant protein comprises a DogCatcher protein. In various embodiments, the recombinant protein comprises SEQ ID NO: 13. In various embodiments, the recombinant protein comprises SEQ ID NO: 21. In various embodiments, the recombinant protein comprises SEQ ID NO: 14. In various embodiments, the recombinant protein comprises SEQ ID NO: 15. In various embodiments, the recombinant protein comprises SEQ ID NO: 16. In various embodiments, the recombinant protein comprises SEQ ID NO: 17.
  • the recombinant protein comprises SEQ ID NO: 18. In various embodiments, the recombinant protein comprises SEQ ID NO: 19. In various embodiments, the recombinant protein comprises SEQ ID NO: 20. In various embodiments, the recombinant protein comprises SEQ ID NO: 22. In various embodiments, the recombinant protein comprises SEQ ID NO: 25. In various embodiments, the recombinant protein comprises SEQ ID NO: 26. In various embodiments, the recombinant protein comprises SEQ ID NO: 27.
  • the peptide tag is incorporated into capsid protein.
  • the peptide tag is encoded into an Ad5 fiber protein.
  • the peptide tag is incorporated into an Ad5 HVR5 protein.
  • the capsid protein with an incorporated peptide tag comprises SEQ ID No. 1.
  • the capsid protein with an incorporated peptide tag comprises SEQ ID No. 2.
  • the capsid protein with an incorporated peptide tag comprises SEQ ID No. 3.
  • the capsid protein with an incorporated peptide tag comprises SEQ ID No. 4.
  • the capsid protein with an incorporated peptide tag comprises SEQ ID No. 5.
  • the capsid protein with an incorporated peptide tag comprises SEQ ID No. 6. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 7. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 8. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 9. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 10.
  • the recombinant protein further comprises a portion capable of recognizing and interacting with a protein expressed on the surface of a B cell.
  • recombinant protein comprises a binding domain, such as an scFv, ligand, antibody, receptor, or fragment thereof which allows the recombinant protein to target specific target cells by binding to a protein expressed on the cell surface.
  • the recombinant protein is capable of binding to a cell surface protein unique to a particular subclass of B cells.
  • plasma cells may be targeted by cell surface protein expression patterns using standard flow cytometry methods.
  • terminally differentiated plasma cells express relatively surface antigens, and downregulate expression of markers such as CD20.
  • Some terminally differentiated plasma cells also express lower levels of CD 19.
  • plasma cells may be identified by expression of CD38 and CD138.
  • CD27 may also be used to identify plasma cells as naive B cells are CD27-, memory B cells are CD27+ and plasma cells are CD27++. Plasma cells express high levels of CD38 and CD138 compared to B cells earlier in differentiation.
  • the recombinant protein is capable of binding CD20, CD 19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD138, PD273, or an Ig receptor.
  • the recombinant protein is capable of binding CD 19, CD20, CD27, CD38 or CD 138. In various embodiments, more than one recombinant protein may be used.
  • the engineered B cell comprises a therapeutic protein to be delivered to a patient in need thereof.
  • therapeutic protein means any protein that may contribute to the treatment, reduction of symptoms, prevention or cure of a disease or disorder in a patient.
  • the therapeutic protein may be suitable for treatment of a rare disease or an orphan disease, where said therapy can be achieved by the replacement of a particular protein and/or enzyme.
  • a therapeutic protein may include but is not limited to an enzyme, a ligand, a naturally occurring, engineered and/or chimeric receptor, a cytokine or a chemokine, or an antibody.
  • the therapeutic protein is for treatment of Lysosomal acid lipase deficiency (LAL), Acid sphingomyelinase deficiency or Niemann-Pick disease (ASM), Mucopolysaccharidosis IVA (GALNS).
  • the therapeutic protein is for the treatment of HIV.
  • the therapeutic protein is an anti -HIV antibody.
  • the anti -HIV antibody comprises SEQ ID No. 23.
  • the anti-HIV antibody comprises SEQ ID NO. 24.
  • the anti-HIV antibody comprises a light chain comprising SEQ ID NO. 23 and a heavy change antibody comprises SEQ ID NO. 24.
  • the therapeutic protein is a receptor protein to be expressed on the surface of a B cell.
  • the receptor is a chimeric antigen receptor or a “CAR-B” receptor.
  • the CAR-B receptor has an intracellular domain that includes CD79a (Immunoglobulin a).
  • the CAR-B receptor has an intracellular domain that includes CD79b (Immunoglobulin P).
  • the extracellular domain comprises an extracellular binding domain and a hinge domain.
  • the extracellular binding domain(s) recognizes at least one antigen or protein expressed on the surface of a target cell.
  • the target cell is selected from the group consisting of a tumor cell, cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell, and an endothelial cell.
  • the extracellular binding domain is a single chain variable fragment (scFv), or a full-length antibody, or the extracellular domain of a receptor or ligand.
  • the extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GPC3, ASGR1, ASGR2, Sarcoglycan, Corin, FAP (fibroblast activation protein) and Her2.
  • the hinge domain is derived from the group consisting of IgG, CD28 and CD8.
  • the cytoplasmic domain comprises at least one signaling domain native to B cell receptors.
  • the cytoplasmic domain comprises a domain that is selected from the group comprising of: CD79a (Immunoglobulin a), CD79b (Immunoglobulin P), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, CD3( ⁇ and BLNK.
  • the cytoplasmic domain further comprises a costimulatory domain.
  • the therapeutic protein is Factor VIII.
  • the therapeutic protein is alpha-galactosidase (aGAL or GLA).
  • the therapeutic protein is selected from the group comprising: IL- 10, TGFP, IL- 21, IL- 12, fFNy, INF a, IL-4, CCR7, IP 10 and FLT3L.
  • the therapeutic protein is an anti-inflammatory antibody.
  • the therapeutic protein is selected from the group consisting of a-4-P-7 integrin, TNFa and IL23R.
  • the therapeutic protein is an oncology antibody.
  • the therapeutic protein is selected from the group consisting of HER2, MSLN, GPC3, PD-1, CTLA-4, LAG-3, BTLA, HVEM, CD20, CD 19, BCMA, CD39, CD73, CCR8, CD33, CD25, CD30, CD32, Trop-2.
  • the therapeutic protein is for the treatment of an infectious disease.
  • the therapeutic protein is for the treatment of CMV, EBV, HSV, HAV, HBV, HCV, RSV, Influenza etapneumovirus (HMPV), human parainfluenza vims types one (HPIV1) and three (HPIV3), Dengue, SARs, SAR-CoV2, MERS, Pan coronavirus, Norovirus.
  • Gene editing is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell.
  • Targeted gene editing enables insertion, deletion and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence).
  • the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration.
  • Targeted editing may be used to disrupt endogenous gene expression.
  • “Targeted integration” refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present.
  • a “disrupted gene” refers to a gene comprising an insertion, deletion, or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited.
  • disrupting a gene refers to a method of inserting, deleting or substituting at least one nucleotide/nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art and described herein.
  • Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease - dependent approach.
  • nuclease-independent targeted editing approach homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell.
  • the exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence.
  • nuclease - dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare - cutting nucleases (e.g., endonucleases).
  • DSBs double strand breaks
  • endonucleases e.g., endonucleases
  • Such nuclease - dependent targeted editing also utilizes DNA repair mechanisms, for example, non - homologous end joining (NHEJ), which occurs in response to DSBs.
  • NHEJ non - homologous end joining
  • DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides.
  • HDR homology directed repair
  • Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), meganucleases, transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases may also be used for targeted integration.
  • ZFN zinc-finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR/Cas9 Clustered Regular Interspaced Short Palindromic Repeats Associated 9
  • DICE dual integrase cassette exchange
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence specific manner through on more zinc fingers.
  • ZFBD zinc finger DNA binding domain
  • a zinc finger is a domain of about 30 amino acids within the zinc finger-binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • a designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.
  • a selected zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection.
  • ZFNs are described in greater detail in U.S. Pat. Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.
  • a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
  • a “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA.
  • TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains.
  • TAL effector DNA binding domain specificity depends on an effector - variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable diresidues (RVD).
  • RVD repeat variable diresidues
  • TALENs are described in greater detail in US Patent Application No. 2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.
  • targeted nucleases suitable for use as provided herein include, but are not limited to, Bxbl, phiC31, R4, PhiBTl, and WB/SPBc/TP901-l, whether used individually or in combination.
  • targeted nucleases include naturally - occurring and recombinant nucleases, e.g., CRISPR/Cas9, restriction endonucleases, meganucleases homing endonucleases, and the like.
  • the CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA - targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (CrRNA) and trans-activating RNA (tracrRNA), to target the cleavage of DNA.
  • PrRNA crisprRNA
  • tracrRNA trans-activating RNA
  • CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon reintroduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule comprising the spacer sequence, which associates with and targets Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).
  • crRNA drives sequence recognition and specificity of the CRISPR - Cas9 complex through Watson - Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5 ' 20nt in the crRNA allows targeting of the CRISPR - Cas9 complex to specific loci.
  • the CRISPR - Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single - guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).
  • sgRNA single - guide RNA
  • PAM protospacer adjacent motif
  • TracrRNA hybridizes with the 3' end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.
  • NHEI non - homologous end - joining
  • HDR homology - directed repair
  • NHEI is a robust repair mechanism that appears highly active in the majority of cell types, including nondividing cells. NHEI is error-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications are typically ⁇ 20 nt. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes.
  • HDR uses a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells, and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as the repair operant.
  • the Cas9 (CRISPR associated protein 9) endonuclease is from Streptococcus pyogenes, although other Cas9 homologs may be used. It should be understood, that wild - type Cas9 may be used or modified versions of Cas9 may be used (e.g., evolved versions of Cas9, or Cas9 orthologues or variants), as provided herein. In some embodiments, Cas9 may be substituted with another RNA- guided endonuclease, such as Cpfl (of a class II CRISPR/Cas system).
  • Cpfl RNA- guided endonuclease
  • the CRISPR/Cas system comprise components derived from a Type-1, Type-II, or Type-III system.
  • Updated classification schemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cas systems, having Types Ito V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov etal., (2015)) Mol Cell, 60:385- 397).
  • Class 2 CRISPR / Cas systems have single protein effectors.
  • Cas proteins of Types II, V, and VI are single - protein, RNA - guided endonucleases, herein called “Class 2 Cas nucleases.”
  • Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, and C2c3 proteins.
  • the Cpfl nuclease (Zetsche et al., (2015) Cell 163: 1-13) is homologous to Cas9, and contains a RuvC - like nuclease domain.
  • the Cas nuclease is from a Type - II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system).
  • the Cas nuclease is from a Class 2 CRISPR Cas system (a single protein Cas nuclease such as a Cas9 protein or a Cpfl protein).
  • the Cas9 and Cpfl family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein.
  • a Cas nuclease may comprise more than one nuclease domain.
  • a Cas9 nuclease may comprise at least one RuvC-like nuclease domain (e.g., Cpfl) and at least one HNH-like nuclease domain (e.g., Cas9).
  • the Cas9 nuclease introduces a DSB in the target sequence.
  • the Cas9 nuclease is modified to contain only one functional nuclease domain.
  • the Cas9 nuclease is modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.
  • the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain.
  • the Cas9 nuclease is modified to contain no functional HNH- like nuclease domain.
  • the Cas9 nuclease is a nickase that is capable of introducing a single-stranded break (a “nick”) into the target sequence.
  • a conserved amino acid within a Cas9 nuclease domain is substituted to reduce or alter a nuclease activity.
  • the Cas nuclease nickase comprises an amino acid substitution in the RuvC- like nuclease domain.
  • Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 nuclease).
  • the nickase comprises an amino acid substitution in the HNH-like nuclease domain.
  • Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863 A, H983 A, and D986A (based on the S. pyogenes Cas9 nuclease).
  • the Cas nuclease is from a Type-I CRISPR/Cas system.
  • the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system.
  • the Cas nuclease is a Cas3 nuclease.
  • the Cas nuclease is derived from a Type-III CRISPR/Cas system.
  • the Cas nuclease is derived from Type-IV CRISPR/Cas system.
  • the Cas nuclease is derived from a Type-V CRISPR/Cas system.
  • the Cas nuclease is derived from a Type- VI CRISPR/Cas system.
  • the present disclosure provides a genome-targeting nucleic acid that can direct the activities of an associated polypeptide (e.g., a site-directed polypeptide) to a specific target sequence within a target nucleic acid.
  • the genome-targeting nucleic acid can be an RNA.
  • a genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein.
  • a guide RNA comprises at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence.
  • the gRNA also comprises a second RNA called the tracrRNA sequence.
  • the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex.
  • the crRNA forms a duplex.
  • the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex.
  • the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.
  • each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva etal., Nature, 471, 602-607 (2011).
  • the genome-targeting nucleic acid e.g., gRNA
  • the genome-targeting nucleic acid is a single-molecule guide RNA.
  • a double-molecule guide RNA comprises two strands of RNA.
  • the first strand comprises in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence.
  • the second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence and an optional tracrRNA extension sequence.
  • a single-molecule guide RNA in a Type II system comprises, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA.
  • the single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension comprises one or more hairpins.
  • a single-molecule guide RNA in a Type V system comprises, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.
  • the sgRNA comprises a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a less than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a more than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence.
  • the sgRNA comprises comprise no uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA comprises comprise one or more uracil at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 1 uracil (U) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 2 uracil (UU) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 3 uracil (UUU) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 4 uracil (UUUU) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 5 uracil (UUUUU) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 6 uracil (UUUUUU) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 7 uracil (UUUUUUU) at the 3' end of the sgRNA sequence.
  • the sgRNA can comprise 8 uracil (UUUUUUUUU) at the 3' end of the sgRNA sequence.
  • the sgRNA can be unmodified or modified.
  • modified sgRNAs can comprise one or more 2'-O-methyl phosphorothioate nucleotides.
  • RNAs used in the CRISPR/Cas/Cpfl system can be readily synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
  • HPLC high performance liquid chromatography
  • One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpfl endonuclease, are more readily generated enzymatically.
  • RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
  • indel frequency may be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules.
  • a highly efficient gRNA yields a gene editing frequency of higher than 80%.
  • a gRNA is considered to be highly efficient if it yields a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • gene disruption may occur by deletion of a genomic sequence using two guide RNAs.
  • Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell are known (Bauer D E et al. Vis. Exp. 2015; 95;e52118).
  • a gRNA comprises a spacer sequence.
  • a spacer sequence is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target nucleic acid of interest.
  • the spacer sequence is 15 to 30 nucleotides.
  • the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • a spacer sequence is 20 nucleotides.
  • the “target sequence” is adjacent to a PAM sequence and is the sequence modified by an RNA-guided nuclease (e.g., Cas9).
  • the “target nucleic acid” is a doublestranded molecule: one strand comprises the target sequence and is referred to as the “PAM strand,” and the other complementary strand is referred to as the “non-PAM strand.”
  • PAM strand the target sequence
  • non-PAM strand complementary strand
  • the gRNA spacer sequence hybridizes to the reverse complement of the target sequence, which is located in the non-PAM strand of the target nucleic acid of interest.
  • the gRNA spacer sequence is the RNA equivalent of the target sequence.
  • the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC-3'.
  • the spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (z.e., base pairing).
  • the nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.
  • the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5' of a PAM of the Cas9 enzyme used in the system.
  • the spacer may perfectly match the target sequence or may have mismatches.
  • Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA.
  • S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.
  • the target nucleic acid sequence comprises 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises more than 20 nucleotides. In some embodiments, the target nucleic acid comprises at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid comprises at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence comprises 20 bases immediately 5' of the first nucleotide of the PAM.
  • the target nucleic acid comprises the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.
  • the gRNAs of the present disclosure are produced by a suitable means available in the art, including but not limited to in vitro transcription (IVT), synthetic and/or chemical synthesis methods, or a combination thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods are utilized. In one embodiment, the gRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT/US2013/30062. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors are used to in vitro transcribe a gRNA described herein.
  • non-natural modified nucleobases are introduced into polynucleotides, e.g., gRNA, during synthesis or post-synthesis.
  • modifications are on internucleoside linkages, purine or pyrimidine bases, or sugar.
  • a modification is introduced at the terminal of a polynucleotide; with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT application No. PCT/US2012/058519. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).
  • enzymatic or chemical ligation methods are used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.
  • Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).
  • nucleic acids e.g., vectors, encoding gRNAs described herein.
  • the nucleic acid is a DNA molecule.
  • the nucleic acid is an RNA molecule.
  • the nucleic acid comprises a nucleotide sequence encoding a crRNA.
  • the nucleotide sequence encoding the crRNA comprises a spacer flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system.
  • the nucleic acid comprises a nucleotide sequence encoding a tracrRNA.
  • the crRNA and the tracrRNA is encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracrRNA is encoded by a single nucleic acid. In some embodiments, the crRNA and the tracrRNA is encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracrRNA is encoded by the same strand of a single nucleic acid.
  • the gRNAs provided by the disclosure are chemically synthesized by any means described in the art (see e.g., WO/2005/01248). While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
  • HPLC high performance liquid chromatography
  • One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together.
  • the gRNAs provided by the disclosure are synthesized by enzymatic methods (e.g., in vitro transcription, IVT).
  • RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
  • more than one guide RNA can be used with a CRISPR/Cas nuclease system.
  • Each guide RNA may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid.
  • one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex.
  • each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA is the same or different.
  • the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1-6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.
  • the length of the targeting sequence may depend on the CRISPR/Cas9 system and components used. For example, different Cas9 proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence may comprise 18-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.
  • a CRISPR/Cas nuclease system includes at least one guide RNA.
  • the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex.
  • the guide RNA may guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), where the Cas protein cleaves the target nucleic acid.
  • the CRISPR/Cas complex is a Cpfl /guide RNA complex.
  • the CRISPR complex is a Type-II CRISPR/Cas9 complex.
  • the Cas protein is a Cas9 protein.
  • the CRISPR/Cas9 complex is a Cas9/guide RNA complex.
  • a gRNA and an RNA-guided nuclease are delivered to a cell separately, either simultaneously or sequentially. In some embodiments, a gRNA and an RNA-guided nuclease are delivered to a cell together. In some embodiments, a gRNA and an RNA-guided nuclease are pre-complexed together to form a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • RNPs are useful for gene editing, at least because they minimize the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protect the RNA from degradation.
  • Methods for forming RNPs are known in the art.
  • an RNP containing an RNA-guided nuclease e.g., a Cas nuclease, such as a Cas9 nuclease
  • a gRNA targeting a gene of interest is delivered a cell (e.g.: a B cell).
  • an RNP is delivered to a B cell by electroporation.
  • a “AAVS1 targeting RNP” refers to a gRNA that targets the AAVS1 genes pre-complexed with an RNA-guided nuclease.
  • a AAVS1 targeting RNP is delivered to a cell.
  • more than one RNP is delivered to a cell.
  • more than one RNA is delivered to a cell separately.
  • more than one RNP is delivered to the cell simultaneously.
  • an RNA-guided nuclease is delivered to a cell in a DNA vector that expresses the RNA-guided nuclease, an RNA that encodes the RNA-guided nuclease, or a protein.
  • a gRNA targeting a gene is delivered to a cell as an RNA, or a DNA vector that expresses the gRNA.
  • RNA-guided nuclease may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used.
  • Genome editing systems can be delivered together or separately and simultaneously or nonsimultaneously. Separate and/or asynchronous delivery of genome editing system components may be particularly desirable to provide temporal or spatial control over the function of genome editing systems and to limit certain effects caused by their activity.
  • Different or differential modes as used herein refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g, a RNA-guided nuclease molecule, gRNA, template nucleic acid, or payload.
  • the modes of delivery can result in different tissue distribution, different halflife, or different temporal distribution, e.g, in a selected compartment, tissue, or organ.
  • Some modes of delivery e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component.
  • examples include viral, e.g., AAV, Adenovirus, or lentivirus, delivery.
  • the components of a genome editing system can be delivered by modes that differ in terms of resulting halflife or persistent of the delivered component the body, or in a particular compartment, tissue or organ.
  • a gRNA can be delivered by such modes.
  • the RNA-guided nuclease molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.
  • a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component.
  • the first mode of delivery confers a first pharmacodynamic or pharmacokinetic property.
  • the first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
  • the second mode of delivery confers a second pharmacodynamic or pharmacokinetic property.
  • the second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
  • the first pharmacodynamic or pharmacokinetic property e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.
  • the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
  • the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
  • the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus.
  • a relatively persistent element e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus.
  • a relatively persistent element e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus.
  • the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
  • the first component comprises gRNA
  • the delivery mode is relatively persistent, e.g, the gRNA is transcribed from a plasmid or viral vector, e.g, an AAV, adenovirus or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation.
  • the second component a RNA-guided nuclease molecule, is delivered in a transient manner, for example as mRNA encoding the protein or as protein, ensuring that the full RNA-guided nuclease molecule/gRNA complex is only present and active for a short period of time.
  • the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.
  • differential delivery modes can enhance performance, safety, and/or efficacy, e.g., the likelihood of an eventual off-target modification can be reduced.
  • Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by WIC molecules.
  • a two-part delivery system can alleviate these drawbacks.
  • a first component e.g., a gRNA is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution.
  • a second component e.g., a RNA-guided nuclease molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution.
  • the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector.
  • the second mode comprises a second element selected from the group.
  • the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element.
  • the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
  • the adenovirus particles will be delivered as a therapeutic to a patient in need thereof.
  • the adenovirus particles will be capable of engineering B cells in vivo and will be capable of treating or preventing various diseases or disorders including, but not limited to infectious diseases, oncology, allergy, and autoimmunity.
  • the invention comprises a pharmaceutical composition comprising an adenoviral particles for the in vivo editing of B cells as described herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition further comprises an additional active agent.
  • said B cells are contacted with an activating agent prior to editing.
  • said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.
  • an activating agent is administered a priori to administering said adenovirus and said recombinant protein.
  • the patient will be preconditioned prior to administration of said pharmaceutical composition.
  • the preconditioning step involves an immunization.
  • the edited B cells will be delivered as a therapeutic to a patient in need thereof.
  • the edited B cells will be capable of treating or preventing various diseases or disorders including, but not limited to infectious diseases, oncology, allergy, and autoimmunity.
  • the invention comprises a pharmaceutical composition comprising edited of B cells as described herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition further comprises an additional active agent.
  • said B cells are contacted with an activating agent prior to editing.
  • said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.
  • an activating agent is administered a priori to administering said adenovirus and said recombinant protein.
  • the activation agent is an immunization.
  • the patient will be preconditioned prior to administration of said pharmaceutical composition.
  • the preconditioning step involves an immunization.
  • compositions according to the invention are administered to a subject in a dosage sufficient to achieve a therapeutic effect.
  • therapeutic effect or action includes an effect or action of a pharmaceutical composition of the invention intended to cure, mitigate, treat, or prevent disease, disorder or condition, or affect the structure or any function of the body of a subject. It will be appreciated that appropriate doses and dosing schedules may vary according to the subject and the intended therapeutic effect. Such appropriate dose levels and dosing schedules can be determined by the healthcare provider as needed. Additionally, multiple doses of cells can be provided in accordance with the invention.
  • the expanded population of engineered B cells are autologous B cells.
  • the modified B cells are allogeneic B cells.
  • the modified B cells are heterologous B cells.
  • the modified B cells of the present application are transfected or transduced in vivo.
  • the engineered cells are transfected or transduced ex vivo.
  • the term "subject" or “patient” means an individual.
  • a subject is a mammal such as a human.
  • a subject can be a nonhuman primate.
  • Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few.
  • the term "subject” also includes domesticated animals, such as cats, dogs, etc., livestock (e.g., llama, horses, cows), wild animals (e.g., deer, elk, moose, etc.,), laboratory animals (e.g., mouse, rabbit, rat, gerbil, guinea pig, etc.) and avian species (e.g, chickens, turkeys, ducks, etc.).
  • livestock e.g., llama, horses, cows
  • wild animals e.g., deer, elk, moose, etc.
  • laboratory animals e.g., mouse, rabbit, rat, gerbil, guinea pig, etc.
  • avian species e.g, chickens, turkeys, ducks, etc.
  • the methods disclosed herein are for the treatment of at least one of a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and a metabolic condition.
  • the patient has Lysosomal acid lipase deficiency (LAL), Acid sphingomyelinase deficiency or Niemann-Pick disease (ASM), Mucopolysaccharidosis IVA (GALNS).
  • LAL Lysosomal acid lipase deficiency
  • ASM Acid sphingomyelinase deficiency
  • Niemann-Pick disease ASM
  • Mucopolysaccharidosis IVA GALNS
  • the methods disclosed herein are for the treatment of HIV.
  • the composition comprising gene edited B cells can be administered with an anti-inflammatory agent.
  • Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triam
  • Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates.
  • Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride.
  • Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.
  • Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors.
  • TNF antagonists e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®
  • the biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules.
  • DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
  • cytokine as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoin
  • FSH follicle
  • a method for treating a subject having a cancer comprising administering an effective amount of an antibody or an isolated polypeptide of the embodiments to the subject.
  • a method for treating a cancer comprises administering an effective amount of a polypeptide (e.g., a glycosylated PD-1 polypeptide) to a subject.
  • a method of treating a cancer comprises administering an effective amount of an antibody of the embodiments (e.g., an antibody selectively binds to glycosylated PD-1 relative to unglycosylated PD-1), such as, but not limited to humanized or chimeric forms of STM418 or STM432, or antibodies that compete for binding to glycosylated PD-1 with STM418 or STM432, to a subject, or anti-PD-1 antibodies that are designed to bind preferentially at low pH.
  • an antibody of the embodiments e.g., an antibody selectively binds to glycosylated PD-1 relative to unglycosylated PD-1
  • an antibody of the embodiments e.g., an antibody selectively binds to glycosylated PD-1 relative to unglycosylated PD-1
  • an antibody of the embodiments e.g., an antibody selectively binds to glycosylated PD-1 relative to unglycosylated PD-1
  • the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.
  • the cancer is an adrenal cancer, an anal cancer, a bile duct cancer, a bladder cancer, a bone cancer, a brain/CNS tumor in an adult, a brain/CNS tumor in a child, a breast cancer, a breast cancer in a man, cancer in an adolescent, cancer in a child, cancer in a young adult, cancer of unknown primary, Castleman disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal or hypopharyngeal cancer, leukemia (e.g., adult acute lymphocytic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), chronic myelomonocytic
  • ALL
  • the method further comprises administering at least a second anticancer therapy to the subject.
  • the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.
  • Adenovirus delivery of CRISPR/Cas9 results in n on-homologous end joining (NHEJ) and homology directed repair (HDR) gene editing at a targeted genetic locus.
  • NHEJ n on-homologous end joining
  • HDR homology directed repair
  • T o achieve long term gene expression via adenoviral vectors
  • a strategy of exploiting CRISPR/Cas9 gene editing for in vivo knock-in of therapeutic genes was pursued.
  • gRNA guide RNA
  • hAAT human alpha- 1 antitrypsin
  • mice receiving the integration system had 6.5x (1 : 1 groups) and 2.9x (3: 1 groups) greater levels of hAAT expression compared with their non-integrating counterparts, even though all treated mice received equivalent amounts of total and hAAT-expressing virus (FIG. 5A).
  • the 1 :3 Ad5EFlahAAT to Ad5Cas9gRNA group receiving the least amount of hAAT -encoding vector of any group, maintained nearly equal or higher levels of hAAT than any non-integrative group.
  • Western blotting of plasma samples from sixteen weeks postinjection showed increased AAT expression among integrative mice relative to equivalent non-integrative mice, further confirming ELISA results (FIG. 5B).
  • a similar study achieved genetic correction of a murine model of FIX deficiency hemophilia.
  • the efficient in vivo gene delivery mediated by the adenoviral vectors feasibilized in vivo gene editing. This gene editing accomplished knock-in of a therapeutic gene at a targeted genome locale.
  • the editing-based knock-in allowed long term expression of the delivered gene with a therapeutic outcome.
  • This section describes approaches for modifying B cells. Procedures for editing human and murine B cells were extensively optimized. These optimizations include development and production of a non-standard Cast 2a ortholog (Mb2Casl2a) whose PAM recognition sequences are compatible with the murine J4 and human J6 regions, improvement of AAV and single-stranded DNA-based HDRTs, optimization of electroporation and ex vivo amplification of primary B cells, and development and production of SOSIP multimers. These efforts have allowed for the improvement upon existing techniques for editing B cells. Previous state-of-the-art approaches are diagrammed in FIGs. 6A and 6B, and our approaches are shown in FIGs. 6C and 6D.
  • Mb2Casl2a non-standard Cast 2a ortholog
  • FIG. 8 shows that the double-editing approach can edit >3% of cells to express the bNAb VRC26.25 variable chains, inserted again in their native murine loci.
  • sera from some engrafted mice neutralized more efficiently than the same bNAbs doped into sera at 2 pg/ml (VRC26.25). These concentrations are 500-1000-fold higher than the median ICso of each bNAb determined by CATNAP.
  • FIG. 9 highlights neutralization with HCDR3-only approach. The HCDR3 of murine cells were reprogrammed, as described for human primary B cells.
  • the murine J4 gene with Mb2Casl2A was cleaved and edited with HDRT with homology arms complementary to conserved sequences at the 3’ ends of most VI- and V3-family variable genes.
  • HCDR3 of the V2-glycan/apex bNAbs PG9, PG16, and CHOI were introduced into human cells.
  • the HCDR3 of VRC26.25 was used. Mice engrafted with HCDR3-edited B cells were immunized 3x with an Env trimer (S0SIP.v8 based on the BG505 isolate) conjugated to the mi3 60mer scaffold.
  • NGS revealed successful integration of the VRC26.25 HCDR3 in >20 distinct VH germline sequences. HCDR3 and the adjacent variable sequences showed evidence of extensive somatic hypermutation).
  • a Q100E mutation was consistently recovered from engrafted animals. Notably, this mutation was also identified in our recently published cell-based screen and observed in some relatives of VRC26.25 found in the human donor CAP256.
  • mice engrafted with the Q100E variant cells exhibited significantly greater neutralization potency than sera from mice engrafted with the unmodified HCDR3 cells, exceeding the equivalent of 2 pg/ml VRC26.25 doped into naive mouse serum.
  • Example 4 Vector retargeting using plug-and-play Adenovirus with molecular glue
  • Spy Catcher- Spy Tag family molecular glues are protein-peptide partners that form spontaneous covalent bonds under gentle conditions, creating new opportunities for synthetic protein design. We hypothesized that we might leverage this technology to decorate the virus capsid with antibodies targeting cell surface markers to achieve targeted gene delivery.
  • B lymphocytes were selected as a model cell, which we have become interested in as a cellular source for protein production, and which are generally challenging to infect with Ads. These cells possess several advantages for gene therapy, including their ability to produce high levels of protein, their ability to proliferate upon appropriate stimulation, and their potential to last the life of an organism as plasma cells.
  • Ad5FDgT Human Adenovirus 5 fiber
  • FIG. 18A Adenoviruses encoding GFP and mixed with F8DgC preferentially expressed GFP in B lymphocytes, where adenoviruses mixed with YTS169DgC did not.
  • FIG. 18B adenoviruses encoding GFP and mixed with YTS169DgC preferentially expressed GFP in T lymphocytes, where adenoviruses mixed with F8DgC did not.
  • FIG. 18B Adenoviruses encoding GFP and mixed with YTS169DgC preferentially expressed GFP in T lymphocytes, where adenoviruses mixed with F8DgC did not.
  • B lymphocytes B cells
  • B cells B lymphocytes
  • ex vivo engineering typically involves taking a sample from the patient, purifying and culturing the cell of interest, modifying the cells using a vector, and finally returning the cells to the patient.
  • direct in vivo cell modification involves the delivery of a targeted vector to the cells of interest in the patient.
  • the vector carries the necessary cell engineering machinery (such as the Cas9 protein, guide RNA and donor template DNA) required for cell modification and generates the desired cell changes in situ. This technique would circumvent the technical and infrastructure requirements involved in ex vivo cell engineering and allow novel medicines to be developed and delivered to populations previously unreachable by cell therapy.
  • Ads Adenoviral vectors
  • a series of promoters were selected as driving strong and/or specific gene expression in B cells or other hematopoietic cells, including the spleen focus forming virus (SFFV) promoter, previously described synthetic promoters EEK and MH based on endogenous human immunoglobulin promoters, and Epstein Barr Virus Wpl 168 promoter (EBVW).
  • SFFV spleen focus forming virus
  • EBVW Epstein Barr Virus Wpl 168 promoter
  • the EEK promoter appeared the standout targeted promoter, having the strongest gene expression of the group.
  • each vector showed strong gene expression in the liver, followed by the spleen, lung, heart and kidneys (FIG. 12D).
  • the AdRGD.EEK vector showed significantly reduced gene expression in B cells - a roughly 6-fold decrease compared to AdRGD.CMV.
  • AdRGD.EEK remained the standout promoter-modified vector, again showing a roughly 6- fold decrease in B cell gene expression compared to AdRGD.CMV and little-to-no off-target gene expression, including in T cells, highlighting the exceptional selectivity of this promoter, especially at the lower vector dose.
  • Ad5 the backbone all our vectors are based on
  • infection of cells involves a two-step entry mechanism - the virus first binds to cells via high-affinity interactions between the fiber protein and its receptor (the coxsackie and adenovirus receptor, CAR).
  • the penton base protein then binds avP3 and avP5 integrins through an RGD containing loop, triggering uptake of the virus particle into the cell.
  • This penton RGD interaction might be responsible for Ad infection of B cells in vivo, explaining the lack of differences between Ad5.CMV and AdRGD.CMV, which carry identical penton base proteins.
  • Ad5.CMV we first pre-treated murine B cells with varying amounts of recombinant Ad5 fiber protein, then infected the cells with Ad5.CMV or AdRGD.CMV (FIG. 13 A). With Ad5.CMV we observed a modest decrease in gene transfer in the pre-treated groups, dependent on the fiber dose.
  • AdRGD.EEK possessed a much higher ratio of eGFP+ cells in the germinal center and plasmablast groups compared to Ad5.CMV or AdRGD.CMV.
  • AdRGD.EEK possessed a much higher ratio of eGFP+ cells in the germinal center and plasmablast groups compared to Ad5.CMV or AdRGD.CMV.
  • Ad vectors for specific gene transfer to B cells in vivo We leveraged our triple targeting platform as a basis for these vectors, utilizing the RGD fiber modification to enhance infectivity, tissue specific promoters for specificity, and liver un-targeting.
  • RGD fiber modification to enhance infectivity
  • tissue specific promoters for specificity we leveraged our triple targeting platform as a basis for these vectors, utilizing the RGD fiber modification to enhance infectivity, tissue specific promoters for specificity, and liver un-targeting.
  • primary activated murine B cells we found ex vivo that the fiber modification enhanced gene transfer to B cells, and a variety of B cell targeted promoters showed good results compared to the CMV control. Importantly, these results crossed to human primary B cells as well, potentially enabling further development of these vectors for clinical use.
  • CD36 was recently identified as one of the major scavenger receptors involved in sequestration of Ad5 in Kupffer cells via binding to IgM on the virus capsid. Intriguingly, it has also been shown that marginal zone B cells express high levels of CD36.
  • the EEK promoter is a synthetic promoter based on the immunoglobulin kappa light chain promoter with enhancer elements. This promoter is likely more dependent on the B cells actively dividing and producing immunoglobulin than the ubiquitous viral CMV promoter, which aligns with its strong expression in the plasmablast and germinal center groups.
  • Ads were capable of infecting B cells in vivo and could be engineered to restrict gene expression using tissue specific promoters. Gene transfer did not appear to be dependent on the identity of the fiber protein but was likely rather mediated by the penton base protein and other as-of-yet undefined pathways. The susceptibility of B cells to expression of the virus transgene appeared to be influenced by the phenotype of the cells and was strongly correlated with B cell activation markers.
  • HEK293 ATCC CRL-1573, Manassas, VA
  • A549 ATCC CCL-185, Manassas, VA
  • F12 1 1 mixture supplemented with L-glutamine, 15mM HEPES, 10% fetal bovine serum (FBS) and lOOU/mL penicillin-streptomycin (Gibco 11330-032, Waltham, MA).
  • FBS fetal bovine serum
  • lOOU/mL penicillin-streptomycin Gibco 11330-032, Waltham, MA
  • Viruses Vectors developed were all E1/E3 deleted Adenoviruses based on the Human Adenovirus C5 genome (HAdV-C5). HAdV-C5 and AdRGD have been previously described. Viral genomes were released from plasmids via digestion with PacI (New England Biolabs, Ipswich, MA) or AbsI (Sibenzyme, Russia) and transfected into HEK293 cells for upscale. Viruses were purified and the concentration of viral particles was determined as previously described by our group.
  • PacI New England Biolabs, Ipswich, MA
  • AbsI Sibenzyme, Russia
  • Promoters To generate promoter-modified vectors, prAd5 and prAdRGD were digested with Swal (New England Biolabs, Ipswich, MA) to release the spacer region. DNA fragments encoding each promoter, eGFP and the SV40 polyadenylation tail were then assembled with the vector backbone via NEB HiFi DNA Assembly.
  • Swal New England Biolabs, Ipswich, MA
  • CMV ubiquitous cytomegalovirus
  • SFFV spleen focus forming virus
  • Epstein Barr Virus “Wpl 168” fragment has been previously described and was synthesized in the pUC57m carrier plasmid by Genscript (Piscataway, NJ) 27 .
  • the promoter was liberated from the carrier by EcoRV digestion and assembled with prAdRGD via NEB HiFi DNA Assembly.
  • Purified murine B cells were cultured and activated at 37C for 30h prior to virus infections. Unless otherwise indicated, infections were conducted by resuspending 5xl0 5 B cells in 40 pL base B cell media with 5% FBS and without activation. lOpL virus diluted in IX PBS to the indicated MOI was then added and cells were infected overnight at 37C, then transferred to 450 pL B cell media with 10% FBS and 50 pg/mL LPS. Cells were cultured for approximately 48 hours after infection then assessed via flow cytometry.
  • PBMCs peripheral blood mononuclear cells
  • B cells were then magnetically isolated from 1.5xl0 8 PBMCs using a kit according to the manufacturer’s instructions (Stem Cell Technologies 17954, Canada). B cells were cultured and differentiated according to previously published methods for 72h prior to infection 43,45 ’ 47 .
  • For infections 2.5xl0 5 cells were resuspended in 20 pL StemMACS HSC Expansion media supplemented with 0.2% FBS. Vectors were diluted in PBS so that the appropriate virus number would be delivered in 20 pL. Virus was added to cells and incubated for 3 hours at 37C, then transferred to 1 mL complete B cell media and incubated for approximately 48 hours prior to flow cytometry analysis.
  • spleens were processed into a single cell suspension as described above. Splenocytes were incubated with lOpL Fc Blocking Reagent (Miltenyi 130- 092-575, Gaithersburg MD) for 10 minutes prior to staining. All B cell subsets were passed through a Live/CD3-/CD19+ gate prior to further analysis.
  • lOpL Fc Blocking Reagent Miltenyi 130- 092-575, Gaithersburg MD
  • Germinal center B cells were defined as GL7+/CD95+/IgDlo/CD381o, marginal zone as IgM+/IgDlo, follicular as IgMlo/IgD+/CD38+, memory as IgDlo/GL7-/CD38+, proliferating as CD71hi, and plasmablasts as CD138+/IgD-.
  • Antibodies used are as follows: CD3 (Invitrogen 46-0032-82, Waltham, MA), CD45 (Invitrogen 47-0451-82, Waltham, MA), CD19, CD71 (BioLegend 113813, San Diego, CA), CD95 (BD Biosciences 563646, Franklin Lakes, NJ), CD138 (BioLegend 142515, San Diego, CA), GL7 (BioLegend 144607, San Diego, CA), CD38 (Miltenyi 130- 125-522, Gaithersburg MD), IgD (Invitrogen 56-5993-80, Waltham, MA), IgM (Invitrogen 47-5790-82, Waltham, MA), CD51 (BioLegend 104105, San Diego, CA), CD61 (BioLegend 104313, San Diego, CA). Live-dead discrimination was carried out using Sytox Red or Fixable Far Red stains, or Zombie UV (BioLegend 423107, San Diego, CA) for integr
  • mice Male C57B1/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in a pathogen-free environment. Mice aged 7-10 weeks were injected with IxlO 11 or 5xl0 10 viral particles retroorbitally and sacrificed approximately 72 hours later. In the indicated studies spleen, lungs, heart, kidney and liver were harvested. One half of each spleen was used for flow cytometry analysis as described above, while the remaining half and other organs were frozen in liquid nitrogen. Organs were then thawed and processed for eGFP quantification using a commercial kit according to the manufacturer’s instructions (Cell BioLabs AKR-120, San Diego, CA). Tissues were homogenized with an Omniprep 96 in the provided lysis buffer with 10% Proteinase Inhibitor Cocktail added (Sigma P8340, Saint Louis, MO).
  • SpyCatcher/SpyTag protein-peptide partners spontaneously form a covalent bond under physiological conditions and have been used in numerous protein engineering studies.
  • Simian Adenovirus serotype 36 (SAd36) could be derivatized with SpyTag and linked with a Cas9- Spy Catch er fusion protein to achieve gene editing in vitro.
  • a similar approach was useful for targeting - by linking an antibody on the virus capsid through SpyCatcher/SpyTag chemistry, we might drive viral uptake through binding of the antibody to the appropriate antigen on a cell surface.
  • B cells were selected as a first choice for targeting due to our previous work on this cell type and standing interest in achieving in vivo engineering for control of infectious diseases.
  • SpyCatcher fusion protein was initially developed based on a single-domain antibody (sdAb) previously studied in our lab which binds to murine CD40 (F8SpC). It was found that linkage of this antibody to an SAd36 derivative with SpyTag incorporated in the hexon protein resulted in strong gene transfer enhancement into mCD40+ murine primary B cells (FIG. 23 A). Interestingly, this enhancement was not found when F8SpC was linked to the fiber or pIX proteins, which is surprising given that the fiber is typically used by Ads to attach to cells, and thus would be expected to be the ideal site for targeting modifications. This may represent new biology specific to SAd36 or may be a result of our particular engineering strategy for incorporating SpyTag in the fiber.
  • the HI loop was selected within the fiber for DogTag insertion, as this site has previously been used for insertion of peptides (FIG. 19A and 19B). It was found that this vector was easily upscaled in standard HEK293 cells and yielded titers comparable to the isogenic unmodified Ad5 vector. SDS-PAGE analysis revealed identical protein band patterns between the Ad5 and Ad5FDgT, with the exception of the expected band shift from the insertion of DogTag in the fiber (FIG. 19D).
  • Adenovirus-antibody (Ad- Ab) complexes To assess the ability of our Adenovirus-antibody (Ad- Ab) complexes to achieve targeted gene transfer, primary murine B and T cells were infected with Ab-Ab complexes decorated with antibodies targeting B or T cell restricted cell markers (CD40, CD 19, CD20 and CD8a). As expected, it was found that gene transfer enhancement occurred only when the appropriate antibody was conjugated on the virus - infectivity did not change when B cells were treated with a virus targeting CD8a, while T cell infection rates were not impacted by conjugation of the virus with CD40, CD 19, or CD20 targeting antibodies (FIG. 20B). It was also assessed this effect in primary human B cells, and similarly found that gene transfer enhancements only occurred in the presence of the appropriate targeting agent.
  • B or T cell restricted cell markers CD40, CD 19, CD20 and CD8a
  • Ad-Ab complexes were a promising platform for controlling gene transfer and expression both in vitro and in vivo.
  • a single component vector without excess antibody fusion protein is desirable. This would also allow us to remove any potential confounding effects of excess antibody in our experiments. It was therefore attempted to develop a workflow to purify and store purified Ad-Ab complexes (FIG 21.A).
  • density gradient centrifugation is often used to prepare purified Ads. From cell lysates, a single ultracentrifugation with cesium chloride gradients was first used to purify Ad5FDgT.
  • This vector was dialyzed against IX PBS to prevent any negative effects towards the virus conjugation from excess cesium chloride.
  • This sample was split into equal thirds, and treated one fraction each with PBS, a-mCD19 lD3DgC, or a-mCD8a YTS169DgC. Conjugation was carried out at 25C for 1 hour, and vectors were ultracentrifuged a second time to separate free antibody from the virus conjugates. Importantly, observation of the vector bands did not reveal any obvious differences between virus treated with PBS and virus conjugated with scFvs (FIG. 21. B). After spinning vector samples were dialyzed again against our standard IX PBS with 10% glycerol and aliquoted and frozen at -80C.
  • liver eGFP expression was assessed and selected two mice per group for tissue immunohistochemistry. It was again observed a weak, non-statistical decrease in liver gene expression in mice injected with Ad5FDgT-lD3, but this result was not true for mice injected with Ad5FDgT-YTS169. There were not significant differences in eGFP expression in male and female mice (FIG. 26). It was further confirmed these results using tissue immunohistochemistry and demonstrated that scFv vector functionalization did not result in any radical changes in overall gene expression across all organs (FIG. 27).
  • HEK293 (ATCC CRL-1573) cells were grown in Dulbecco’s Modified Eagle Medium/Ham’s F12 1 : 1 mixture supplemented with L-glutamine, 15mM HEPES, 10% fetal bovine serum (FBS) and lOOU/mL penicillin-streptomycin. Cells were grown at 37C with 5% CO2 under sterile conditions.
  • a previously described first-generation E1ZE3 deleted HAdV-C5 vector with the cytomegalovirus (CMV) promoter driving eGFP (Ad5.CMVeGFP) was used to generate Ad5FDgT.
  • the parent plasmid was cut with Bari (Sibenzyme) and BstBI (New England Biolabs) to release the fiber protein.
  • Two PCR fragments were generated encoding the regions upstream and downstream of the HI loop domain with overlaps for the Bari and BstBI cut sites.
  • a synthetic fragment encoding DogTag with short linkers and overlaps for the PCR fragment was synthesized by Integrated DNA Technologies (IDT).
  • Ad5.CMVeGFP backbone using NEB HiFi DNA Assembly (New England Biolabs), generating Ad5FDgT.
  • This backbone was linearized using PacI and transfected into HEK293 cells for upscale. Viruses were purified and analyzed for viral particle concentration as previously reported.
  • the single domain antibody JPP-F8 was derived from immunization of alpacas according to previously described methods.
  • pDEST14-F8DgC was derived by cloning of a synthesized DNA fragment (IDT) containing the camelid JPP-F8 single domain antibody followed by a (G4S)3 flexible linker into the Sfol site of pDEST14-DogCatcher (Addgene #171772), and the resultant sequences encoded “6xHis-TEV-JPPF8-DogCatcher”.
  • pcDNA3.4-18B12DgC and pcDNA3.4-RtxvlDgC were derived by cloning of synthesized DNA fragments (IDT) containing 18B12 and Rtxvl scFvs together with a DgC fragment into the EcoRI and Hindlll sites of pcDNA3.4-c-Fos scFv [N486/76] (Addgene #190560), and the resultant sequences encoded “IL-2 signal sequence- 18B 12 scFV-(G4S)3-DgC-6xHis” and “IL- 2 signal sequence-Rtxvl scFV-(G4S)3-DgC-6xHis”.
  • pcDNA3.4-lD3DgC and pcDNA3.4- FMC163DgC were derived by cloning of synthesized DNA fragments containing 1D3 scFv and FMC163 scFv into the EcoRI and BamHI sites of pcDNA3.4-RtxvlDgC, and the resultant sequences encoded “IL-2 signal sequence-lD3 scFv-(G4S)3-DgC-6xHis” and “IL-2 signal sequence-FMC63 scFv-(G4S)3-DgC-6xHis”.
  • pcDNA3.4-HA22DgC pcDNA3.4- lYTS169DgC pcDNA3.4-2.43DgC were derived by cloning of synthesized DNA fragments containing IGHV1-46 signal sequence followed by HA22 svFv, YTS169 scFv, and 2.43 scFv into Xbal and BamHI sites of pcDNA3.4-lD3DgC, and resultant sequences encoded “IGHV1- 46 signal sequence-HA22 scFv-(G4S)3-DgC-6xHis”, “IGHV1-46 signal sequence-YTS169 scFv-(G4S)3-DgC-6xHis”, and“IGHVl-46 signal sequence-2.43 scFv-(G4S)3-DgC-6xHis”.
  • the plasmids pDEST14-F8DgC and pDEST14-DogCatcher were introduced into protein expression BL21(DE3)-RIPL E. coli cells. Single colonies were used to inoculate 25 mL starter LB containing 100 pg/mL carbenicillin and 50 pg/ml chloramphenicol grown at 37 °C overnight. The starter cultures were added to 500 ml fresh media without antibiotics, and cultures were grown at 37 °C with shaking at 250 rpm for 2.5 hours. Protein expression was induced with 1 mM IPTG, and the cultures were incubated at 30 °C with shaking at 250 rpm for 4 hours.
  • lysis buffer 0.5 mM Tris, 0.3 M NaCl, 10 mM imidazole, 0.2% Triton X-100, 1 mg/ml lysozyme, 20 units/ml DNase I, 1 mM PMSF, and one complete mini EDTA-free protease inhibitor cocktail tablet per 10 ml
  • lysis buffer 0.5 mM Tris, 0.3 M NaCl, 10 mM imidazole, 0.2% Triton X-100, 1 mg/ml lysozyme, 20 units/ml DNase I, 1 mM PMSF, and one complete mini EDTA-free protease inhibitor cocktail tablet per 10 ml
  • the 6xHis-tagged recombinant proteins produced in bacterial and mammalian systems were purified using a HisPur Ni-NTA column with 20 to 40 mM imidazole washing buffer and 300 mM imidazole elution buffer, and eluted proteins were dialyzed in 10% glycerol in PBS with three buffer changes using 3.5KDa molecular weight cut-off Slide- A-Lyzer Dialysis Cassettes. Protein concentration was measured using BCA according to the manufacturer’s instructions (Thermo Scientific #23227).
  • the iBlot 2 Dry Blotting System (Invitrogen #IB21001) was used to transfer electrophoretically resolved viral proteins from the gel to PVDF membrane (Invitrogen #IB24002) as recommended by the manufacturer.
  • We employed the iBind Western System (Invitrogen #SLF1000) for immunodetection of Ad5FDgT fiber proteins using primary mouse monoclonal antibody 4D2 (PMID: 1962447) against the N-terminal fiber tail domain and secondary anti-mouse IgG conjugated with Alkaline Phosphatase (AP) (Millipore Sigma #A3688).
  • the protein bands bound with both primary and secondary antibody were developed with colorimetric AP substrate reagent kit (Bio-Rad Laboratories #1706432) as recommended by the manufacturer.
  • mouse B cells were magnetically isolated from splenocytes and cultured for 3 Oh in RPMI 1640 supplemented with 10% FBS, IX Nonessential Amino Acids, IX sodium pyruvate and IX 2-mercapto-ethanol. 50 pg/mL LPS was used as an activation agent. Infections were carried out overnight with 5x105 cells in 50 pL LPS-free media with 5% FBS. Infected cells were then returned to 500 pL total volume with complete media and incubated for a total of 48 hours after infection prior to flow cytometry analysis.
  • Human B cells were magnetically isolated from healthy donor peripheral blood mononuclear cells and cultured for approximately 72 hours prior to infection according to previously described protocols. For infections 2.5x105 cells were infected in a total volume of 40 pL STEMMacs HSC Expansion media (Stem Cell Technologies) with 0.2% FBS for 3 hours, then transferred to ImL complete media. Flow cytometry analysis was carried out 48 hours after infection.
  • Mouse T cells were isolated from splenocytes using the Miltenyi Pan T Cell Isolation Kit II according to the manufacturer’s instructions. Cells were activated for 1 hour prior to infection using the Miltenyi T Cell Activation/Expansion kit according to the manufacturer’s instructions. 1-2x106 T cells were incubated with 200 pL anti-CD3/CD28 microbeads in lOmL RPMI 1640 supplemented as above and with lOng/mL mouse IL-4. For infections 5x105 cells were resuspended in 100 pL culture media and infected for 16-20 hours. An additional 150 pL complete media was then added and cells were analyzed via flow cytometry 48 hours after infection.
  • virus was prepared at 2X the concentration required for infection and mixed with an equal volume of the required amount of antibody fusion protein diluted in IX PBS. This mixture was incubated at room temperature or 25 °C for about 2 hours prior to infection.
  • splenocyctes isolated from C57B1/6J mice were stained in 100 pL FACS buffer (IX PBS containing 0.5% BSA) with 0.5 pg of DgC, lD3DgC, 18B12DgC, 2.43DgC, or PBS control at 4 °C for 30 minutes.
  • Samples were then washed with 2 ml FACS buffer and resuspended in 100 pl FACS buffer containing 0.5 pl Alexa FITC anti-His Tag antibody (BioLegend) and 0.5 pl Alexa Fluor® 594 anti -mouse B220 antibody (BioLegend) or 0.5 pl Alexa Fluor® 594 anti-mouse CD8a antibody (BioLegend). Samples were incubated at 4 °C for 30 minutes, washed again in 2 ml FACS buffer then resuspended in 100 pl FACS buffer. For blocking studies, splenocytes were pretreated with 7.5 pg or 15.0 pg full length 1D3 antibody (BioLegend) at 4 °C for 30 minutes before the addition of lD3DgC.
  • Germinal center B cells were gated as GL7+/CD95+/IgDlo/CD381o, marginal zone as IgM+/IgDlo, follicular as IgMlo/IgD+, memory as IgDlo/GL7-/CD38+, and plasmablasts as CD138+/IgD-. All B cell subsets were first passed through a singlets/live/CD19+ gate prior to further analysis.
  • B cells were gated as singlets/live/CD19+/CD3-/CD22+/CD4-/CD8-.
  • CD8a+ T cells were gated as singlets/live/CD8a+/CD19-/CD3+/CD22-/CD4-.
  • CD4+ T cells were gated as singlets/live/CD4+/CD19-/CD3+/CD22-/CD8a-.
  • Mouse antibodies used were as follows: CD19 (Invitrogen RM7717 or Miltenyi 130-111-887), CD8a (BioLegend 100762), CD3 (Introgen 46-0032-82 or 47-0031-82), GL7 (BioLegend 144608), CD95 (Invitrogen 46-0951-82) IgD (Invitrogen 47-5993-82 or 56-5993- 82), CD38 (Miltenyi 130-125-522), IgM (Invitrogen 47-5790-82), CD138 (Invitrogen MAS- 23527), CD22 (BioLegend 126111), CD4 (Invitrogen 56-0041-82).
  • C57B1/6J mice were acquired from the Jackson Laboratory and housed in a pathogen-free environment. Mice aged 6-9 weeks were injected retroorbitally with 5x1010 viral particles in 150 pL total volume and sacrificed roughly 72 hours later via anesthetization with Avertin followed by cervical dislocation. In the first in vivo study, one-half of each spleen was used for flow cytometry analysis, while the remaining half, liver, lungs, kidneys and heart were snap frozen in liquid nitrogen. Organs were thawed on ice and processed for eGFP quantification using the Cell BioLabs Fluorimetric GFP Quantitation Kit (Cell Biolabs #AKR- 120) per the manufacturer’ s instructions.
  • Tissues were homogenized in the included lysis buffer supplemented with 10% Proteinase Inhibitor Cocktail (Sigma #P8340) using an Omniprep 96 automated homogenizer. Tissue lysated were then centrifuged at 4000-5000 rpm to remove cell debris and supernatants were transferred to clean tubes. Supernatants were diluted as appropriate then aliquoted into 96-well plates for eGFP quantification. Total solution eGFP was normalized against total solution protein from a BCA assay of the lysates performed according to the manufacturer’s instructions (Thermo Scientific #23227).
  • Virus was then split into three equal aliquots and treated with either PBS, 2-fold molar excess of lD3DgC, or two-fold molar excess of YTS169DgC for 1 hour at 25 °C. Conjugated viruses were ultracentrifuged again on cesium chloride gradients for 1.5 hours at 25,000 rpm at 4 °C. Viral bands were harvested and dialyzed three times against IX PBS with 10% glycerol, then frozen at -80 °C.
  • C57B1/6J mice were obtained from Jackson Labs. Mice were injected retroorbitally and sacrificed approximately 72 hours later as described in the main. Flow cytometry analysis of splenocytes was carried out as described in the main.
  • mice were obtained from Jackson Labs and injected with the indicated vectors as described in the main (one male and one female mouse per group). Three days after virus administration, mice were anesthetized with Avertin and perfused via the left ventricle with 10% neutral -buffered formalin. Lung was further inflated by injecting formalin solution into the trachea, closing the trachea by ligature, and then processing as below. Harvested organs were postfixed in formalin at room temperature for 2-4 h and cryopreserved in 30% sucrose in PBS at 4 °C overnight. Treated tissues were embedded in NEG50 (Thermo Scientific) and frozen on dry ice. All mouse tissues were cryosectioned at 16 pm.
  • Example 9 Use of off-the-shelf whole antibodies to functionalize adenovirus for targeted cell delivery.
  • BPA forms a covalent bond with adjacent Ig residues upon exposure to 365nm light. See, e.g., Tsourkas et al., Bioconjug Chem 2015. The DogCatcher-AbBD fusion proteins were successfully able to covalently bind to off the shelf mouse CD 19 antibodies (FIG. 29).
  • Functionalized off-the-shelf native antibodies using this strategy showed improved gene transfer in murine primary B cells.
  • FIG. 30A-B This improvement was seen across a number of different commercially available B cell targeting antibodies.
  • FIG. 30A-B This improvement was seen across a number of different commercially available B cell targeting antibodies.
  • MSTNSFDGSIVS S YLTTRMPPWAGVRQNVMGS SIDGRPVLP ANSTTLTYET VS GTPLETAASAAASAAAATARGIVTDFAFLSPLASSAASRSSARDDKLTALLAQ

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Abstract

Sont divulguées de nouvelles technologies de ciblage pour administrer des thérapies géniques à des types de cellules spécifiques à la fois in vivo et ex vivo, comprenant un système qui comprend une particule adénovirale modifiée pour incorporer une ou plusieurs étiquettes peptidiques dans un ou plusieurs composants de la protéine de capside, et une protéine recombinante qui comprend une première partie pouvant former une liaison à affinité élevée avec ladite étiquette peptidique, et une seconde partie comprenant un ligand ou une protéine de liaison à l'antigène pouvant cibler un type de cellule spécifique (par exemple, une cellule B).
PCT/US2024/019736 2023-03-17 2024-03-13 Procédés d'édition in vivo de cellules b Pending WO2024196669A2 (fr)

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