EP4680727A2

EP4680727A2 - Methods for in vivo editing of b cells

Info

Publication number: EP4680727A2
Application number: EP24775387.4A
Authority: EP
Inventors: Mark Selby; Hangil Park; Rosa Romano; David T. Curiel; Paul Boucher; Zhi Hong LU
Original assignee: Washington University in St Louis WUSTL; Walking Fish Therapeutics Inc
Current assignee: Washington University in St Louis WUSTL; Walking Fish Therapeutics Inc
Priority date: 2023-03-17
Filing date: 2024-03-13
Publication date: 2026-01-21
Also published as: WO2024196669A2; WO2024196669A3

Abstract

Disclosed herein are novel targeting technologies to deliver gene therapies to specific cell types both in vivo and ex vivo, comprising a system that includes 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).

Description

METHODS FOR IN VIVO EDITING OF B CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit and priority of U.S. Provisional Application No. 63/452,960, filed on March 17, 2023, U.S. Provisional Application No. 63/495,050, filed on April 7, 2023, and U.S. Provisional Application No. 63/510,822, filed on June 28, 2023, the contents of each of which are incorporated herein by reference in their entireties.

[0002] This invention was made with government support under TR002851, CA211096, and HL007088 awarded by the National Institutes of Health. The government has certain rights in the invention.

INTRODUCTION

[0003] B cells are naturally hardwired to present antigens and secrete immunoglobulins. [0004] Theoretically, B cells should have great potential as a cellular therapy for targeting certain diseased cell types and expressing therapeutic proteins. There thus exists a need for 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.

[0005] 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. Importantly, B cells can be matured to long-lived cells that are ideal for provision of therapeutic proteins that are required for extended periods. Indeed, injected B cells can traffic to their normal tissue niches including spleen, lymph nodes and the bone marrow where they can persist. Typically, ex vivo modification has been contemplated to avoid in vivo use of recombinant viruses to which immune responses can inactivate. A need exists for targeted technologies to more efficiently deliver gene therapies to specific cell types both in vivo and ex vivo.

[0006] Successful gene therapy requires the efficient and safe delivery of genetic material to cells of interest. 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. However, off-target gene expression and viral particle sequestration may lead to safety concerns in vivo. Despite decades of work, targeting to specific cell markers remains a challenge, and vectors capable of using existing antibodies for targeting would be of strong utility.

SUMMARY

[0007] Disclosed herein are novel targeting technologies to deliver gene therapies to specific cell types in vivo.

[0008] In various embodiments, 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.

[0009] 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. 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. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond.

[0010] In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0011] In various embodiments, 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.

[0012] In various embodiments, said second recombinant peptide comprises a Cas 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said therapeutic protein is a recombinant antibody. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38. In various embodiments 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. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0013] In various embodiments, 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.

[0014] In various embodiments, 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said wherein said therapeutic protein is a recombinant antibody. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0015] In various embodiments, 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.

[0016] In various embodiments, said second recombinant peptide comprises a Cas 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said wherein said therapeutic protein is a recombinant antibody. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0017] In various embodiments, 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. 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said wherein said therapeutic protein is a recombinant antibody. [0018] In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0019] In various embodiments, 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.

[0020] 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said wherein said therapeutic protein is a recombinant antibody. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0021] In various embodiments, 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.

[0022] 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, therapeutic protein is a recombinant antibody.

[0023] In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0024] In various embodiments, 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.

[0025] 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said therapeutic protein is a recombinant antibody. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

[0026] In various embodiments, 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.

[0027] 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. In various embodiments, the native tropism of said adenovirus has been ablated. In various embodiments, said therapeutic protein is a recombinant antibody. In various embodiments, 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. In various embodiments, said peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, said peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, said high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the recombinant protein comprises a ligand. In various embodiments, the ligand is selected from the group consisting of CD40, CD 19, CD20, and CD38. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell. In various embodiments, said B cell is contacted with an activating agent, prior to contacting sad B cell with said adenovirus and said recombinant protein. In various embodiments said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin. In various embodiments, said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein. In various embodiments, said preconditioning is an immunization or vaccination.

In various embodiments, 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.

[0028] In various embodiments, the method for editing said cell is ex vivo. In various embodiments, the method for editing said cell is in vivo. In various embodiments, 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. In various embodiments, 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. In various embodiments, the peptide tag is expressed in the Hl loop domain of the Ad fiber. In various embodiments, the peptide tag is expressed in the hexon protein of said adenovirus. In various embodiments, the peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein. In various embodiments, the high affinity interaction has a KD of 1 nM. In various embodiments, said high affinity interaction is a covalent bond. In various embodiments, said covalent bond is an isopeptide bond. In various embodiments, the first or second recombinant protein comprises a ligand. In various embodiments, 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. In various embodiments, the recombinant protein comprises an antigen binding domain. In various embodiments, the first or second recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody. In various embodiments, the adenovirus is contacted with said recombinant protein, prior to contacting with said one or more cells. In various embodiments, the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said one or more cells. In various embodiments, the therapeutic protein is an anti-HIV antibody. In various embodiments, the anti-HIV antibody comprises SEQ ID No. 23. In various embodiments, the anti-HIV antibody comprises SEQ ID NO. 24.

BRIEF DESCRIPTION OF FIGURES

[0029] 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. 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

[0030] 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.OxlO¹¹ 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. Magnification: 100X, Red: endomucin/CD31, Green: eGFP [0031] 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. 48h post injection splenocytes were harvested and B cells were assessed for GFP expression as above. 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

[0032] 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. (4A) Analysis of adenoviral vectors with altered tropism. Comparison was made between 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)

[0033] 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.

[0034] 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. 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). (B) The double-editing strategy used by Huang et al. replaces the 3’ J segments of the IgH and IgK loci with an exogenous template sequence. The endogenous VDJ/VJ sequences are left intact and express along with the introduced variable gene. Note that in both (A) and (8), native variable genes are also expressed and the engineered bNAb is distant from the optimal location for somatic hypermutation. (C) We recently developed an editing strategy that directly replaces the endogenous variable regions. Note that the position of the homology arms allows for replacement of nearly any endogenous variable region, and the final heavy- and light-chain variable genes are placed in their native loci. See FIGs 7 and 8 for neutralization results from this strategy. (D) As described in Ou et al., we have also developed another means of engineering the native heavy-chain locus, namely by introducing only an exogenous HCDR3 region. This HCDR3-based strategy preserves the diversity of the edited repertoire and can be used with V2-glycan apex antibodies whose binding is strongly dependent on the HCDR3 loop alone.

[0035] FIG. 7 shows physiologic BCR editing enables robust in vivo CAR B cell responses. Two groups of n=3 mice were engrafted with B cells edited to express the bNAb 10-1074. Murine B cells were edited by intron editing (represented in FIG. 6, view A) or our double editing approach (FIG. 6, view C). After receiving CAR B cells, mice were vaccinated twice with a 60-meric I3-B505 gpl20 construct. One week after each challenge, 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). Note: gpl20 vaccination elicits binding but not neutralizing antibody responses in mice (see grey lines in FIG. 8, for example); therefore, all of the plasma neutralizing activity can be attributed to CAR B antibody expression

[0036] FIG. 8 shows native locus editing of murine heavy- and light-chain genes to express the variable chains of the bNAb VRC26.25. (A) 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. Specifically, 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. (B) The editing process used to introduce heavy- and light-chain genes into their native loci is efficient. Primary mouse B cells were reprogrammed to express VRC26.25 and stained with a soluble Env trimer. Roughly 7.5% of edited cells expressed BCRs capable of recognizing SOSIP, compared to <0.5% of mock-edited controls. (C) shows the ex vivo neutralizing activity of serum taken from the mice against the HIV-1 isolate CRF250. Yellow, orange, and red lines show the serum neutralizing activity of three different tgB animals after each challenge. Note the low intra-assay variability. A multiply vaccinated control animal (un-engrafted; gray lines) failed to mount any neutralizing response. The dotted black line shows the neutralizing activity of naive mouse serum spiked with 2 pg/mL of recombinant VRC26.25.

[0037] 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). Bar heights show the percentage of sequences mutated at the indicated sites and the colored segments indicate individual amino acid substitutions. The large red-brown bar corresponds to a Q100E mutation that arose independently in all three mice. In panel (D), mice mice were engrafted with either wild type VRC26.25 HCDR3 (n=3, green) or the Q100E variant (n=3, blue) and vaccinated twice with soluble trimer. Plasma neutralizing activity of these two variants is shown compared to that of un-engrafted animals (n=3; -ctl; black) and 2 pg/mL of recombinant VRC26.25 added to naive mouse serum (+ Ctrl, red). Note that the diverse repertoire of cells expressing the Q100E HCDR3 variant exceeded the neutralizing activity of the 2 pg/ml VRC26.25 control (red).

[0038] FIGs. 10A-10B shows vector retargeting. A) Immunohistochemistry demonstrating in vivo retargeting of Ad5FDgT-F8. Blue = DAPI, Green = eGFP from the vector. B) Flow cytometry data showing enhanced gene transfer to human primary B cells ex vivo. Activated cells were infected with virus and assessed for eGFP expression forty-eight hours later.

[0039] 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.

[0040] FIG. 12 shows in vivo assessment of promoter modified vectors. A) Experimental setup. Six mice per group were injected with IxlO¹¹ vp of the vectors indicated in FIG. 11, view A, plus the hexon modified AdH5/H3RGD.EEK vector. 72 hours later mice were sacrificed and major organs were harvested. One-half of the spleen was processed for flow cytometry scoring of eGFP expression in B and T cells, while the remaining half and heart, lungs, liver and kidneys were prepared for total tissue eGFP quantification using a fluorometric assay. B cells were scored as CD45+/CD19+/CD3- and T cells were scored as CD45+/CD19-/CD3+. B) Flow cytometry scoring of eGFP expression in B and T cells after IxlO¹¹ vp delivery. All samples were normalized to the PBS control. Left; percent of total eGFP+ cells. Right; mean fluorescent intensity in eGFP+ cells. Data are expressed as means ± SD. C) Statistical analysis of B cell (left) and liver (right) eGFP expression using ordinary one-way ANOVA. Both datasets failed the Shapiro-Wilk test for normality of residuals upon analysis, so the data were log transformed for further statistical analyses. Bars represent means. D) Tissue eGFP expression after IxlO¹¹ vp delivery via quantitative assay. Tissues were homogenized in lysis buffer and assessed for eGFP expression using a fluorometric kit. E) Flow scoring of eGFP expression in B and T cells after 5xlO¹⁰ vp delivery, normalized to PBS. Left; percent of total eGFP+ cells. Right; mean fluorescent intensity in eGFP+ cells. Data are expressed as means ± SD. F) Tissue eGFP expression after 5xlO¹⁰ vp delivery. [0041] FIG. 13 shows analysis of the mechanism of gene transfer to B cells via Ad vectors. A). Blocking of Ad infection in murine B cells via recombinant fiber. 5xl0⁵ 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. B) Blocking of Ad infection in A549 cells via recombinant fiber. 5xl0⁵ A549 cells were seeded in a 12-well plate and incubated overnight. Recombinant fiber was added and cells were infected as above. Recombinant Ad5 fiber has been previously described⁴⁸. C). Comparison of gene transfer to B cells and A549 cells by a CAR-binding ablated vector. 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. 5xl0⁴ 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) has been used previously to analyze penton binding to integrins and GRADSP (Sigma SCP0156, Saint Louis, MO) is a nonbinding control. 5xl0⁵ 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. In A-D flow cytometry was used to score for eGFP+ cells approximately 48 hours after infection. E) Analysis of integrin expression in eGFP+ B cells after in vivo delivery of 5xl0¹⁰ viral particles of the indicated vectors.

[0042] FIG. 14 shows immunophenotyping of murine B cells after in vivo vector administration. A) Gating strategy to define individual B cell populations. All populations were initially gated as singlets/live/CD19+/CD3-. GC = germinal center, FO = follicular, MZ = marginal zone, CD71hi = proliferating, PBs = plasmablasts B) eGFP expression in defined cell populations. C) Fold change in eGFP expressing cells in individual B cell populations compared to all B cells. D) Representative analysis of cell marker expression in eGFP+ and eGFP- cells. All data are expressed as means ± SD.

[0043] 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. All infections were carried out at 2000 MOI. [0044] 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.

[0045] 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.

[0046] 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. The molar ratio of antibody to virus (10X, 5X, 2.5X, 1.25X, 0.625X) was determined by calculating DogCatcher (1 copy per antibody fusion molecule) versus DogTag (36 copies per virus capsid). Cells were infected and scored for gene expression approximately 40 hours later using flow cytometry. B cells are CD40+ and T cells are CD8+.

[0047] 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. SDS-PAGE analysis of control Ad5 and Ad5FDgT. E. Gel shift analysis of Ad5FDgT binding to DogCatcher and antibody -DogCatcher fusions. Left, binding to bacterially produced DogCatcher and an sdAb-DogCatcher fusion. Middle and right, binding to representative scFv-DogCatcher fusions. F. Flow cytometry analysis of representative scFvs in murine splenocytes. lD3DgC and 18B12DgC stains were gated on B220+ populations, while 2.43DgC staining was gated on the CD8+ population. lD3DgC specificity was assessed by blocking with excess full length 1D3 antibody.

[0048] 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.

[0049] FIGs. 21 A-21C show in vivo characterization of Ad-Ab targeting. A. Experiment design. C57BL/6J mice were injected RO with 5x1010 vp/mouse of either Ad5FDgT or Ad5FDgT conjugated with lD3DgC. Major organs were harvested 72h later for eGFP expression analyses. B. Flow cytometry results. Left, GFP expression in B and T cells. Right, GFP expression in B cell subsets. GC = germinal center, MEM = memory, MZ = marginal zone, FO = follicular, PB = plasmablast. In all cases non-conjugated and conjugated groups were compared in each population using a standard t-test. Welch’s correction was used in cases where the F-test revealed significant differences in variances. C. Quantitative GFP analysis in major tissues. Tissues were homogenized in lysis buffer and eGFP expression was analyzed via fluorimetry. eGFP was normalized to total tissue protein content as assessed by BCA. In all cases ordinary one-way ANOVA was used to compare groups. In the liver, lung and kidney values were log transformed to normalize variances. Icons generated by BioRender.

[0050] 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.

[0051] FIGs. 23A-23C show Initial proof-of-concept Ad-Ab targeting using SAd36 reagents. A. Screening of SAd36-SpyTag variants conjugated with bacterially produced antibodies in murine B cells. Left, screening of SAd36 with SpyTag incorporated in the hexon (H-SpT) or protein IX (pIX-SpT). SpC-F8 and SpC-Gl are sdAbs that target murine CD40 while SpC-lD3 is an scFv that targets murine CD19. Right, assessment of SAd36 with SpyTag incorporated in a fiber-fibritin configuration (FF-SpT). 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. B. Analysis of targeting reliance on the specific conjugation of SpyTag and SpyCatcher. SAd36-H-SpT was incubated with either SpC-F8, F8 with bacterial thioredoxin fused in place of SpC (Trx-F8), or SpC-H7. 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.

[0052] 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).

[0053] FIGs. 25A-25B show in vitro analysis of purified Ad-Ab complexes. A. Infection of primary murine B cells and T cells with purified or freshly prepared Ad-Ab complexes. Purified complexes are denoted as “scFv name (pure)” while freshly prepared complexes are denoted as “scFv name (molar ratio DgT:DgC). [0054] FIGs. 26A-26D show in vivo analysis of Ad-Ab targeting by sex. A. Data from Figure 3 was re-analyzed by sex using multiple unpaired t-tests with the Holm-Sidak correction for multiple comparisons.

[0055] FIGs. 27A-27B show in vivo analysis of Ad-Ab targeting by sex. A. Data from Figure 3 was re-analyzed by sex using multiple unpaired t-tests with the Holm-Sidak correction for multiple comparisons.

[0056] 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.

[0057] FIG. 29 shows a western blot of adenovirus particles functionalized with an off- the-shelf whole antibody using a pGA24BPA PG fusion. In the presence of the pG DogCatcher fusion and increasing concentrations of an anti-mouse CD 19 antibody (alpha- mCD19 Rab mAb), a heavy band (Heavy+binder) is observed on the Western Blot indicating the ability of the DogCatcher fusion protein to bind the native antibody.

[0058] 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.

DETAILED DESCRIPTION

[0059] Disclosed herein are novel targeting technologies to deliver gene therapies to specific cell types both in vivo and ex vivo. In various embodiments, 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). See, e.g., FIG. 1. In various embodiments, the adenoviral particles carry the genetic machinery to edit the target cells to express a therapeutic protein. In various embodiments, the adenoviral particle may express more than one distinct peptide tag and more than one distinct recombinant protein. In various embodiments, 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. In various embodiments at least one recombinant protein comprises an RNA binding protein. The benefits of the various embodiments disclosed herein include durable and consistent therapeutic protein expression (reducing required dosing therapy). The methods disclosed herein have the benefit of allowing the skilled artisan to specifically edit a B cell or a specific subclass of B cell using either ex vivo or in vivo methods.

[0060] In various embodiment 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.

[0061] In other embodiments, 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.

[0062] In various embodiments, the invention relates toa 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. [0063] In various embodiments, 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..

[0064] In various embodiments, 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.

[0065] In various embodiments, 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.

[0066] In various embodiments, 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.

[0067] In various embodiments, 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.

[0068] In various embodiments 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.

A. Definitions

[0069] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0070] In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0071] The term “polynucleotide”, “nucleotide”, or “nucleic acid” 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. [0072] The term “oligonucleotide” refers to a polynucleotide comprising 200 or fewer nucleotides. 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.

[0073] The term “control sequence” refers to a polynucleotide sequence that affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism. In particular embodiments, control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, 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.

[0074] As used herein, “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.

[0075] The term “vector” means any molecule or entity e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell. The term “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.

[0076] 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.

[0077] The term “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. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques.

Following transfection or transduction, 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.

[0078] The term “transfection” refers to the uptake of foreign or exogenous DNA by a cell. A number of 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.

[0079] The term “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.

[0080] The terms “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. The terms “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. The term “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.

[0081] A “variant” of a polypeptide e.g., an antigen-binding molecule) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins. [0082] The term “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. When used with reference to a cell, 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. For example, the sample may be blood, bone marrow, or a tissue sample. For example, 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. For example, B cells are isolated by flow cytometry, magnetic cell isolation and cell separation (MACS), 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.

[0083] The term “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”).

[0084] To calculate percent identity, 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.). The computer algorithm 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). In certain embodiments, 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.

[0085] As used herein, 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) of the twenty conventional 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). In the polypeptide notation used herein, 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.

[0086] Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. 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.

[0087] For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.

[0088] According to certain embodiments, the hydropathic index of amino acids can be considered in making protein modifications. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). See, e.g., Kyte etal., 1982, ./. Mol. BioL, 157, 105-131. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. Exemplary amino acid substitutions are set forth in Table 1.

Table 1

[0089] The term “derivative” refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain embodiments, a chemically modified antigen-binding molecule can have a greater circulating half-life than an antigen-binding molecule that is not chemically modified. In some embodiments, 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.

[0090] 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.

[0091] The term “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.

[0092] The terms “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.

[0093] The term “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. The term “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.

[0094] 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.

[0095] As used herein, 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. In one embodiment, 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.

[0096] As used herein, 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.

[0097] As used herein, 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. In one embodiment, an event is not appreciable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, or less of the time. [0098] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms “include,” “has,” “contains,” and “comprise” are used synonymously.

[0099] As used herein, “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. [0100] By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0101] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0102] As used herein, 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. In particular embodiments, 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.

[0103] As used herein, the term “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.

[0104] As used herein, the term “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. As the context may require , the term “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. In various embodiments, 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. In particular, 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.

B. Modified Adenoviral Particles

[0105] In various embodiments, the invention comprises a method for editing a B cell comprising contacting said B cell with an adenovirus. In various embodiments the adenovirus is a human adenovirus. In various embodiments the adenovirus is a primate adenovirus. In various embodiments, the adenovirus is a derivative adenovirus. As used herein, “human adenovirus”, “serotype” or “human adenovirus serotype” means any of the 51 human adenovirus serotypes currently known or isolated in the future. To do. See, for example, Strauss, "Adenovirus infections in humans," in The Adenoviruses, Ginsberg, ea., Plenum Press, New York, NY, pp. 451-596 (1984). These serotypes are classified into subgroup AF (as shown in Table 1 below, Shenk, “Adenoviridae: The Viruses and Their Replication,” in Fields Virology, Vol. 2, Fourth Edition, Knipe, ea. , Lippincott Williams & Wilkins, pp. 2265-2267 (2001)).

In various embodiments, the native tropism of said adenovirus has been ablated.

[0106] As used herein, “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. As used herein, “parent adenovirus serotype” means an adenovirus serotype that represents a serotype from which the majority of the genome of the chimeric adenovirus is derived. As used herein, the term “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). For example, 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.

[0107] As used herein, 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.

As used herein, tropism with reference to an adenovirus 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.

[0108] As used herein, 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. As used 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. Thus, 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. The production of such particles require that the genome be replicated and that those proteins necessary for assembling an infectious virus are produced. The particles also can require certain proteins necessary for the maturation of the viral particle. Such proteins can be provided by the vector or by the packaging cell. As used herein, 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.

As used herein, 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. One example, is the production of an adenovirus 5 vector particle containing an Ad37 or Ad35 fiber protein. This can be accomplished by producing the adenoviral vector in packaging cell lines expressing different fiber proteins.

[0109] In various embodiments, 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. The capsid shell of an adenovirus (Ad) comprises multiple copies of three major capsid proteins (MCPs; hexon, penton base, and fiber) and four minor/cement proteins (Illa, VI, VIII, and IX) that are organized with pseudo-T = 25 icosahedral symmetry. In various embodiments, one or more peptide tags are expressed on the capsid shell of said adenovirus. In various embodiments, 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.

C. Recombinant Proteins With High Affinity for Peptide Tags

[0110] In various embodiments, the invention further comprises a recombinant protein capable of recognizing and forming a high affinity interaction with one or more peptide tags. In various embodiments, said interaction is a covalent bond. In various embodiments, said interaction is an isopeptide bond. A number of known systems are suitable for use with the claimed invention.

[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).

[0112] Another example is the in-frame addition of nucleotides encoding one or more cysteines to the C- or N- termini of polypeptides. When such free cysteine containing polypeptides are mixed under oxidizing conditions, they will form disulfide bridges. Such systems, however, suffer from the synthesis of many side-products and from instability of the disulfide bridge under reducing conditions.

[0113] Another example is the Spy Tag/Spy Catcher (Reddington et al., 2015) system. Here, the concept of spontaneous isopeptide formation in naturally occurring proteins has been used to covalently attach one polypeptide to another. A domain from the Streptococcus pyogenes protein FbaB, which contains such isopeptide bond is split into two parts. One part, the SpyTag, is a 13 amino acid peptide that contains part (e.g., an aspartic acid) of the autocatalytic center. 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. Mixing those two polypeptides restores the autocatalytic center and leads to formation of the isopeptide bond, thereby covalently linking the SpyTag to the SpyCatcher (Zakeri et al ., 2012). Further engineering has led to a shorter version of SpyCatcher with only 84 amino acids as well as an optimized version, SpyTag002 and SpyCatcher002 with accelerated reaction (Li et al, 2014 and Keeble el al., 2017); both of which are hereby incorporated by reference in their entirety. More engineering has led to another optimized version, SpyTag003 (SEQ ID NO. 10) and SpyCatcher003, with a reaction close to the diffusion limit (Keeble et al., 2019), which is hereby incorporated by reference in entirety. A further modification of the system was the invention of SpyLigase (Fierer et al, 2014), which was achieved by splitting the FbaB domain into three parts, the SpyTag, the K- tag and the SpyLigase. 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.

[0114] Applications of such systems includes stabilization of proteins by circularization, vaccine generation, multimerization of proteins by integrating streptavidin/biotin with SpyTag/SpyCatcher (Reddington et al, 2015), affibody and Fab multimerization (Fierer et al, 2014), generation of antibodies from modules (Alam et al, 2017) as well as creation of antibody-drug conjugates (Siegmund et al, 2016), and generation of bispecific antibodies (Yumura et al, 2017). A similar system using the adhesin RrgA from Streptococcus pneumoniae was developed and termed SnoopTag/SnoopCatcher (Veggiani et al, 2016), with a later development of a SnoopLigase system (Bui dun et al, 2018). The 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). 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 Isopeptag/Split Spy0128 and SdyTag/SdyCatcherDANG short technologies are hereby incorporated by reference in their entirety.

[0115] In various embodiments, 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. In various embodiments, 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.

[0116] In various embodiments, the peptide tag is incorporated into capsid protein. In various embodiments the peptide tag is encoded into an Ad5 fiber protein. In various embodiments, the peptide tag is incorporated into an Ad5 HVR5 protein. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 1. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 2. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 3. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 4. In various embodiments, the capsid protein with an incorporated peptide tag comprises SEQ ID No. 5. In various embodiments, 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.

D. B Cell Targeting Moieties

[0117] In various embodiments, the recombinant protein further comprises a portion capable of recognizing and interacting with a protein expressed on the surface of a B cell. In various embodiments, 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. In various embodiments, the recombinant protein is capable of binding to a cell surface protein unique to a particular subclass of B cells.

[0118] For example, as would be recognized by the skilled person, plasma cells may be targeted by cell surface protein expression patterns using standard flow cytometry methods. For example, 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. Instead, 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. In various embodiments, 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. In various embodiments, 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.

E. Therapeutic Proteins

[0119] In various embodiments, the engineered B cell comprises a therapeutic protein to be delivered to a patient in need thereof. As used herein, the term “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. In certain embodiments, 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. In various embodiments, the therapeutic protein is for treatment of Lysosomal acid lipase deficiency (LAL), Acid sphingomyelinase deficiency or Niemann-Pick disease (ASM), Mucopolysaccharidosis IVA (GALNS). In various embodiments, the therapeutic protein is for the treatment of HIV. In various embodiments, the therapeutic protein is an anti -HIV antibody. In various embodiments, the anti -HIV antibody comprises SEQ ID No. 23. In various embodiments, the anti-HIV antibody comprises SEQ ID NO. 24. In various embodiments, the anti-HIV antibody comprises a light chain comprising SEQ ID NO. 23 and a heavy change antibody comprises SEQ ID NO. 24.

[0120] In various embodiments, the therapeutic protein is a receptor protein to be expressed on the surface of a B cell. In various embodiments the receptor is a chimeric antigen receptor or a “CAR-B” receptor. In various embodiments, the CAR-B receptor has an intracellular domain that includes CD79a (Immunoglobulin a). IN various embodiments the CAR-B receptor has an intracellular domain that includes CD79b (Immunoglobulin P). In various embodiments, the extracellular domain comprises an extracellular binding domain and a hinge domain. In various embodiments, the extracellular binding domain(s) recognizes at least one antigen or protein expressed on the surface of a target cell. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, the hinge domain is derived from the group consisting of IgG, CD28 and CD8. In various embodiments, the cytoplasmic domain comprises at least one signaling domain native to B cell receptors. In various embodiments, 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. In various embodiments, the cytoplasmic domain further comprises a costimulatory domain.

[0121] In various embodiments the therapeutic protein is Factor VIII. In various embodiments the therapeutic protein is alpha-galactosidase (aGAL or GLA). In various embodiments 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. In various embodiments, the therapeutic protein is an anti-inflammatory antibody. In various embodiments the therapeutic protein is selected from the group consisting of a-4-P-7 integrin, TNFa and IL23R. In various embodiments, the therapeutic protein is an oncology antibody. In various embodiments, 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. In various embodiments, the therapeutic protein is for the treatment of an infectious disease. In various embodiments, 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.

F. Methods of Engineering B Cells

[0122] Gene editing (including genomic 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). When a sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s)/nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, 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. As used herein, 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. As used herein, “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.

[0123] Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease - dependent approach. In the 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.

[0124] Alternatively, the 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). Such nuclease - dependent targeted editing also utilizes DNA repair mechanisms, for example, non - homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

[0125] 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.

[0126] 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. 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. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496. 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.

[0127] 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). 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.

[0128] Additional examples of 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.

[0129] Other non - limiting examples of targeted nucleases include naturally - occurring and recombinant nucleases, e.g., CRISPR/Cas9, restriction endonucleases, meganucleases homing endonucleases, and the like.

1. CRISPR-Cas9 Gene Editing

[0130] 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. 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).

[0131] 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).

[0132] 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.

[0133] Once the CRISPR - Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).

[0134] After binding of CRISPR - Cas9 complex to DNA at a specific target site and formation of the site - specific DSB, the next key step is repair of the DSB. Cells use two main DNA repair pathways to repair the DSB: non - homologous end - joining (NHEI) and homology - directed repair (HDR).

[0135] 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. Alternatively, 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.

[0136] In some embodiments, 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).

[0137] In some embodiments, 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.

[0138] In some embodiments, the Cas nuclease is from a Type - II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, 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.

[0139] In some embodiments, a Cas nuclease may comprise more than one nuclease domain. For example, 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). In some embodiments, the Cas9 nuclease introduces a DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, 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. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH- like nuclease domain. In some embodiments in which only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that is capable of introducing a single-stranded break (a “nick”) into the target sequence. In some embodiments, a conserved amino acid within a Cas9 nuclease domain is substituted to reduce or alter a nuclease activity. In some embodiments, 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). In some embodiments, 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).

[0140] In some embodiments, the Cas nuclease is from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from Type-IV CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type-V CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type- VI CRISPR/Cas system.

2. Guide RNAs

[0141] 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. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. In some embodiments, 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.

[0142] As is understood by the person of ordinary skill in the art, 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). [0143] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-molecule guide RNA. In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA.

[0144] 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.

[0145] A single-molecule guide RNA (referred to as a “sgRNA”) 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.

[0146] 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.

[0147] In some embodiments, 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.

[0148] In some embodiments, 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. For example, 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 (UUUUUUUU) at the 3' end of the sgRNA sequence. [0149] The sgRNA can be unmodified or modified. For example, modified sgRNAs can comprise one or more 2'-O-methyl phosphorothioate nucleotides.

[0150] By way of illustration, guide RNAs used in the CRISPR/Cas/Cpfl system, or other smaller RNAs 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. 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. Various types of 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. [0151] In some embodiments, indel frequency (editing frequency) may be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules. In some embodiments, a highly efficient gRNA yields a gene editing frequency of higher than 80%. For example, 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%.

[0152] In some embodiments, 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 (e.g., to knock out a gene in a cell) are known (Bauer D E et al. Vis. Exp. 2015; 95;e52118).

3. Spacer Sequence

[0153] In some embodiments, 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. In some embodiments, the spacer sequence is 15 to 30 nucleotides. In some embodiments, the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence is 20 nucleotides.

[0154] 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.” One of skill in the art recognizes that 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. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. For example, if the target sequence is 5'-AGAGCAACAGTGCTGTGGCC-3', then 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.

[0155] In a CRISPR/Cas system herein, 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. For example, 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.

[0156] In some embodiments, 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. For example, in a sequence comprising 5'- NNNNNNNNNNNNNNNNNNNNNRG-3', 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.

4. Methods of Making gRNAs

[0157] 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.

[0158] In some embodiments, non-natural modified nucleobases are introduced into polynucleotides, e.g., gRNA, during synthesis or post-synthesis. In certain embodiments, modifications are on internucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, 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).

[0159] In some embodiments, 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).

[0160] Certain embodiments of the invention also provide nucleic acids, e.g., vectors, encoding gRNAs described herein. In some embodiments, the nucleic acid is a DNA molecule. In other embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a crRNA. In some embodiments, 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. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a tracrRNA. In some embodiments, 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.

[0161] In some embodiments, 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. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together.

[0162] In some embodiments, the gRNAs provided by the disclosure are synthesized by enzymatic methods (e.g., in vitro transcription, IVT).

[0163] Various types of 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.

[0164] In certain embodiments, 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. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA is used, 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.

[0165] The guide RNA may target any sequence of interest via the targeting sequence (e.g., spacer sequence) of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, 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.

[0166] 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.

[0167] In some embodiments of the present disclosure, a CRISPR/Cas nuclease system includes at least one guide RNA. In some embodiments, 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. In some embodiments, the CRISPR/Cas complex is a Cpfl /guide RNA complex. In some embodiments, the CRISPR complex is a Type-II CRISPR/Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR/Cas9 complex is a Cas9/guide RNA complex.

5. Delivery of guide RNA and Nuclease

[0168] In some embodiments, 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).

[0169] 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. In some embodiments, an RNP containing an RNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) and a gRNA targeting a gene of interest is delivered a cell (e.g.: a B cell). In some embodiments, an RNP is delivered to a B cell by electroporation.

[0170] As used herein, a “AAVS1 targeting RNP” refers to a gRNA that targets the AAVS1 genes pre-complexed with an RNA-guided nuclease. In some embodiments, a AAVS1 targeting RNP is delivered to a cell. In some embodiments, more than one RNP is delivered to a cell. In some embodiments, more than one RNA is delivered to a cell separately. In some embodiments, more than one RNP is delivered to the cell simultaneously.

[0171] In some embodiments, 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. In some embodiments, a gRNA targeting a gene is delivered to a cell as an RNA, or a DNA vector that expresses the gRNA.

[0172] Delivery of an RNA-guided nuclease, gRNA, and/or an RNP may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used.

6. Multi-Modal or Differential Delivery of Components

[0173] Skilled artisans will appreciate that different components of 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.

[0174] 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. For example, 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.

[0175] 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.

[0176] By way of example, the components of a genome editing system, e.g., a RNA- guided nuclease and a gRNA, 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. In an embodiment, 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.

[0177] More generally, in an embodiment, 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.

[0178] In certain embodiments, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.

[0179] In certain embodiments, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

[0180] In certain embodiments, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

[0181] In certain embodiments, 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. As such vectors are relatively persistent product transcribed from them would be relatively persistent.

[0182] In certain embodiments, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.

[0183] In certain embodiments, the first component comprises gRNA, and 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.

[0184] Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.

[0185] Use of 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.

[0186] Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, 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. In an embodiment 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. In an embodiment, 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. In certain embodiments, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody. [0187] When the RNA-guided nuclease molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA and the RNA-guided nuclease molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.

G. Methods of Treatment

[0188] In various aspects of the invention, the adenovirus particles will be delivered as a therapeutic to a patient in need thereof. In various embodiments, 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.

[0189] In other respects, 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. In some embodiments, the pharmaceutical composition further comprises an additional active agent. In various embodiments, said B cells are contacted with an activating agent prior to editing. In some embodiments, said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin. In various embodiments, an activating agent is administered a priori to administering said adenovirus and said recombinant protein. In various embodiments, the patient will be preconditioned prior to administration of said pharmaceutical composition. In various embodiments, the preconditioning step involves an immunization.

[0190] In various aspects of the invention, the edited B cells will be delivered as a therapeutic to a patient in need thereof. In various embodiments, 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.

[0191] In other respects, the invention comprises a pharmaceutical composition comprising edited of B cells as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent. In various embodiments, said B cells are contacted with an activating agent prior to editing. In some embodiments, said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin. In various embodiments, an activating agent is administered a priori to administering said adenovirus and said recombinant protein. In various embodiments, wherein said B cell is edited in vivo, the activation agent is an immunization. In various embodiments, the patient will be preconditioned prior to administration of said pharmaceutical composition. In various embodiments, the preconditioning step involves an immunization.

[0192] In one aspect, pharmaceutical compositions according to the invention are administered to a subject in a dosage sufficient to achieve a therapeutic effect. As used herein “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.

[0193] In some embodiments, the expanded population of engineered B cells are autologous B cells. In some embodiments, the modified B cells are allogeneic B cells. In some embodiments, the modified B cells are heterologous B cells. In some embodiments, the modified B cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo. [0194] As used herein, the term "subject" or “patient” means an individual. In some aspect, a subject is a mammal such as a human. In some aspect, 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.). Preferably, the subject is a human subject. More preferably, the subject is a human patient.

[0195] In various embodiments, 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. In various embodiments the patient has Lysosomal acid lipase deficiency (LAL), Acid sphingomyelinase deficiency or Niemann-Pick disease (ASM), Mucopolysaccharidosis IVA (GALNS). In various embodiments the methods disclosed herein are for the treatment of HIV.

[0196] In additional embodiments, 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. 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. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline. [0197] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. “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. Included among the cytokines are 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); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0198] In still a further embodiment provided herein is 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. In certain aspects, a method for treating a cancer comprises administering an effective amount of a polypeptide (e.g., a glycosylated PD-1 polypeptide) to a subject. In further aspects, 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. In some aspects, 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. In certain aspects, 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 (CMML), childhood leukemia), liver cancer, lung cancer (e.g., non-small cell, small cell), lung carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, naval cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma in a child, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., adult soft tissue cancer), skin cancer (e.g., basal and squamous cell, melanoma, merkel cell), small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor. In certain aspects, the antibody is in a pharmaceutically acceptable composition. In further aspects, the antibody is administered systemically. In particular aspects, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously or locally.

[0199] In some aspects, the method further comprises administering at least a second anticancer therapy to the subject. In certain aspects, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.

EXAMPLES

[0200] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that may be changed or modified to yield essentially similar results.

Example 1 - Targeted In Vivo Gene Delivery

[0201] Cell-specific gene delivery in vivo. This example capitalizes on the unique molecular promiscuities of Ad, as well as its unparalleled ability to achieve efficient in vivo gene delivery, to accomplish highly specific modification of target cells. The triple targeting approach is based upon: (1) transcriptional targeting via tissue selective promoters (TSP); (2) transductional targeting via viral capsid modification to incorporate target cell binding ligands; and (3) liver untargeting via modification of the major capsid protein hexon to mitigate native viral hepatotropism. The combination of these targeting strategies operates in a synergistic manner to allow highly efficient and specific in vivo gene delivery (FIG. 1).

[0202] Here we show data validating in vivo gene delivery achieved for vascular endothelial cell targets. The principles embodied for this specific “vectorology” goal, however, are generalizable. The proof-of-principle underscored by this data is our ability to achieve efficient and selective in vivo gene delivery to a defined target via our vector modification methods (FIG. 2).

[0203] These studies highlight our ability to accomplish highly efficient and selective gene delivery to specific target cells in vivo. The modular design of the triple targeting approach allows for adapting this strategy to other target cells of interest, using these same methods and technologies, which are currently operative in our laboratory.

[0204] Gene delivery to B cells. Initially it was sought to determine gains in B cell gene transfer via adenovirus that could accrue vector capsid engineering. In this regard, lymphocytes are relatively deficient in the primary adenovirus receptor CAR (coxsackie-and- adenovirus receptor). On this basis, vectors with modified tropism, that embody “CAR- independent” gene delivery, have shown benefit for application to otherwise Ad-refractory lymphocytes. One of these modifications that we have defined is chimerism for the adenovirus capsid fiber with replacement of the native fiber knob with the corresponding domain from porcine adenovirus serotype 4 (PK4). These studies thus validate the feasibility of engineering adenovirus to achieve infection of B cells.

[0205] Of note, the levels of in vivo targeting achieved in this initial proof-of-principle data (FIG. 3) allow us to already test the key delivery hypothesis with respect to our vectored immunotherapy approach. Clearly, the vector engineering we propose will yield even further gains vis-a-vis targeted genetic modification of B cells in vivo in our murine model. To extend these principles, vector engineering for gene transfer was applied to human B cell targets (FIG. 4). In these studies, modification of vector binding dramatically enhanced B cell susceptibility to adenoviral vectors. Of further note, the utilization of B cell selective tissue specific promoters (TSPs) facilitated additional enhancements in expression. These studies highlight the facile means by which one can transition our vector design for cross-species application in our models as well as for ultimate human employment.

Example 2 - In Vivo Gene Editing-Based Knock-In for Long Term Gene Expression

[0206] 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. 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. To test if adenoviral-mediated delivery of CRISPR/Cas9 enables gene editing, a vector encoding Cas9 and guide RNA (gRNA) targeted to murine ‘safe harbor’ ROSA26 was produced (Ad5.Cas9- gRNA).

[0207] To determine if editing based knock-in provides long-term expression of a therapeutic serum protein, a human alpha- 1 antitrypsin (hAAT) expression in mice as a model was employeed. Various ratios of Ad5.EFla-hAAT (donor) to Ad5.CMV-EGFP mixtures (sham) or an equivalent amount of Ad5.EFla-hAAT (donor) to Ad5.Cas9-gRNA (CRISPR) were injected into mice (10¹¹ total virus for all mice). Plasma was collected intermittently to determine hAAT levels using a human specific ELISA. Approximately 100 days post-infection, non-integrative groups’ (donor + sham) hAAT levels decreased to levels below their integrative counterparts (donor + CRISPR). This trend continued to a point whereby both the 3: 1 and 1 : 1 Ad5.EFla-hAAT to Ad5.Cas9gRNA mice had significantly higher gene expression than any non-integrative group (FIG. 5 A). At 210 days post-infection, 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).

Additionally, 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). [0208] A similar study achieved genetic correction of a murine model of FIX deficiency hemophilia. Importantly, 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. Importantly, the editing-based knock-in allowed long term expression of the delivered gene with a therapeutic outcome.

Example 3 - Genetic Modification of B Cells to Express bnAbs as a Novel AntiHIV Immunotherapy

[0209] 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. The main distinction between the approaches disclosed herein and the prior art is that the approaches disclosed herein simply replace both the heavy- and light-chain variable genes, and only the HCDR3, with their human bNAb analogues. In contrast to other approaches, our approach does not disrupt the genomic organization or regulation of the BCR locus. Thus, when both approaches are used to introduce the broadly neutralizing antibody (bNAbs) 10-1074, only those in which heavy and light chain were introduced into their respective native loci underwent expansion and affinity maturation necessary to generate potent neutralizing sera (FIG. 7).

[0210] 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. After three immunizations with soluble Env trimers, 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. In that published study, HCDR3 of the V2-glycan/apex bNAbs PG9, PG16, and CHOI, were introduced into human cells. In the murine study, 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. After vaccination, 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). As shown, 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. We then edited mouse primary B cells with the Q100E variant of the VRC26.25 HCDR3 and compared it to the wild-type HCDR3. Sera from 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.

[0211] In summary, robust methods for editing B cells ex vivo have been developed using approaches that retain the natural organization of the B cell receptor genes. These methods allow us to introduce human variable chains into murine BCR, or just the HCDR3 region. When these edited B cells are engrafted into mice and stimulated with appropriate antigen, they expand and undergo extensive hypermutation and affinity maturation, generating sera that protects from or control viruses sensitive to the edited bNAb.

[0212] The data show that: (1) efficient and specific gene delivery in vivo via adenoviral vectors modified via our triple targeting approach can be accomplished; (2) efficient gene transfer to B cell targets in vivo in a murine model can be achieved; (3) adenovirus can be used to achieve in vivo gene editing based upon efficient delivery of CRISPR/Cas9; (4) B cells can be genetically modified to provide a novel means to realize anti-HIV immunotherapy.

Example 4 - Vector retargeting using plug-and-play Adenovirus with molecular glue

[0213] 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. To demonstrate proof-of-concept vector retargeting, we leveraged a Simian Adenovirus 36 vector with SpyTag inserted in the hexon protein (SAd36HSpT) (SEQ ID NO. 6). We first utilized a single domain antibody (sdAb) previously characterized in our lab, F8, which targets murine CD40. We developed a soluble SpyCatcher fusion protein with this sdAb and showed that the purified fusion successfully conjugated to the SAd36HSpT hexon. We assessed gene transfer using primary CD40+ murine B cells and found that gene transfer only occurred in the presence of both SAd36HSpT and F8SpC. We iterated on this design by incorporating a loop-friendly glue, DogTag, into the HI loop domain of the Human Adenovirus 5 fiber (SEQ ID NO. 1), resulting in a vector (Ad5FDgT), which was rescuable to high titers comparable to the control virus. The infectivity of this vector was not impacted by DogCatcher conjugation as gene transfer to murine primary B cells was strongly enhanced by conjugation with the F8 antibody fused to DogCatcher (F8DgC). In vivo we observed that systemically delivered Ad5FDgT conjugated with F8DgC redirected vector tropism from the liver towards endothelial cells in the lung, presumably to a CD40 expressing subset.

[0214] Having demonstrated success with this sdAb system, we next hypothesized that we might extend the targeting concept to other antibody species. To this end, we developed single-chain variable fragment antibodies targeting mouse CD 19 and CD20 and human CD 19 (1D3, 18B12 and FMC63, respectively) as DogCatcher fusions, and found that like the sdAb system, they could be conjugated to the Ad5FDgT capsid with high efficiency. In both mouse and human primary B cells, delivery of the conjugated vector resulted in dramatic enhancement of gene transfer. See, FIG. 10.

[0215] Next, we developed a system to differentially target either B cells or T cells using the same DogTag molecular glue peptide, using either a DogCatcher specific to B cells (F8Dgc), or a catcher specific to T cells (YTS169DgC). The DogTag was inserted into the virus fiber protein at the HI loop domain to form Ad5FDgT, while antibodies, sdAB (F8DgC, specific to B cells) or scFv (YTS169DGC, specific to T cells) were expressed as fusions with DogCatcher partner protein. See FIG 17. Adenoviruses encoding GFP and mixed with F8DgC preferentially expressed GFP in B lymphocytes, where adenoviruses mixed with YTS169DgC did not. FIG. 18A. To the contrary, adenoviruses encoding GFP and mixed with YTS169DgC preferentially expressed GFP in T lymphocytes, where adenoviruses mixed with F8DgC did not. FIG. 18B.

Example 5 - Development of transcriptionally targeted Ad vectors [0216] Cellular therapy using genetically modified T lymphocytes (CAR-T cells) has resulted in several clinical successes in the treatment of cancer, generating interest in using other cell types as therapies. Several alternatives have been explored, including NK cells, non-canonical T cells such as y5 T cells, and others. In recent years, B lymphocytes (B cells) have become the subject of active investigation. B cells are part of the adaptive immune system and are defined by the expression of a membrane tethered antibody, known as the B cell receptor or BCR. Upon binding of the BCR to a cognate antigen in conjunction with various co-stimulatory signals, B cells proliferate and differentiate into memory B cells and plasma cells. These cell populations possess several features that are critical to pathogen control, namely 1) the capacity to be maintained throughout the life of the organism, 2) the ability to secrete large amounts of antibodies, and 3) the ability to rapidly respond to antigen re-exposure via massive proliferation of memory B cells and production of antibodies.

[0217] These studies have largely leveraged ex vivo techniques to achieve BCR modification. Of note, 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. In contrast, 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.

[0218] With these goals in mind, the use of Adenoviral vectors (Ads) to achieve gene transfer to B cells in vivo. Ads possess several features that make them well suited for in vivo cell modification was explored, including their large packaging capacity (up to 35kB), their ability to be rationally engineered to infect targeted cell types, and their proven safety record in the clinic with systemic and local administration. Additionally, the SARS-CoV-2 pandemic demonstrated the ability of the current infrastructure to produce Ads at a global scale at low cost, making them an ideal vector for infectious disease control. In this study, we genetically engineered Ads for gene transfer to B cells and demonstrated their ability to achieve highly specific gene expression in vivo.

[0219] Results. A “triple targeting” strategy for the generation of highly targeted vectors was developed, which involves 1) fiber modifications to enhance gene transfer to the cell type of interest, 2) the use of a tissue specific promoter, and 3) liver un-targeting of the vector through hexon modifications. To initiate development of such a system for B cells, we first screened several vectors developed in our lab for their ability to infect both resting and LPS activated murine primary B cells. We found that the previously described vectors AdRGD and AdPK4, which infect cells through integrins and glycans, respectively, were both capable of enhancing gene transfer into B cells compared to the Ad5 control (FIG. 14). We selected the AdRGD backbone for further development due to its well-characterized cell entry biology, ease of upscale, and previous use in the clinic²⁴.

[0220] 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). (FIG. 11 A). We generated and attempted to rescue and upscale AdRGD vectors with each promoter driving eGFP from the El region of the vector. We were able to purify vectors containing all the described promoters at similar yields to the cytomegalovirus promoter (CMV) control.

[0221] We initially assessed these vectors ex vivo in activated primary mouse and human B cells and found that vectors carrying the targeted promoters were all capable of driving reasonable gene expression, often > 50% of the CMV control (FIG. 1 IB, C). Interestingly, in human B cells a much higher concentration of viral particles were required to achieve similar gene expression. This may be due to an intrinsic difference between the cells, or due to differences in the activation and culturing protocols used (LPS in mouse versus CD40 + interleukins + CpG DNA in human). Importantly, no vector showed signs of toxicity in mouse or human cells even at high MOIs, at least based on flow cytometry viability dye binding. In both mouse and human cells, the EEK promoter appeared the standout targeted promoter, having the strongest gene expression of the group. We therefore developed a fully triple targeted vector based on this backbone, inserting the previously described H5/H3 hexon modification for liver un-targeting.

[0222] We next injected C57BL/6J mice retro-orbitally with IxlO¹¹ vp/mouse and harvested major organs (liver, spleen, lungs, heart and kidneys) 72 hours post-injection. One- half of each spleen was assessed for B cell eGFP expression using flow cytometry, while the other half and remaining organs were assessed for tissue gene expression using a quantitation assay (FIG 12A). In vivo, we were surprised to find that the Ad5.CMV control achieved similar B cell gene expression levels to the targeted AdRGD.CMV vector - additionally, quantitative tissue data showed no difference between either vector (FIG 12B-D). As expected, each vector showed strong gene expression in the liver, followed by the spleen, lung, heart and kidneys (FIG. 12D). In contrast, the AdRGD.EEK vector showed significantly reduced gene expression in B cells - a roughly 6-fold decrease compared to AdRGD.CMV. However, we observed dramatically reduced eGFP expression in off-target tissues, including near-undetectable expression in the liver, ultimately resulting in a much higher targeting ratio. We also assessed gene expression in T cells via flow cytometry. Although we did detect eGFP+ cells in the AdRGD.EEK group, the MFI was much lower than in control groups. We did not observe any differences between the AdRGD.EEK and AdH5/H3RGD.EEK vectors, potentially indicating the liver sequestration of vector particles does not impact gene transfer in the spleen, at least to B cells.

[0223] Next we decided to test the remaining promoters in vivo as well. We selected a lower virus dose, 5xlO¹⁰ vp/mouse, hypothesizing that the similarity in gene expression Ad5.CMV and AdRGD.CMV may have been due to saturation of the mouse with the high virus dose. We saw similar results to our previous assay, with Ad5.CMV and AdRGD.CMV showing very similar gene expression profiles in B cells and elsewhere (FIG. 12E-F). 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.

[0224] Analysis of the mechanism of gene transfer to B cells. Intrigued that the AdRGD.CMV and control Ad5.CMV vectors showed comparable in vivo gene transfer to B cells, we hypothesized that Ad mediated gene transfer to B cells might be driven by virus-cell interactions different than the classic fiber protein-receptor pathway. In this regard, 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. We hypothesized that 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. [0225] To assess this hypothesis, 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. However, the magnitude of this response was much lower than in A549 cells, which express CAR and bind Ad5 through fiber-receptor interactions (FIG. 13B). We did not observe a similar decrease with the AdRGD.CMV vector as expected. We also infected murine B cells and A549 cells with Ad5E3gfpFFc3.1uc, which contains a premature stop codon prior to the fiber knob region, resulting in a vector completely incapable of binding cells through CAR (FIG. 13C). In B cells this vector was nearly as infective as the Ad5.CMV control, but in A549 the vector was almost incapable of gene transfer. Finally, to determine directly if integrins are responsible for Ad5 gene transfer to B cells, we pre-incubated cells with varying amounts of an integrin blocking peptide, GRGDSP. We observed a dose-dependent reduction in gene transfer, which was not observed when cells were pre-incubated with GRADSP, a non-binding irrelevant peptide control.

[0226] We also attempted to indirectly analyze the mechanism of gene transfer in vivo using flow cytometry. We assessed B cells for av and P3 integrin expression after vector delivery and found that these populations were highly enriched with eGFP+ cells compared to the overall CD 19+ group (FIG. 13E). This effect was especially pronounced in the av group, suggesting a strong correlation between expression of this receptor and susceptibility to gene transfer from the Ad vectors.

[0227] Immunophenotyping of B cells. We next asked if certain B cell sub-populations in the spleen might be more susceptible to Ad gene transfer. We developed a flow cytometry panel to assess marginal zone, follicular, germinal center, and memory B cells, as well as plasmablasts. We also included a marker for proliferation, hypothesizing that actively growing B cells might be more likely to express the reporter gene. Compared to the overall CD 19+ group, we found enriched eGFP+ cells, in order, in the plasmablast, germinal center, proliferating, and marginal zone groups. We did not observe any major differences between Ad5.CMV and AdRGD.CMV in these groups. However, AdRGD.EEK possessed a much higher ratio of eGFP+ cells in the germinal center and plasmablast groups compared to Ad5.CMV or AdRGD.CMV. We also compared expression of individual markers in eGFP+ and eGFP- B cells and found that the eGFP+ population was highly enriched for CD95 expression and moderately enriched for GL7 and CD38 expression. This population also showed lower levels of IgD in comparison to the eGFP- group.

[0228] In this study we developed and characterized 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. In 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.

[0229] In vivo, we found that the lead promoter modified vector, AdRGD.EEK, showed significantly weaker eGFP expression in all B cells (in terms of number of cells expressing the reporter and mean fluorescence intensity) than the control groups, possibly due to its dependence on immunoglobulin production (discussed further below). However, in off-target tissues including the liver we were unable to detect any significant eGFP expression, highlighting the selectivity of the promoter. Similar results were obtained for the other tissuespecific promoters used in this study. This selectivity may be especially critical for in vivo cell engineering applications where off-target expression of the gene editing machinery is undesirable. The low number of B cells expressing the gene of interest may be overcome through a boosting strategy as described by other groups whereby vaccination induces B cell expansion, or by further vector engineering strategies to enhance B cell gene transfer, such as the penton modifications suggested above.

[0230] Interestingly, we did not find that incorporating the H5/H3 hexon modification increased the number of eGFP+ B cells in vivo, counter to our hypothesis that reducing liver viral particle sequestration might improve splenocyte gene transfer. Alternatively, our phenotyping data indicates that expression of the viral transgene is correlated with B cell activation status. It is possible that increased numbers of B cells were infected by the H5/H3 modified vector but did not express eGFP.

[0231] Similarly, when delivered systemically in vivo, we found that the standard Ad5.CMV vector and the AdRGD.CMV vector achieved the same levels of gene transfer to B cells. This result was surprising to us, given that historical studies have indicated that the murine spleen and B cells express very low or no levels of the receptor used by Ad5, CAR. We therefore attempted to determine the mechanism of gene transfer to B cells by Ad5 using a variety of methods. We found that gene transfer was only slightly reduced by pre-treatment of B cells with recombinant fiber, and that a vector totally deficient in its ability to bind CAR was nearly as infectious as the control, potentially indicating that the primary mode of gene transfer is fiber-independent. In comparison, blocking murine B cells with a synthetic peptide that binds to integrins showed a strong dose-dependent reduction in gene transfer. This data correlates with our finding that eGFP+ B cells in vivo are highly enriched for av and P3 integrin expression. Furthermore, one study previously reported that penton base mutated vectors lacking the binding RGD motif accumulated much less in the spleen than the control, demonstrating that this motif is critical for splenic gene transfer in vivo.

[0232] In total, these data suggest that Ads infect B cells primarily through the penton base protein in vivo. Further engineering efforts may focus on penton base engineering to replace the RGD motif with other motifs targeting integrins expressed exclusively and at high levels on B cells. Modification of the penton base with a peptide binding integrin oc4[31 has already been described, making this a high priority target for further vector engineering. However, we cannot rule out additional mechanisms influencing gene transfer both in vivo and ex vivo. Although the majority of B cells expressing eGFP in vivo did show high levels of av integrin, a significant population were negative, indicating there may be other mechanisms of virus sequestration occurring - for example, natural IgMs are known to bind Ad5 in naive mice. Tissue macrophage populations are believed to then interact with the IgM bound virus through various receptors. In particular, 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. This finding is in alignment with immunohistochemical imaging of the spleen after virus administration - although we were able to detect rare populations such as germinal center B cells expressing eGFP using flow cytometry in this study, it is clear that the vast majority of eGFP+ splenocytes are in the marginal zone (FIG. 15). This pathway may thus represent another mechanism by which B cells are infected in vivo.

[0233] We also assessed the phenotype of B cells expressing eGFP after in vivo vector delivery. We found several populations were enriched for eGFP+ cells compared to the whole B cell group, including plasmablasts, germinal center B cells, and marginal zone B cells, and that eGFP+ B cells generally appeared to possess an activated phenotype, including strong enrichment of GL7 and CD95 markers. A straight-forward hypothesis is that activated and/or rapidly proliferating B cells are more susceptible to either Ad virus uptake or transgene expression. Such a phenomenon has been shown with adeno-associated vectors, and our own preliminary data indicates culturing B cells with activating agents strongly enhances gene expression (FIG. 15). This finding is important for our future goals of in vivo B cell engineering, as homology directed repair generally requires the cell to be proliferating in the S/G2 cell cycle phases. However, it is also possible that Ad infection triggers cellular pathways in B cells that lead to an activated phenotype. To assess these hypotheses in the future, we aim to pre-condition mice via immunization prior to vector delivery and assess if the number of infected B cells increases.

[0234] We found few if any qualitative differences between B cell sub-sets infected by Ad5.CMV or AdRGD.CMV or markers expressed by eGFP+ cells infected by these vectors. However, we did observe clear differences between those groups and the AdRGD.EEK group, which showed a much higher ratio of plasmablast and germinal center eGFP+ cells compared to all B cells than Ad5 or AdRGD.CMV. 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.

[0235] In total, we found that 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.

[0236] Methods. Cells. HEK293 (ATCC CRL-1573, Manassas, VA) and A549 (ATCC CCL-185, Manassas, VA) 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 (Gibco 11330-032, Waltham, MA). Cells were grown at 37C with 5% CO2 under sterile conditions.

[0237] 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.

[0238] To facilitate rapid cloning of fiber and promoter modified vectors, we utilized an Ad backbone with unique restriction sites surrounding the fiber (JC201 : pMVP/Ad/5-35- DEST was a gift from Christopher Newgard (Addgene plasmid # 121847; http://n2t.nct/addgene: 121847; RRID:Addgene_121847, Watertown, MA))⁴⁰. We made several modifications to this backbone to further suit our purposes. We restored the U exon region downstream of the fiber by digesting the backbone with Srfl and SgrDI (New England Biolabs, Ipswich, MA) and inserting the corresponding genomic region with the U exon from pAdEasy 1 via NEB HiFi DNA Assembly (New England Biolabs, Ipswich, MA), as U exon deficient vectors have been shown to have reduced yields⁴¹.

[0239] We then replaced the El region with a synthetic spacer region flanked by unique Swal/Clal restriction sites. We accomplished this swap by cloning the spacer (synthetized by Integrated DNA Technologies, Coralville, IA) into pShuttle via NEB HiFi DNA Assembly. This plasmid was then linearized via Pmel (New England Biolabs, Ipswich, MA) and swapped into the Ad genome via homologous recombination in BJ5183 bacteria (Agilent, Santa Clara, CA). Digestion of the vector backbone with either enzyme thus released the spacer region and allowed Gibson Assembly of desired DNA fragments directly into the vector genome.

[0240] We also modified the fiber region by digesting the backbone with Bari (SibEnzyme, Russia) and BstBI (New England Biolabs, Ipswich MA). Standard HAdV-C5 fiber was PCR amplified from pAdEasy 1 and assembled into the digested backbone via NEB HiFi DNA Assembly. The AdRGD fiber was assembled with the backbone in the same manner after amplification from the previously described backbone. These new plasmids were termed “prAd5” and “prAdRGD” or plasmid rapid Ad5/AdRGD

[0241] 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.

[0242] The ubiquitous cytomegalovirus (CMV) promoter was used as a control and was amplified from the previously described pTrackCMVLuc³⁸. The spleen focus forming virus (SFFV) promoter was obtained from pHR-SFFV-dCas9-BFP, a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid # 46910; http://n2t.nct/addgene:46910; RRID:Addgene_46910, Watertown, MA)⁴². The synthetic promoters EEK and MH have been described previously and were synthesized by Integrated DNA Technologies (Coralville, IA)²⁶. The 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.

[0243] Primary B Cell Culture. Isolation and culture of murine primary B cells has been described previously. Briefly, spleens were crushed on a 70 pm filter using a syringe plunger and washed through using IX PBS + 2% FBS (B cell isolation media, BCIM). Cells were pelleted via centrifugation at 1200 rpm or 500g for 5 minutes and resuspended in 3mL ACK Lysis Buffer (Gibco Al 0492-01, Waltham, MA). After 3 minutes incubation 7mL BCIM was added, and cells were pelleted again. Cells were resuspended in 3mL BCIM and washed through a second 70 pm filter to obtain a single cell suspension. B cells were purified from this suspension using a magnetic isolation kit according to the manufacturer’s instructions (Miltenyi 130-090-862, Gaithersburg, MD).

[0244] Purified murine B cells were cultured and activated at 37C for 30h prior to virus infections. Unless otherwise indicated, infections were conducted by resuspending 5xl0⁵ 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.

[0245] Human primary B cells were obtained from healthy donors. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from LRS chambers and frozen in liquid nitrogen prior to B cell isolation. B cells were then magnetically isolated from 1.5xl0⁸ 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 2.5xl0⁵ 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.

[0246] Flow Cytometry. For ex vivo analysis, murine B cells were stained with CD19 (Invitrogen RM7717, Waltham, MA) and Sytox Red or Fixable Far Red stains for live/dead discrimination (Invitrogen S34859 and L10120, Waltham, MA). Human B cells were stained with CD19 (BioLegend 302251, San Diego, CA) and Fixable Far Red stain.

[0247] For in vivo studies, 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. 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-.

[0248] 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 integrin staining experiments.

[0249] Animal Studies. 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 IxlO¹¹ or 5xl0¹⁰ 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).

Example 6 - Development of molecular glue driven antibody conjugation to the virus capsid

[0250] 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.

[0251] 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.

[0252] It was next shown that this gene transfer enhancement only occurred when the specific combination of virus modified with SpyTag and antibody modified with SpyCatcher was present, and that this enhancement was reduced by the presence of competing antibody (FIG. 23B and 23C). It was found in a pilot study in vivo that SAd36 and its derivatives were completely unable to achieve gene transfer to splenic B cells (FIG. 24). Next an Ad5 vector was designed incorporating molecular glue. Furthermore, concurrent with this work a study describing Ads engineered with the loop-friendly molecular glue “DogTag” was published. It was thus elected to move forward with the design of a vector incorporating DogTag in the Ad5 fiber protein (Ad5FDgT). 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).

[0253] To pair with this vector, several antibodies were developed as DogCatcher fusions. The previously mentioned F8 anti-mCD40 sdAb was initially developed as a DogCatcher fusion, but also developed single-chain fragment variable (scFv) fusions of commercial antibodies targeting murine and human B and T cell markers, including CD 19, CD20, and CD8a (FIG. 19C). To assess if these reagents were able to conjugate the virus fiber capsid protein, Ad5FDgT and different antibody fusions were co-incubated at room-temperature for approximately 2 hours, then ran SDS-PAGE gels to assess the degree of shifting in the fiber protein. It was found that the DogTag-DogCatcher pair was highly reactive and that all or nearly all of the fiber-DogTag protein reacted with the antibody fusions (FIG. 19E). Furthermore, these antibodies retained their ability to bind to the appropriate cell type, as determined by flow cytometry (FIG. 19F).

Example 7 - In vitro and in vivo analysis of Adenovirus-antibody complexes

[0254] 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. In all cases a variety of molar ratios of antibody-DogCatcher to virus-DogTag were tested, and often observed a bell curve where very high and very low ratios of antibody resulted in lower gene transfer enhancements. This is consistent with the idea that too much antibody fragment may act as a competitive inhibitor, but too little antibody may not saturate the binding sites on the virus. Several notable exceptions to this bell curve rule were observed, especially in the human B cell samples where the highest ratio of antibody-DogCatcher to virus-DogTag resulted in the highest gene transfer enhancement. Some antibodies might require higher excesses to achieve full saturation of the virus surface. Further work is needed to fully elucidate this effect. Our results were particular striking in murine T cells and human B cells, where Ad5FDgT without enhancing antibody was almost completely incapable of gene transfer.

[0255] Next it was determined if antibody conjugation could enhance specific gene transfer in vivo. C57BL/6J fully immunocompetent mice were injected retro-orbitally with Ad5FDgT alone or Ad5FDgT conjugated with lD3DgC, an scFv targeting murine CD 19, a B cell restricted cell marker. Splenic B and T cells were scored for reporter gene expression three days later, and found a statistically significant enhancement of gene transfer in the B cell compartment, but not in the T cell compartment (FIG. 2 IB). This enhancement was found across several of the B cell subpopulations that were scored, including memory B cells, marginal zone B cells, and follicular B cells. No enhancement was observed in the plasmablast population, which may be due to lower CD 19 expression on this cell type. Interestingly, a non- statistical trend towards increased gene transfer in splenic T cells, as well as a non-statistical trend towards decreased liver gene transfer, and a significant trend in decreased overall spleen gene transfer was observed (FIG. 21C). The increased T cell infection may therefore be due to less virus sequestration in sites such as the liver and splenic macrophages.

Example 8 - Development and analysis of purified single-component Ad-Ab complexes:

[0256] Taken together, the data suggested that Ad-Ab complexes were a promising platform for controlling gene transfer and expression both in vitro and in vivo. However, for clinical translation and industrial production 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). At the lab scale, 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.

[0257] Several assays were carried out to determine the quality of these purified Ad-Ab complexes. Encouragingly, SDS-PAGE analysis revealed that the antibody-conjugated samples retained an identical protein band pattern to the PBS treated vector, with the exception of the expected complete shift in the fiber-DogTag band (FIG. 21C). Critically, we also were unable to observe any signs of free antibody. This result was further confirmed by a western blot analysis of the fiber protein, which revealed that the vast majority of fiber-DogTag reacted with the antibody -DogCatcher fusions (FIG. 2 ID). In vitro analyses of these viruses were carried out and found that gene transfer enhancement from the purified and stored viruses was comparable to freshly prepared Ad-Ab complexes (FIG. 25). [0258] Interestingly, it was observed that different antibodies required different molar ratios of DogTag:DogCatcher to achieve optimal gene transfer (e.g., lD3DgC was most potent in the 10X-2.5X range while YTS169DgC was most potent at 0.625X - see FIG. 20). With purified Ad5FDgT completely functionalized with YTS169DgC, we observed that gene transfer was lower than in our previous experiments with a 0.625X DogTag:DogCatcher ratio. These results indicate that, at least for mCD8a targeting antibodies, an un-saturated capsid is superior to a saturated one. The Ad fiber presents as a trimer, with three copies of DogTag displayed in close proximity. Full functionalization of this complex apparently results in lower gene transfer, at least in these contexts.

[0259] Next, it was endeavored to assess these purified complexes in vivo (FIG. 2 IE). As before, injecting C57BL/6J mice retro-orbitally with either non-conjugated Ad5FDgT, Ad5FDgT-lD3, or Ad5FDgT-YTS169. Three separate experiments were conducted first in female mice, then male, and finally a mixed group. It was again found that functionalization of the virus with lD3DgC resulted in a roughly two-fold increase in gene expression to B cells compared to the parent vector. It was found a similar enhancement in the CD8a+ T cell population, with YTS169DgC functionalization resulting in an approximately two-fold increase in gene expression. Critically, neither modification resulted in an increase in gene expression in off-target cell types - lD3DgC functionalization did not increase gene expression in CD8a+ T cells, and YTS169DgC did not increase gene expression in B cells. It was also not observed any statistical differences in groups separated by sex, confirming that our technology is applicable in both male and female mice (FIG. 26).

[0260] Interestingly, a slight, non-significant increase in gene expression in CD4+ T cells in mice injected with Ad5FDgT-lD3 and Ad5FDgT-YTS169 was observed. The potential mechanism of this result is not clear - activated CD4+ T cells do express an Fc receptor which could conceivably bind to the antibodies used to functionalize our vector, but our study uses scFvs lacking any Fc receptor binding. Another explanation could be that incorporation of scFvs into the fiber increases the circulation time of the virus and allows slight increases in transduction of off-target cells. There may also be as-of-yet uncharacterized interactions between the various hematopoietic cells and scFvs or DogCatcher itself that are responsible. Regardless, if the increase in gene expression in CD4+ T cells is not an artifact it is ultimately very weak, not rising to the level of statistical significance, and is likely not a concern.

[0261] To further characterize the distribution of gene expression in mice injected with targeted or non-targeted vectors, 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).

Methods for Examples 6-8

[0262] Cell Lines:

[0263] 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.

[0264] Viruses:

[0265] 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). These three fragments were assembled with the cut 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.

[0266] Protein Constructs:

[0267] 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”. [0268] Recombinant Protein Production:

[0269] 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. Cultures were centrifuged, and cell pellets were resuspended in 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) and incubated at 37 °C for 30 minutes. The cell lysates were clarified by centrifugation at 32,000 ref at 4 °C for 30 minutes.

[0270] For mammalian recombinant protein production, 80-90% confluent 293T cells were transfected with pcDNA3.4-based protein expression plasmids in the presence of transporter 5 reagent (Polysciences, Inc.). The transfected cells were cultivated in DMEM medium containing 10% fetal bovine serum for 4 to 6 hours and switched to FreeStyle 293 medium for additional 4 to 5 days. The culture supernatants were collected and concentrated with Amicon Ultra- 15 with 10K NMWL.

[0271] 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).

[0272] SDS-PAGE and Western Blot:

[0273] Whole virus protein analysis and conjugation analysis was carried out using SDS- PAGE. Viruses and proteins were incubated at a 2: 1 ratio with 3x SDS sample buffer containing 187.5 mM Tris-HCl, 6% SDS, 30% glycerol, 0.125M dithiothreitol and 0.03% bromophenol blue at pH 6.8 for 15 minutes at 100 °C. Samples were then loaded on a 4-15% gradient gel and resolved using a Criterion electrophoresis system (Bio-Rad). Staining was carried out using GelCode Blue according to the manufacturer’s protocol (Thermo Scientific #1860957).

[0274] Western blot analysis was carried out to detect the Ad5FDgT fiber protein and its modified/conjugated derivatives. The samples containing about 6 x toi l viral particles were mixed 1 : 1 with the 2X Laemmli SDS-PAGE loading buffer containing 2-mercaptoethanol (Millipore Sigma #S3401) and heated in boiling water for 5 minutes to denature the viral proteins. The denatured samples were run on 4 - 20% Tris-Glycine Mini protein gel (Invitrogen #XP04202BOX) using Novex Tris-Glycine SDS Running Buffer (Invitrogen #LC26755) as recommended by the manufacturer. 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.

[0275] Primary Cell Culture:

[0276] Primary mouse and human B cell culture and infection was carried out as previously described by our group. Briefly, 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. [0277] 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.

[0278] 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.

[0279] Virus Conjugation:

[0280] For in vitro experiments, 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.

[0281] For in vivo experiments, the required amount of virus for all injections was incubated with the required amount of antibody without further dilution for 2 hours at room temperature or 25 °C. IX PBS was then added to bring the conjugated virus to the appropriate final volume.

[0282] Flow Cytometry:

[0283] For analysis of antibody-DogCatcher fusions, splenocyctes isolated from C57B1/6J mice (about 1 million cells) 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.

[0284] For analysis of in vitro infected murine B cells, samples were stained with anti- CD19 (Invitrogen #RM7717) and Fixable Far Red live/dead dye (Invitrogen #L10120). Samples were gated as singlets/live/CD19+. Human B cells were stained with anti-CD19 (BioLegend #302251) and Fixable Far Red dye. Samples were gated as above. Mouse T cells were stained with anti-CD8a (BioLegend #100762) and Fixable Far Red dye or Sytox Red dye ( Invitrogen #S34859). Samples were gated as singlets/live/CD8a+.

[0285] For in vivo studies, spleens were processed into single cell suspensions and resuspended in ImL IX PBS with 2% FBS. IOOUL of the suspension was used for analysis and was pre-incubated with 10 pL Fc Blocking Reagent (Miltenyi 130-092-575) for 10 minutes prior to staining. In all cases live/dead discrimination was carried out using Fixable Far Red dye. For the first in vivo assay (Fig. 3), B cells were gated as singlets/live/CD19+/CD3-, while T cells were gated as singlets/live/CD3+/CD19-. 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.

[0286] For the second in vivo assay (Fig. 4), 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-.

[0287] 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).

[0288] Animal Studies:

[0289] 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).

[0290] In the second in vivo study, the whole spleen of each animal was used for flow cytometry and whole livers were used for tissue eGFP quantitation as above. All experiments were approved by the Institutional Animal Care and Use Committee of the Washington University in St. Louis School of Medicine (Protocol #22-0360) and were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. All efforts were made to minimize suffering and the total number of animals used in the study.

[0291] Purification of Ad- Ab complexes:

[0292] Cells from 20X infected T175 flasks were harvested after the development of cytopathic effect and centrifuged at 1200 rpm for 5 minutes. Supernatants were removed and cell pellets were frozen at -80 °C. Samples were subjected to 3 freeze-thaw cycles in roomtemperature water and dry ice to lyse the cells, then centrifuged at 4000 rpm for 10 minutes to clarify the lysates. Supernatants were then ultracentrifuged on cesium chloride gradients for 2 hours at 25,000 rpm at 4 °C. Viral bands were harvested and dialyzed twice against IX PBS. Virus was removed from dialysis and viral particles were quantified as above. 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.

[0293] Statistical Analyses:

[0294] Specific methods used for analysis are noted in the corresponding Figure legends. In all cases statistical analysis was carried out using GraphPad Prism 10. A p value of < 0.05 was used and significance is indicated as *: p < 0.0322, **: p < 0.0021, ***: p < 0.0002, **** p < 0.0001, ns: not significant. [0295] Viruses and Recombinant Proteins:

[0296] Simian Adenovirus 36 (SAd36) SpyTag derivatives have been described previously. All single-domain antibody fusions and single-chain fragment variable antibody fusions were produced in bacteria as described in the main. Conjugations were carried out as described in the main.

[0297] In vivo pilot study:

[0298] 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.

[0299] Tissue Immunohistochemistry:

[0300] C57B1/6J 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. Frozen section slides were air-dried for 10 min, washed three times in PBS, blocked with protein block solution (5% donkey serum and 0.1% Triton X-100 in PBS) for 1 h, incubated at room temperature in protein block containing chicken anti-GFP 1 :400 (A10262, ThermoFisher) for 1 hour, washed three times in PBS, incubated with corresponding Alexa Fluor 488-conjugated secondary antibody 1 :400 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and counterstained with SlowFade Gold Antifade mounting reagent with 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies). Immunofluorescence images were collected using an Olympus BX63 microscope equipped with an FVII digital camera (Olympus America, Center Valley, PA, USA).

Example 9 Use of off-the-shelf whole antibodies to functionalize adenovirus for targeted cell delivery.

[0301] Off-the-shelf whole antibodies were used to functionalize adenoviruses for targeting specifically to B cells using the strategy described in FIG. 28. The fiber nob of the adenoviral particles was replaced with an Ad5.FiberDogTag. The modified adenoviral particles were incubated with a DogCatcher-AbBD fusion protein. The AbBD domain of the DogCatcher-AbBD fusion protein comprised either pZQ32 or pGA24 — both protein fragments known to target the Fc domain of native antibodies. The AbBD incorporated photocrosslinking amino acids (pZQ32BPA or pGA24BPA) for covalent attachment to the IgG residues of the native antibody. 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). [0302] 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.

SEQ ID NO: 1

Ad5 Fiber-HI-DogTag

MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLSLRLSEP LVTSNGMLALKMGNGLSLDEAGNLTSQNVTTVSPPLKKTKSNINLEISAPLTV TSEALTVAAAAPLMVAGNTLTMQSQAPLTVHDSKLSIATQGPLTVSEGKLAL QTSGPLTTTDSSTLTITASPPLTTATGSLGIDLKEPIYTQNGKLGLKYGAPLHVT DDLNTLTVATGPGVTINNTSLQTKVTGALGFDSQGNMQLNVAGGLRIDSQNR RLILDVSYPFDAQNQLNLRLGQGPLFINSAHNLDINYNKGLYLFTASNNSKKL EVNLSTAKGLMFDATAIAINAGDGLEFGSPNAPNTNPLKTKIGHGLEFDSNKA MVPKLGTGLSFDSTGAITVGNKNNDKLTLWTTPAPSPNCRLNAEKDAKLTLV LTKCGSQILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDPEYW NFRNGDLTEGTAYTNAVGFMPNLSAYPKSHGKTAKSNIVSQVYLNGDKTKP VTLTITLNGTQETGDTTGSGGSGDIPATYEFTDGKHYITNEPIPPKGSGGSGPS

AYSMSF S WDW SGHN YINEIF AT S S YTF S YIAQE

SEQ ID NO: 2

Ad5 FiberY477A-HI-DogTag

MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLSLRLSEP LVTSNGMLALKMGNGLSLDEAGNLTSQNVTTVSPPLKKTKSNINLEISAPLTV TSEALTVAAAAPLMVAGNTLTMQSQAPLTVHDSKLSIATQGPLTVSEGKLAL QTSGPLTTTDSSTLTITASPPLTTATGSLGIDLKEPIYTQNGKLGLKYGAPLHVT DDLNTLTVATGPGVTINNTSLQTKVTGALGFDSQGNMQLNVAGGLRIDSQNR RLILDVSYPFDAQNQLNLRLGQGPLFINSAHNLDINYNKGLYLFTASNNSKKL EVNLSTAKGLMFDATAIAINAGDGLEFGSPNAPNTNPLKTKIGHGLEFDSNKA MVPKLGTGLSFDSTGAITVGNKNNDKLTLWTTPAPSPNCRLNAEKDAKLTLV LTKCGSQILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDPEAW NFRNGDLTEGTAYTNAVGFMPNLSAYPKSHGKTAKSNIVSQVYLNGDKTKP VTLTITLNGTQETGDTTGSGGSGDIPATYEFTDGKHYITNEPIPPKGSGGSGPS

AYSMSF S WDW SGHN YINEIF AT S S YTF S YIAQE

SEQ ID NO: 3

Ad5 fiber-fibritin-DogTag

MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLSLRLSEP LVTSNGMLALKMGNGLSLDEAGNLTSQNVTTVSPPLKKTKSNINLEISAPLTV TSEALTVAAAAPLMVAGNTLTMQSQAPLTVHDSKLSIATQGPLTVSEGKLAL QTSGPLTTTDSSTLTITASPPLTTATGSLGIDLKEPIYTQNGKLGLKYGAPLHVT DDLNTLTVATGPGVTINNTSLQTKVTGALGFDSQGNMQLNVAGGLRIDSQNR RLILDVSYPFDAQNQLNLRLGQGPLFINSAHNLDINYNKGLYLFTASNNSKKL EVNLSTAKGLMFDATAIAINAGDGLEFGSPNAPNTNPLKTKIGHGLEFDSNKA MVPKLGTGLSFDSTGAITVGNKNNDKGSGYIPEAPRDGQAYVRKDGEWVLL STFLSPAGGGGSGGGGSGGGGSDIPATYEFTDGKHYITNEPIPPK

SEQ ID NO: 4

Ad5-HVR5-DogTag MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVA PTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDI RGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATGGGGSGGGGSRGVP HIVMVDAYKRYKGGGGSGGGGSQQKTHVFGQAPYSGINITKEGIQIGVEGQT PKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNEN GGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLYSEDVDIETP DTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVL AGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVR IIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEI RVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYM NKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGR YVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGA SIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPAN ATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTF YLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKD WFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQ QVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCD RTLWRIPF S SNFMSMGALTDLGQNLL YANS AHALDMTFEVDPMDEPTLL YV LFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT*

SEQ ID NO: 5

Ad5-HVRl-G4S2-SpyTag

MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDV TTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTF KPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVF GQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKT TPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSGSGGSGDIPATYEFTD GKHYITNEPIPPKGSGGSGPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSM PNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDS IGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKT GQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSP SNVKISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLR YRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGND LRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIP ANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLN HTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQML ANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSG FVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGA LTDLGQNLL YANS AHALDMTFEVDPMDEPTLL YVLFEVFDVVRVHQPHRG VIET VY LRTPFSAGNATT*

SEQ ID NO: 6

Ad5-HVR7-SpyTag

MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVA PTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDI RGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNED EVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGES QWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLES QVEMQFFSTTEAAAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRE LMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQD RNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFP LGGVINTETLTKVKGSGGSGRGVPHIVMVDAYKRYKGSGGSGWEKDATEFS DKNEZRVGNNFAMEFNLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNT YDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSML LGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDL

RVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANML YPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPY LDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQC NMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTK YKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQK KFLCDRTLWRIPF S SNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEP TLL YVLFEVFDVVRVHQPHRG VIET VYLRTPFSAGN ATT*

SEQ ID NO: 7

Ad5-pIX-SpyTag

MSTNSFDGSIVS S YLTTRMPPWAGVRQNVMGS SIDGRPVLP ANSTTLTYET VS GTPLETAASAAASAAAATARGIVTDFAFLSPLASSAASRSSARDDKLTALLAQ LDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVSADYKDDDDKLEETRARL SKELQAAQARLGADMEDVCGRLVQYRGEVQAAAGSGGSGRGVPHIVMVDA YKRYKSG*SAd36-HVR5-SpyTag

SEQ ID NO: 8

SAd36-FF-SpyTag

MSKKRVRVDDDFDPVYPYDADNAPTVPFINPPFVSSDGFQEKPLGVLSLRLA DPVTTKNGEITLKLGEGVDLDSSGKLISNTATKAAAPLSFSNNTISLNMDHPF YTKDGKLSLQVSPPLNILRTSILNTLALGFGSGLGLRGSALAVQLVSPLTFDTD GNIKLTLDRGLHVTTGDAIESNISWAKGLKFEDGAIATNIGNGLEFGSSSTETG VDDAYPIQVKLGSGLSFDSTGAIMAGNKEDDKIKGQVVALNTLVNGTNPNGS TVEERGLTNSIKANETNIASVTQEVNTAKGNISSLQGDVQALQEAGYIPEAPR DGQAYVRKDGEWVLLSTFLSPAGGGGSGGGGSGGGGSSRGVPHIVMVDAY KRYKSG*

SEQ ID NO: 9

SAd36-pIX-SpyTag

MSGSASFEGGVFSPYLTGRLPSWAGVRQNVMGSTVDGRPVQPANSSTLTYA TLS S SS VD AAAAAAAAS AAS AVRGMALGAGYYS SLVANSS STNNPASLNEE KLLLLMAQLEALTQRLGELTQQVAQLQAETRAAVATVKTKVGGGGSGGGG SGGGGSGSGSGSGSGSGESGRGVPHIVMVDAYKRYKL

SEQ ID NO: 10 DOGTAG

DIPATYEFTDGKHYITNEPIPPK

SEQ ID NO: 11

SPYTAG003

RGVPHIVMVDAYKRYK

SEQ ID NO: 12:

F8 VHH-SpyCatcher003

QVQLVESGGGLVQPGGSLRLSCAASGGTFSSYPMGWFRQAPGKEREFVAAIS WSGGSTYYADSTKGRFTISRDSARNTVYLQMNSLKPEDTAVYYCAANTHFS GAFEHARTYDYWGQGTQVTVSSGGGGSGGGGSGGGGSAMVTTLSGLSGEQ GPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVK DFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSS GSHHHHHH

SEQ ID NO: 13

F8 VHH-DogCatcher

HHHHHHD YDIPTTENL YFQGQ VQLVESGGGL VQPGGSLRLSC AASGGTF S S Y PMGWFRQAPGKEREFVAAISWSGGSTYYADSTKGRFTISRDSARNTVYLQM NSLKPEDT AVYYC AANTHF SGAFEHARTYD YWGQGTQ VTVS SGGGGSGGG GSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNG TYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVR DVTSIVPQ

SEQ ID NO: 14

F8 VHH-DogCatcher

HHHHHHD YDIPTTENL YFQGQ VQLVESGGGL VQPGGSLRLSC AASGGTF S SY PMGWFRQAPGKEREFVAAISWSGGSTYYADSTKGRFTISRDSARNTVYLQM NSLKPEDT AVYYC AANTHF SGAFEHARTYD YWGQGTQ VTVS SGGGGSGGG GSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNG TYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVR DVTSIVPQ

SEQ ID NO: 15

1D3 scFv-DogCatcher

DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYGASD LQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLEL KGGGGSGGGGSGGGGSEVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMH FVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTS EDTATYFCIYGGYYFDYWGQGVMVTVSSGGGGSGGGGSGGGGSAMKLGEI EFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLT

FTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSSGSHH HHHH

SEQ ID NO: 16

18B12 scFv-DogCatcher

QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPGQGLEWIGVI DPSDNYTKYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYFCAREGYYG SSPWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQIVMSQSPAILSASPGEKV TMTCRARSSVSYIHWYQQKPGSSPKPWIYATSNLASGVPGRFSGSGSGTSYSL TITRVEAEDAATYYCQQWSSKPPTFGGGTKLEIKGGGGSGGGGSGGGGSAM KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGE

DGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGS SGSHHHHHH

SEQ ID NO: 17

FMC63 scFv-DogCatcher

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT GGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWI RQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSAMK LGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGED

GKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSS GSHHHHHH

SEQ ID NO: 18

Rituximab scFvl-DogCacther

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAI YPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYY GGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGE KVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSA MKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTG

EDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQG S SGSHHHHHH

SEQ ID NO: 19

Rituximab scFv2-DogCacther

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLA SGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKS S STAYMQLS SETS EDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSA MKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTG

EDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQG SSGSHHHHHH

SEQ ID NO: 20 spCas9(3xNLS)-DogCatcher

MASMTGGQQMGRAAPAAKKKKLDGSVDMDKKYSIGLDIGTNSVGWAVITD EYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKN GLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD

FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY LQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQ FYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK EVLDATLIHQSITGLYETRIDLSQLGGDTGGGPGGGAAAGSGSPKKKRKVGS GSKRPAATKKAGQAKKKKLYDIPTTENLYFQGAMKLGEIEFIKVDKTDKKPL RGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIE NSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO. 21

SpyCatcher003

VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGE ATEGDAHT

SEQ ID NO: 22

DogCatcher MKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTG

EDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ

SEQ ID NO: 23

Anti -HIV VRC01 vK:

EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAA GIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK

SEQ ID NO: 24

Anti-HIV VRC01 vH:

QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWL KPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNC D YNWDFEHWGRGTP VI VS S

SEQ ID NO: 25

YTS169 scFv-DogCatcher

EVKLQESGGGLVQPGRSLKLSCAASGFNFNDYWMGWVRQAPGKGLEWIGEI NKDSSTINYTPSLKDKFTISRDNAQNTLYLQMSKLGSEDTAIYYCARARGMM VLIIPHYFDYWGQGVMVTVS SGGGGSGGGGSGGGGSDIVLTQ SPAMAMSPG ERITISCRASESVSTRMHWYQQKPGQQPKLLIYGASNLESGVPARFSGSGSGT DFTLTIDPVEANDTATYFCQQSWYDPWTFGGGTKLELKGGGGSGGGGSGGG GSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDV RTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIV PQGSSGSHHHHHH

SEQ ID NO: 26

2.43 scFv-DogCatcher:

EVQLVESGGGLVQPGRSLKLSCAASGFTFSNYYMAWVRQAPTKGLEWVAYI NTGGGTTYYRDSVKGRFTISRDDAKSTLYLQMDSLRSEDTATYYCTTAIGYY FDYWGQGVMVTVSSGGGGSGGGGSGGGGSDIQLTQSPASLSASLGETVSIEC LASEDIYSYLAWYQQKPGKSPQVLIYAANRLQDGVPSRFSGSGSGTQYSLKIS GMQPEDEGDYFCLQGSKFPYTFGAGTKLELKGGGGSGGGGSGGGGSAMKL GEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDG KLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSSG SHHHHHH

SEQ ID NO: 27

DogCatcher:

MKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTG EDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ

SEQ ID NO: 28 Ad5FDgT Fiber:

AYSMSF S WDW SGHN YINEIF AT S S YTF S YIAQE

SEQ ID NO: 29

6xHis-TEV-DogCatcher:

MSYYHHHHHHDYDIPTTENLYFQGAMKLGEIEFIKVDKTDKKPLRGAVFSLQ KQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKP VQNKPIVSFRIVDGEVRDVTSIVPQ

SEQ ID NO: 30

6xHi s-TEV - JPPF 8-DogCatcher :

MSYYHHHHHHDYDIPTTENLYFQGQVQLVESGGGLVQPGGSLRLSCAASGG TFSSYPMGWFRQAPGKEREFVAAISWSGGSTYYADSTKGRFTISRDSARNTV YLQMNSLKPEDT AVYYC AANTHF SGAFEHART YDYWGQGTQ VTVS SGGGG SGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAI DQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVD GEVRDVTSIVPQ

SEQ ID NO: 31

IL-2 signal sequence- 18B 12 scFV-(G4S)3-DgC-6xHis:

MYRMQLLSCIALSLALVTNSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSY WMHWIKQRPGQGLEWIGVIDPSDNYTKYNQKFKGKATLTVDTSSSTAYMQL SSLTSEDSAVYFCAREGYYGSSPWFAYWGQGTLVTVSSGGGGSGGGGSGGG GSQIVMSQSPAILSASPGEKVTMTCRARSSVSYIHWYQQKPGSSPKPWIYATS NLASGVPGRFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSKPPTFGGGTKLE IKGGGGSGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYP

DIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIV SFRIVDGEVRD VTSIVPQGS SGSHHHHHH

SEQ ID NO: 32

IL-2 signal sequence-Rtxvl scFV-(G4S)3-DgC-6xHis: MYRMQLLSCIALSLALVTNSQVQLQQPGAELVKPGASVKMSCKASGYTFTS YNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGS GGGGSQIVL SQ SP AIL S ASPGEK VTMTCRAS S S VS YMWFQQKPGS SPKPWIYA TSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTK LEIKGGGGSGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPD YPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKP IVSFRIVDGEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO: 33

IL-2 signal sequence-lD3 scFv-(G4S)3-DgC-6xHis:

MYRMQLLSCIALSLALVTNSDIQMTQSPASLSTSLGETVTIQCQASEDIYSGLA WYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVY FCQQGLTYPRTFGGGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRPG TSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKA TLTADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSSGGGG SGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAI DQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVD GEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO: 34

IL-2 signal sequence-FMC63 scFv-(G4S)3-DgC-6xHis:

MYRMQLLSCIALSLALVTNSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSL SVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIK DNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIY GAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFR IVDGEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO: 35

IGHV1-46 signal sequence-HA22 scFv-(G4S)3-DgC-6xHis:

MDWTWRVFCLLAVAPGAHSEVQLVESGGGLVKPGGSLKLSCAASGFAFSIY DMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQM SSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSAGGGGSGGGGS GGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLI YYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGT KLEIKGGGGSGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHP DYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQN KPIVSFRIVDGEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO: 36

IGHV1-46 signal sequence-YTS169 scFv-(G4S)3-DgC-6xHis: MDWTWRVFCLLAVAPGAHSEVKLQESGGGLVQPGRSLKLSCAASGFNFNDY WMGWVRQAPGKGLEWIGEINKDSSTINYTPSLKDKFTISRDNAQNTLYLQMS KLGSEDTAIYYCARARGMMVLIIPHYFDYWGQGVMVTVSSGGGGSGGGGSG GGGSDIVLTQSPAMAMSPGERITISCRASESVSTRMHWYQQKPGQQPKLLIYG ASNLESGVPARFSGSGSGTDFTLTIDPVEANDTATYFCQQSWYDPWTFGGGT KLELKGGGGSGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHP DYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQN KPIVSFRIVDGEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO: 37

IGHV1-46 signal sequence-2.43 scFv-(G4S)3-DgC-6xHis:

MDWTWRVFCLLAVAPGAHSEVQLVESGGGLVQPGRSLKLSCAASGFTFSNY YMAWVRQAPTKGLEWVAYINTGGGTTYYRDSVKGRFTISRDDAKSTLYLQ MDSLRSEDTATYYCTTAIGYYFDYWGQGVMVTVSSGGGGSGGGGSGGGGS DIQLTQSPASLSASLGETVSIECLASEDIYSYLAWYQQKPGKSPQVLIYAANRL QDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQGSKFPYTFGAGTKLELK GGGGSGGGGSGGGGSAMKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDI

YGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSF RIVDGEVRDVTSIVPQGSSGSHHHHHH

SEQ ID NO: 38

JPP-F8-(G4S)3-SpyCatcher003-6xHis:

MAQVQLVESGGGLVQPGGSLRLSCAASGGTFSSYPMGWFRQAPGKEREFVA AISWSGGSTYYADSTKGRFTISRDSARNTVYLQMNSLKPEDTAVYYCAANTH FSGAFEHARTYDYWGQGTQVTVSSGGGGSGGGGSGGGGSAMVTTLSGLSGE QGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHV KDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGS SGSHHHHHH

SEQ ID NO: 39

JPP-Gl-(G4S)3-SpyCatcher003-6xHis:

MAQVQLVESGGRLVQPGGSLRLSCVASGFTFNGDTMIWYRQTPGKTREWVA GIAPIGDIANYANSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCNTVTW GQSNSYWGQGTQVTVSSGGGGSGGGGSGGGGSAMVTTLSGLSGEQGPSGD MTTEED S ATHIKF SKRDEDGRELAGATMELRDS SGKTISTWISDGHVKDF YL YPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGSHH HHHH

SEQ ID NO: 40

JPP-H7-(G4S)3-SpyCatcher003-6xHis

MAQVQLVESGGGLVQPGGSLRLSCLASESDFSADALGWYRQAPGKERDLIA RITRGGSIIYADSVKGRFTISRDSAKNMVYLQMNSLKPEDTAVYYCNRLHRGI LMFGQGTQVTVSSAHHSEDPSGGGGSGGGGSGGGGSAMVTTLSGLSGEQGP SGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDF YLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS

HHHHHH

SEQ ID NO: 41

IL2 signal sequence-lD3 scFv-(G4S)3-SpyCatcher003-6xHis:

MYRMQLLSCIALSLALVTNSDIQMTQSPASLSTSLGETVTIQCQASEDIYSGLA WYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVY FCQQGLTYPRTFGGGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRPG TSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKA TLTADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSSGGGG SGGGGSGGGGSAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGREL

AGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEF TVNEDGQ VTVDGEATEGDAHTGS SGSHHHHHH

SEQ ID NO: 42

IL2 signal sequence- 18B 12 scFv-(G4S)3-SpyCatcher003-6xHis

MYRMQLLSCIALSLALVTNSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSY WMHWIKQRPGQGLEWIGVIDPSDNYTKYNQKFKGKATLTVDTSSSTAYMQL SSLTSEDSAVYFCAREGYYGSSPWFAYWGQGTLVTVSSGGGGSGGGGSGGG GSQIVMSQSPAILSASPGEKVTMTCRARSSVSYIHWYQQKPGSSPKPWIYATS NLASGVPGRFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSKPPTFGGGTKLE IKRTDAAGGGGSGGGGSGGGGSAMVTTLSGLSGEQGPSGDMTTEEDSATHIK

FSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAA PDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGSHHHHHH

SEQ ID NO: 43

PGA24

MTFKLIINGKTLKGEITIEAVDA*EAEKIFKQYANDYGIDGEWTYDDATKTFTV TE

SEQ ID NO: 44

PZQ32

MVDNI<FNI<EQQNAFYEILHLPNLNEEQRNAFESLI<DDPSQSANLLAEAI<I<L NDAQAPK

Claims

What is Claimed

1. 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.

2. The method of claim 1, wherein said method for editing said B cell is ex vivo.

3. The method of claim 1, wherein said method for editing said B cell is in vivo.

4. The method of claim 1, wherein said B cell is selected from a plasma cell or a plasmablast.

5. The method of claim 1, wherein said adenovirus is a human or primate adenovirus.

6. The method of claim 1, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and SAdV-36.

7. The method of claim 1, wherein the native tropism of said adenovirus has been ablated.

8. The method of claim 1, wherein said RNA-guide nuclease is CAS9.

9. The method of claim 1, wherein said therapeutic protein is a recombinant antibody.

10. The method of claim 1, wherein 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.

11. The method of claim 1, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

12. The method of claim 1, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

13. The method of claim 1, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

14. The method of claim 1, wherein said high affinity interaction has a KD of 1 nM.

15. The method of claim 1, wherein said high affinity interaction is a covalent bond.

16. The method of claim 1, wherein said covalent bond is an isopeptide bond.

17. The method of claim 1, wherein the recombinant protein comprises a ligand.

18. The method of claim 17, wherein the ligand is selected from the group consisting of CD20, CD19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD 138, PD273, and an Ig receptor.

19. The method of claim 1, wherein the recombinant protein comprises an antigen binding domain.

20. The method of claim 19, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

21. The method of claim 1, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

22. The method of claim 1, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

23. The method of claim 1, wherein the therapeutic protein is an anti -HIV antibody.

24. The method of claim 23, wherein the anti-HIV antibody comprises SEQ ID No. 23.

25. The method of claim 23, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

26. The method of claim 1, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

27. The method of claim 26, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

28. The method of claim 3, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

29. The method of claim 28, wherein said preconditioning is an immunization or vaccination.

30. 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.

31. The method of claim 30, wherein said second recombinant peptide comprises a Cas protein

32. The method of claim 30, wherein said method for editing said B cell is ex vivo.

33. The method of claim 30, wherein said method for editing said B cell is in vivo.

34. The method of claim 30, wherein said B cell is selected from a plasma cell or a plasmablast.

35. The method of claim 30, wherein said adenovirus is a human or primate adenovirus.

36. The method of claim 30, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

37. The method of claim 30, wherein the native tropism of said adenovirus has been ablated.

38. The method of claim 30, wherein said therapeutic protein is a recombinant antibody.

39. The method of claim 30, wherein 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.

40. The method of claim 30, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

41. The method of claim 30, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

42. The method of claim 30, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

43. The method of claim 30, wherein said high affinity interaction has a KD of 1 nM.

44. The method of claim 30, wherein said high affinity interaction is a covalent bond.

45. The method of claim 30, wherein said covalent bond is an isopeptide bond.

46. The method of claim 30, wherein the recombinant protein comprises a ligand.

47. The method of claim 46, wherein the ligand is selected from the group consisting of CD20, CD19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD 138, PD273, and an Ig receptor.

48. The method of claim 30, wherein the recombinant protein comprises an antigen binding domain.

49. The method of claim 48, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

50. The method of claim 30, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

51. The method of claim 30, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

52. The method of claim 30, wherein the therapeutic protein is an anti-HIV antibody.

53. The method of claim 52, wherein the anti-HIV antibody comprises SEQ ID No. 23.

54. The method of claim 52, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

55. The method of claim 30, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

56. The method of claim 55, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

57. The method of claim 30, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

58. The method of claim 57, wherein said preconditioning is an immunization or vaccination.

59. 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.

60. The method of claim 59, wherein said second recombinant peptide is capable of recognizing and interacting with albumin.

61. The method of claim 59, wherein said second recombinant peptide is capable of recognizing and interacting with serum protein.

62. The method of claim 59, wherein said method for editing said B cell is ex vivo.

63. The method of claim 59, wherein said method for editing said B cell is in vivo.

64. The method of claim 59, wherein said B cell is selected from a plasma cell or a plasmablast.

65. The method of claim 59, wherein said adenovirus is a human or primate adenovirus.

66. The method of claim 59, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

67. The method of claim 59, wherein the native tropism of said adenovirus has been ablated.

68. The method of claim 59, wherein said therapeutic protein is a recombinant antibody.

69. The method of claim 59, wherein 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.

70. The method of claim 59, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

71. The method of claim 59, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

72. The method of claim 59, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

73. The method of claim 59, wherein said high affinity interaction has a KD of 1 nM.

74. The method of claim 59, wherein said high affinity interaction is a covalent bond.

75. The method of claim 59, wherein said covalent bond is an isopeptide bond.

76. The method of claim 59, wherein the recombinant protein comprises a ligand.

77. The method of claim 76, wherein the ligand is selected from the group consisting of

CD20, CD19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD 138, PD273, and an Ig receptor.

78. The method of claim 59, wherein the recombinant protein comprises an antigen binding domain.

79. The method of claim 78, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

80. The method of claim 59, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

81. The method of claim 59, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

82. The method of claim 59, wherein the therapeutic protein is an anti-HIV antibody.

83. The method of claim 82, wherein the anti-HIV antibody comprises SEQ ID No. 23.

84. The method of claim 82, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

85. The method of claim 59, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

86. The method of claim 85, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

87. The method of claim 59, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

88. The method of claim 87, wherein said preconditioning is an immunization or vaaccination.

89. 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..

90. The method of claim 89, wherein said second recombinant peptide comprises a Cas protein.

91. The method of claim 89, wherein said method for editing said B cell is ex vivo.

92. The method of claim 89, wherein said method for editing said B cell is in vivo.

93. The method of claim 89, wherein said B cell is selected from a plasma cell or a plasmablast.

94. The method of claim 89, wherein said adenovirus is a human or primate adenovirus.

95. The method of claim 89, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

96. The method of claim 89, wherein the native tropism of said adenovirus has been ablated.

97. The method of claim 89, wherein said therapeutic protein is a recombinant antibody.

98. The method of claim 89, wherein 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.

99. The method of claim 89, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

100. The method of claim 89, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

101. The method of claim 89, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

102. The method of claim 89, wherein said high affinity interaction has a KD of 1 nM.

103. The method of claim 89, wherein said high affinity interaction is a covalent bond.

104. The method of claim 82, wherein said covalent bond is an isopeptide bond.

105. The method of claim 89, wherein the recombinant protein comprises a ligand.

106. The method of claim 105, wherein the ligand is selected from the group consisting of CD20, CD19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD 138, PD273, and an Ig receptor.

107. The method of claim 89, wherein the recombinant protein comprises an antigen binding domain.

108. The method of claim 89, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

109. The method of claim 89, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

110. The method of claim 89, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

111. The method of claim 89, wherein the therapeutic protein is an anti-HIV antibody.

112. The method of claim 111, wherein the anti-HIV antibody comprises SEQ ID No. 23.

113. The method of claim 111, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

114. The method of claim 89, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

115. The method of claim 114, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

116. The method of claim 89, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

117. The method of claim 116, wherein said preconditioning is an immunization or vaccination.

118. 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.

119. The method of claim 111, wherein said method for editing said B cell is ex vivo.

120. The method of claim 111, wherein said method for editing said B cell is in vivo.

121. The method of claim 111, wherein said B cell is selected from a plasma cell or a plasmablast.

122. The method of claim 111, wherein said adenovirus is a human or primate adenovirus.

123. The method of claim 111, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

124. The method of claim 111, wherein the native tropism of said adenovirus has been ablated.

125. The method of claim 111, wherein said therapeutic protein is a recombinant antibody.

126. The method of claim 111, wherein 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.

127. The method of claim 111, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

128. The method of claim 111, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

129. The method of claim 111, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

130. The method of claim 111, wherein said high affinity interaction has a KD of 1 nM.

131. The method of claim 111, wherein said high affinity interaction is a covalent bond.

132. The method of claim 111, wherein said covalent bond is an isopeptide bond.

133. The method of claim 111, wherein the recombinant protein comprises a ligand.

134. The method of claim 133, wherein the ligand is selected from the group consisting of

135. The method of claim 111, wherein the recombinant protein comprises an antigen binding domain.

136. The method of claim 111, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

137. The method of claim 111, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

138. The method of claim 111, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

139. The method of claim 111, wherein the therapeutic protein is an anti-HIV antibody.

140. The method of claim 139, wherein the anti-HIV antibody comprises SEQ ID No. 23.

141. The method of claim 139, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

142. The method of claim 118, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

143. The method of claim 142, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

144. The method of claim 118, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

145. The method of claim 144, wherein said preconditioning is an immunization or vaccination.

146. 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.

147. The engineered B cell of claim 139, wherein said method for editing said B cell is ex vivo.

148. The engineered B cell of claim 139, wherein said method for editing said B cell is in vivo.

149. The engineered B cell of claim 139, wherein said B cell is selected from a plasma cell or a plasmablast.

150. The engineered B cell of claim 139, wherein said adenovirus is a human or primate adenovirus.

151. The engineered B cell of claim 139, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and SadV-36.

152. The engineered B cell of claim 139, wherein the native tropism of said adenovirus has been ablated.

153. The engineered B cell of claim 139, wherein said therapeutic protein is a recombinant antibody.

154. The engineered B cell of claim 139, wherein 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.

155. The engineered B cell of claim 139, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

156. The engineered B cell of claim 139, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

157. The engineered B cell of claim 139, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

158. The engineered B cell of claim 139, wherein said high affinity interaction has a KD of 1 nM.

159. The engineered B cell of claim 139, wherein said high affinity interaction is a covalent bond.

160. The engineered B cell of claim 139, wherein said covalent bond is an isopeptide bond.

161. The engineered B cell of claim 139, wherein the recombinant protein comprises a ligand.

162. The engineered B cell of claim 161, wherein 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, and an Ig receptor.

163. The engineered B cell of claim 139, wherein the recombinant protein comprises an antigen binding domain.

164. The engineered B cell of claim 163, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

165. The engineered B cell of claim 139, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

166. The engineered B cell of claim 139, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

167. The engineered B cell of claim 139, wherein the therapeutic protein is an anti-HIV antibody.

168. The engineered B cell of claim 167, wherein the anti-HIV antibody comprises SEQ ID No. 23.

169. The engineered B cell of claim 167, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

170. The method of claim 146, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

171. The method of claim 170, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

Ill

172. The method of claim 146, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

173. The method of claim 172, wherein said preconditioning is an immunization or vaccination.

174. 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.

175. The method of claim 167, wherein said method for editing said B cell is ex vivo.

176. The method of claim 167, wherein said method for editing said B cell is in vivo.

177. The method of claim 167, wherein said B cell is selected from a plasma cell or a plasmablast.

178. The method of claim 167, wherein said adenovirus is a human or primate adenovirus.

179. The method of claim 167, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

180. The method of claim 167, wherein the native tropism of said adenovirus has been ablated.

181. The method of claim 167, wherein said therapeutic protein is a recombinant antibody.

182. The method of claim 167, wherein 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.

183. The method of claim 167, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

184. The method of claim 167, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

185. The method of claim 167, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

186. The method of claim 167, wherein said high affinity interaction has a KD of 1 nM.

187. The method of claim 167, wherein said high affinity interaction is a covalent bond.

188. The method of claim 167, wherein said covalent bond is an isopeptide bond.

189. The method of claim 167, wherein the recombinant protein comprises a ligand.

190. The method of claim 167, wherein the ligand is selected from the group consisting of

191. The method of claim 167, wherein the recombinant protein comprises an antigen binding domain.

192. The method of claim 191, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

193. The method of claim 167, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

194. The method of claim 167, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

195. The method of claim 167, wherein the therapeutic protein is an anti -HIV antibody.

196. The method of claim 195, wherein the anti -HIV antibody comprises SEQ ID No. 23.

197. The method of claim 195, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

198. The method of claim 174, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

199. The method of claim 174, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

200. The method of claim 199, wherein said preconditioning is an immunization or vaccination.

201. 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.

202. The composition of claim 201, wherein said method for editing said B cell is ex vivo.

203. The composition of claim 201, wherein said method for editing said B cell is in vivo.

204. The composition of claim 201, wherein said B cell is selected from a plasma cell or a plasmablast.

205. The composition of claim 201, wherein said adenovirus is a human or primate adenovirus.

206. The composition of claim 201, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

207. The composition of claim 201, wherein the native tropism of said adenovirus has been ablated.

208. The composition of claim 201, wherein said therapeutic protein is a recombinant antibody.

209. The composition of claim 201, wherein 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.

210. The composition of claim 201, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

211. The composition of claim 201, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

212. The composition of claim 201, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

213. The composition of claim 201, wherein said high affinity interaction has a KD of 1 nM.

214. The composition of claim 201, wherein said high affinity interaction is a covalent bond.

215. The composition of claim 201, wherein said covalent bond is an isopeptide bond.

216. The composition of claim 201, wherein the recombinant protein comprises a ligand.

217. The composition of claim 216, wherein 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, and an Ig receptor.

218. The composition of claim 201, wherein the recombinant protein comprises an antigen binding domain.

219. The composition of claim 218, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

220. The composition of claim 201, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

221. The composition of claim 201, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

222. The composition of claim 201, wherein the therapeutic protein is an anti -HIV antibody.

223. The composition of claim 222, wherein the anti -HIV antibody comprises SEQ ID No.

23.

224. The composition of claim 222, wherein the anti-HIV antibody comprises SEQ ID NO.

24.

225. The composition of claim 201, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

226. The composition of claim 225, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

227. The composition of claim 201, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

228. The composition of claim 227, wherein said preconditioning is an immunization or vaccination.

229. 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.

230. The method of claim 229, wherein said method for editing said B cell is ex vivo.

231. The method of claim 229, wherein said method for editing said B cell is in vivo.

232. The method of claim 229, wherein said B cell is selected from a plasma cell or a plasmablast.

233. The method of claim 229, wherein said adenovirus is a human or primate adenovirus.

234. The method of claim 229, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

235. The method of claim 229, wherein the native tropism of said adenovirus has been ablated.

236. The method of claim 229, wherein said therapeutic protein is a recombinant antibody.

237. The method of claim 229, wherein 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.

238. The method of claim 229, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

239. The method of claim 229, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

240. The method of claim 229, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

241. The method of claim 229, wherein said high affinity interaction has a KD of 1 nM.

242. The method of claim 229, wherein said high affinity interaction is a covalent bond.

243. The method of claim 229, wherein said covalent bond is an isopeptide bond.

244. The method of claim 229, wherein the recombinant protein comprises a ligand.

245. The method of claim 244, wherein the ligand is selected from the group consisting of

246. The method of claim 229, wherein the recombinant protein comprises an antigen binding domain.

247. The method of claim 246, wherein the recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

248. The method of claim 229, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said B cell.

249. The method of claim 229, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said B cell.

250. The method of claim 229, wherein the therapeutic protein is an anti-HIV antibody.

251. The method of claim 250, wherein the anti-HIV antibody comprises SEQ ID No. 23.

252. The method of claim 250, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

253. The method of claim 229, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.

254. The method of claim 253, wherein said activating agent is a CD40 agonist, IL-4, IL-5, IL6, CD30L, BlyS, April, and leptin.

255. The method of claim 229, wherein said method comprises a preconditioning step, prior to contacting said B cell with said adenovirus and said recombinant protein.

256. The method of claim 255, wherein said preconditioning is an immunization or vaccination.

257. 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.

258. The method of claim 257, wherein said method for editing said cell is ex vivo.

259. The method of claim 257, wherein said method for editing said cell is in vivo.

260. The method of claim 257, wherein said adenovirus is a human or primate adenovirus.

261. The method of claim 257, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

262. The method of claim 257, wherein the native tropism of said adenovirus has been ablated.

263. The method of claim 257, wherein said therapeutic protein is a recombinant antibody.

264. The method of claim 257, wherein 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.

265. The method of claim 257, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

266. The method of claim 257, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

267. The method of claim 257, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

268. The method of claim 257, wherein said high affinity interaction has a KD of 1 nM.

269. The method of claim 257, wherein said high affinity interaction is a covalent bond.

270. The method of claim 257, wherein said covalent bond is an isopeptide bond.

271. The method of claim 257, wherein the first or second recombinant protein comprises a ligand.

272. The method of claim 271, wherein the ligand is selected from the group consisting of CD20, CD19, CD21, CD22, CD23, CD24, CD27, CD30, CD38, CD39, CD40, CD45, CD73, CD78, CD80, CD 138, PD273, and an Ig receptor.

273. The method of claim 257, wherein the recombinant protein comprises an antigen binding domain.

274. The method of claim 273, wherein the first or second recombinant protein comprises an scFv, an IgG antibody, a bispecific antibody, or a diabody.

275. The method of claim 257, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said one or more cells.

276. The method of claim 257, wherein the peptide tag has formed a high affinity interaction to said recombinant protein prior to contacting with said one or more cells.

277. The method of claim 257, wherein the therapeutic protein is an anti-HIV antibody.

278. The method of claim 277, wherein the anti-HIV antibody comprises SEQ ID No. 23.

279. The method of claim 278, wherein the anti-HIV antibody comprises SEQ ID NO. 24.

280. A method for editing a cell comprising contacting with said 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 the Fc domain of an antibody; and an antibody, wherein said antibody binds to a protein on the surface of said cell.

281. The method of claim 280, wherein said method for editing said cell is ex vivo.

282. The method of claim 280, wherein said method for editing said cell is in vivo.

283. The method of claim 280, wherein said adenovirus is a human or primate adenovirus.

284. The method of claim 280, wherein said adenovirus is selected from the group consisting of Ad5, HAdV-C5, and S AdV-36.

285. The method of claim 280, wherein the native tropism of said adenovirus has been ablated.

286. The method of claim 280, wherein said RNA-guide nuclease is CAS9.

287. The method of claim 280, wherein said therapeutic protein is a recombinant antibody.

288. The method of claim 280, wherein 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.

289. The method of claim 280, wherein said peptide tag is expressed in the Hl loop domain of the Ad fiber.

290. The method of claim 280, wherein said peptide tag is expressed in the hexon protein of said adenovirus.

291. The method of claim 280, wherein said peptide tag is expressed on the pIX protein, the penton base protein, a fiber-fibritin recombinant protein.

292. The method of claim 280, wherein said high affinity interaction has a KD of 1 nM.

293. The method of claim 280, wherein said high affinity interaction is a covalent bond.

294. The method of claim 280, wherein said covalent bond is an isopeptide bond.

295. The method of claim 280, wherein the recombinant protein comprises an antibody binding domain (abBD).

296. The method of claim 295, wherein the recombinant protein comprises photocrosslinking amino acids.

297. The method of claim 280, wherein the recombinant protein comprises pZQ32.

298. The method of claim 280, wherein the recombinant protein comprises pGA24.

299. The method of claim 280, wherein the adenovirus is contacted with said recombinant protein, prior to contacting with said cell.

300. The method of claim 280, wherein the cell is a B cell.

301. The method of claim 300, wherein said B cell is contacted with an activating agent, prior to contacting said B cell with said adenovirus and said recombinant protein.