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WO2025188676A1 - Genetic engineering of cells for secretion of therapeutic antibodies - Google Patents

Genetic engineering of cells for secretion of therapeutic antibodies

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Publication number
WO2025188676A1
WO2025188676A1 PCT/US2025/018226 US2025018226W WO2025188676A1 WO 2025188676 A1 WO2025188676 A1 WO 2025188676A1 US 2025018226 W US2025018226 W US 2025018226W WO 2025188676 A1 WO2025188676 A1 WO 2025188676A1
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sequence
seq
cell
locus
sgrna
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French (fr)
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WO2025188676A8 (en
Inventor
Matthew H. PORTEUS
William FEIST
Sofia Elena LUNA
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Leland Stanford Junior University
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Leland Stanford Junior University
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Publication of WO2025188676A8 publication Critical patent/WO2025188676A8/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/13—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/33—Antibodies; T-cell engagers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • HSCT Hematopoietic stem cell transplantation
  • KO CCR5 knockout
  • HSCT with CCR5 KO cells is ineffective in patients carrying CXCR4-tropic (X4- tropic) HIV-1 strains that do not rely on CCR5 for cellular entry. This was demonstrated in the case of the Essen patient, where CCR5 KO HSCT followed by ART interruption resulted in rebound of X4-tropic virus. 39 X4-tropic HIV-1 is estimated to be present in 18% to 52% of patients, highlighting the need for strategies to combat these strains. 40,41 [0007] Autologous HSCT with genetically modified cells is a promising strategy to overcome the risk of GvHD and the need for rare donor cells.
  • the present disclosure provides a method of genetically modifying a cell from a subject.
  • the method comprises introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting a selected locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the selected locus in a genome, whereupon the nuclease cleaves the locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell.
  • sgRNA single guide RNA
  • HDR homology directed recombination
  • the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, the RNA-guided nuclease, and the homologous donor template.
  • the sgRNA comprises chemical modifications at one or more nucleotides.
  • -O-methyl- - phosphorothioate (MS) modifications at one or more nucleotides.
  • the -O-methyl- -phosphorothioate (MS) modifications are present at the three terminal [0010]
  • the selected locus is a safe harbor locus.
  • the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In some embodiments, the safe harbor locus is a CCR5 locus and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof. In some embodiments, the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof.
  • the safe harbor locus is an AAVS1 locus, and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39.
  • the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof.
  • the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof.
  • the selected locus is an immunoglobulin-associated locus.
  • the immunoglobulin-associated locus is an IgH locus, an an locus.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof.
  • the RNA-guided nuclease is a Cas9.
  • the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
  • the RNP is introduced into the cell by electroporation.
  • the transgene is present within an expression cassette.
  • the expression cassette comprises a coding sequence for the therapeutic antibody, operably linked to a promoter, and an exogenous polyadenylation (polyA) fragment.
  • the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain.
  • the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain.
  • the promoter is a B-cell specific promoter.
  • the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof.
  • the EEK promoter comprises a sequence having at least 80% identity to SEQ ID NO: 7.
  • the B29 promoter comprises a sequence having at least 80% identity to SEQ ID NO: 8.
  • the IgH promoter comprises a sequence having at least 80% identity to SEQ ID NO: 9 or 10.
  • the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment.
  • the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic antibody.
  • the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen.
  • the transgene encodes a therapeutic antibody against HIV infection.
  • the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain.
  • the at least one light chain and the at least one heavy chain are linked by a linker.
  • the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6.
  • the therapeutic antibody comprises an amino acid sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18.
  • the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26.
  • the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector.
  • rAAV recombinant adeno-associated virus
  • the recombinant adeno-associated virus is serotype 6 (rAAV6).
  • the cell is a hematopoietic stem and progenitor cell (HSPC).
  • the cell is a primary B cell.
  • the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9.
  • the inducible Caspase 9 is a Caspase 9-FKBPF36V.
  • the transgene encodes a fusion protein comprising the therapeutic antibody fused with a destabilization domain.
  • the destabilization domain is a FKBP12-derived destabilization domain.
  • the present disclosure provides a method of treating a subject in need thereof, comprising (i) genetically modifying a cell from the subject using the method described herein, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject.
  • the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection.
  • the cell is reintroduced into the subject by systemic transplantation.
  • the cell is reintroduced into the subject by local transplantation.
  • the local transplantation is intrafemoral or intrahepatic.
  • the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject.
  • the present disclosure provides a sgRNA that specifically targets a CCR5 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 1.
  • the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 4.
  • the present disclosure provides a sgRNA that specifically targets an AAVS1 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 39.
  • the - O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides. In some embodiments, -O-methyl- -phosphorothioate (MS) modifications In some embodiments, the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47.
  • the present disclosure provides a homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic antibody, operably linked to (b) a promoter and (c) a polyadenylation signal at the 3’ end of the coding sequence; (ii) a first homology region located to one side of the expression cassette within the donor template; and (iii) a second homology region located to the other side of the expression cassette within the donor template.
  • the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic.
  • the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. In some embodiments, the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 30 or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34 or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 38 or a fragment thereof.
  • the expression cassette encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen.
  • the expression cassette encodes a therapeutic antibody against HIV infection.
  • the therapeutic antibody encoded by the expression cassette comprises at least one light chain and at least one heavy chain. In some embodiments, the at least one light chain and the at least one heavy chain are linked by a linker.
  • the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6.
  • the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18.
  • the coding sequence comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 19- 26.
  • the present disclosure provides a genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody.
  • the selected locus is a safe harbor locus.
  • the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus.
  • the selected locus is an immunoglobulin-associated locus.
  • the immunoglobulin-associated locus is an IgH locus, an an In some embodiments, the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11- 18.
  • the cell was modified using the method described herein.
  • the present disclosure provides a pharmaceutical composition comprising a plurality of HSPCs, a plurality of B cells, or a plurality of genetically modified cells described herein.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0030]
  • Figure 1 HIV Inhibiting Antibodies Maintain Function with a Peptide Linker.
  • FIG. 1 C) IC 50 of traditional and linker antibodies against a panel of HIV-1 pseudoviruses measured in vitro with TZM-bl infection assay (IC50 was calculated from technical duplicate infections across serial dilutions of each antibody). Pseudoviruses not shown on a graph were not inhibited within the tested antibody [0031]
  • Figure 2 Efficient targeted integration of antibody expression cassettes at the CCR5 locus in HSPCs.
  • MOI multiplicity of infection
  • C) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel 2B (n 3).
  • Cell were targeted at CCR5
  • G) Number of BFU-E, CFU-GM, and CFU-GEMM colonies formed per 500 cells plated in the CFU assay (n 2, with technical duplicates wells for each donor).
  • H) Relative frequency of BFU-E, CFU-GM, and CFU-GEMM colonies formed within the CFU assay (n 2, with technical duplicate wells for each donor).
  • FIG. 3 Antibody edited HSPCs maintain engraftment capacity and multilineage reconstitution in vivo.
  • B) Distribution frequency of WT, INDEL, and KI alleles in CB CD34 + HSPCs prior to transplantation in NSG mice. Cells were targeted at CCR5 with an AAV6 MOI of 1250 for each antibody construct (n 1 pooled sample from 5 donors).
  • C) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel b (n 1).
  • F Percent of human cells in the bone marrow (n are the same as in D) or G) spleen (n are the same as in E) that are CD19 + (B cell lineage), CD33 + (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited HSPCs (10-1074, blue; Ibalizumab, purple; 10-1074 and Linker Ibalizumab, red).
  • H Percent of human alleles from the bone marrow or spleen with knock-in of the indicated antibody constructs (n are the same as in d and e for bone marrow and spleen, respectively).
  • Figure 4 HSPCs with high frequency knock-in maintain edited alleles following engraftment in vivo.
  • B) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel 4A (n 2).
  • D Percent of human cells in the bone marrow that are CD19 + (B cell lineage), CD33 + (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited (red) HSPCs (n are the same as in C).
  • E Percent of human alleles with knock-in from the bulk bone marrow or in positively selected bone marrow CD19 + cells. Lines connect dots representing measurements from the same mice (n are the same as in c). Two- tailed Mann-Whitney test (ns, p>0.05).
  • F Percent of human alleles from the bone marrow with an INDEL at CCR5 (n are the same as in c). This analysis does not include alleles with KI. All bars represent mean and all error bars represent SD. All dots represent individual mice engrafted with mock HSPCs or HSPCs edited with AAV6 and RNP. [0034] Figure 5: Antibody Engineered B Cells Secrete Functional Linker Antibodies.
  • Figure 7 CCR5 INDEL profile and construct specific ddPCR for integration analysis.
  • Construct specific integration is based on IgG1 (10-1074) or IgG4 (Ibalizumab).
  • D) Percent of KI alleles as measured by ddPCR using bulk integration or construct specific integration primers across dilutions of CB CD34 + HSPCs targeted with an IgG4 antibody construct (Ibalizumab, n 1).
  • Measurement efficiency is determined by dividing the construct specific integration measurement by the bulk integration measurement at the indicated dilutions of targeted cells.
  • Figure 8 Bulk CB CD34+ HSPCs edited with three antibody cassettes simultaneously.
  • A) Allelic integration frequency at CCR5 in CB CD34 + HSPCs targeted with AAV6 cassettes for linker antibodies as indicated (n 1 biological donor).
  • Figure 9 Maintenance of knockout and knock-in in xenografted NSG and NBSGW mice.
  • UBC-GFP is integrated using a different CCR5 sgRNA (sg-CCR5-#2) that cuts downstream within exon 2.
  • B) Percent GFP + cells from adult peripheral blood CD19 + B Cells targeted with AAV6 cassettes as indicated (n 1 biological donor). Each AAV6 was used at an MOI of 25,000.
  • C) Median fluorescence intensity of GFP + B cells targeted as indicated (n 1). Each AAV6 was used at an MOI of 25,000.
  • Figure 12 B cell subsets are not perturbed by expression of exogenous antibodies.
  • FIG. 14 Targeted integration frequency of gene-targeted antibody (GT-Ab).
  • GT-Ab Gene-targeted antibody
  • GT-Abs Targeted integration frequency of GT-Ab B cells on day 3 post-editing measured by droplet digital PCR.
  • E Concentration of therapeutic antibodies in B cell culture supernatant 5 days after re-plating measured by ELISA.
  • Figure 15 Expression of gene-targeted antibodies (GT-Abs) in the engineered cells.
  • B Concentration of therapeutic anti- antibody in the serum of mice 4-16 weeks post-transplantation measured by ELISA.
  • C Total edited alleles in the spleen (SP) and peripheral blood (PB) of mice 16 weeks post-transplantation as measured by droplet digital PCR.
  • Figure 16 Concentration of anti-PCSK9 antibodies in B cell culture supernatant measured by ELISA 5 days after re-plating of cells editing with anti-PCSK9 antibody expression cassette with fused destabilization domain with and without addition of small molecule stabilizer (1uM Shield-1). DETAILED DESCRIPTION 1. Introduction [0046] The present disclosure provides methods and compositions for disease (e.g., cancer) treatment in subjects in need through the introduction and integration at a locus of transgenes encoding therapeutic antibodies.
  • disease e.g., cancer
  • the methods involve the introduction of single guide RNAs (sgRNAs) and RNA-guided nucleases (e.g., Cas9) into cells from the subject, as well as the introduction of homologous donor templates for transgene integration at a target locus.
  • the transgenes encoding therapeutic antibodies can be introduced and integrate in a safe harbor locus (e.g., CCR5) or an immunoglobulin-associated locus (e.g., IgH).
  • the RNP complexes e.g., comprising CCR5 sgRNA and Cas9 protein, are delivered to cells via electroporation, followed by the transduction of the homologous donor template using an AAV6 viral vector.
  • the homologous templates for repair are constructed to have arms of homology centered on the cut site within the target locus, located on either side of the coding sequence for a therapeutic antibody of interest, under the control of a designated promoter. Transcription is terminated using an exogenous polyadenylation signal. Depending on the promoter, the system can achieve, e.g., supraphysiological expression and/or cell- specific expression. This system can be used to modify any human cell, and in particular embodiments HSPCs are used. 2. General [0047] Practicing this disclosure utilizes routine techniques in the field of molecular biology.
  • nucleic acids sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and/or RNA can be given in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • oligonucleotides Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983). 3. Definitions [0050] As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0051] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
  • a cell includes a plurality of such cells, and so forth.
  • the terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • CCR5 locus may refer to the specific location of a C-C chemokine receptor type 5 (CCR5 or CD195) gene, CCR5 DNA sequence, CCR5-encoding sequence, or CCR5 position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides.
  • locus may comprise a regulatory element of a gene, including, for example, an
  • safe harbor locus refers to a locus in the genome which allows for expression of an inserted transgene without the risk of affecting surrounding endogenous genes.
  • Examples of safe harbor loci known to exist within mammalian cells include but not limited to a CCR5 locus, an AAVS1 locus, a ROSA26 locus, and a CLYBL locus.
  • the term “immunoglobulin- associated locus” refers to a locus in the genome which includes an immunoglobulin (Ig) gene and its regulatory elements for expressing the immunoglobulin protein.
  • Examples of Immunoglobulin-associated loci known to exist within mammalian cells include but not limited to an IgH locus, an and an [0056]
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • the promoter can be an endogenous or exogenous promoter.
  • An “endogenous promoter” refers to a promoter that occurs naturally within a cell or tissue.
  • An “exogenous promoter” refers to a promoter that are not normally present in a cell or tissue but introduced into the cell by molecular biological technique.
  • the promoter can also be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013/176772, herein incorporated by reference in its entirety for all purposes.
  • a constitutive promoter is one that is active in all tissues or particular tissues at all developing stages.
  • constitutive promoters examples include the human cytomegalovirus immediate early (hCMV), mouse cytomegalovirus immediate early (mCMV), human elongation factor 1 alpha (hEF1a), mouse elongation factor 1 alpha (mEF1a), mouse phosphoglycerate kinase (PGK), chicken beta actin hybrid (CAG or CBh), SV40 early, and beta 2 tubulin promoters.
  • inducible promoters include, for example, chemically regulated promoters and physically regulated promoters.
  • Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter).
  • alcohol-regulated promoters e.g., an alcohol dehydrogenase (alcA) gene promoter
  • tetracycline-regulated promoters e.g., a tetracycline-responsive promoter, a tetracycl
  • Tissue-specific promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter).
  • Tissue-specific promoters can be, for example, neuron-specific promoters, glia- specific promoters, muscle cell-specific promoters, heart cell-specific promoters, kidney cell-specific promoters, bone cell-specific promoters, endothelial cell-specific promoters, or immune cell-specific promoters (e.g., a B cell promoter or a T cell promoter).
  • developmentally regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell.
  • An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.
  • the promoter can be a heterologous promoter.
  • a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).
  • a first polynucleotide or polypeptide is "heterologous" to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form.
  • a promoter when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).
  • “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • expression and “expressed” refer to the production of a transcriptional and/or translational product, e.g., of a therapeutic antibody and/or a nucleic acid sequence encoding a therapeutic antibody.
  • the term refers to the production of a transcriptional and/or translational product encoded by a polynucleotide or a portion thereof.
  • the level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.
  • the term “therapeutic antibody” relates to any antibody preparation which is intended for use in a subject (e.g., a human being) that results in the alleviation and/or a decrease in the progression of a disease in vivo.
  • the term “antibody” refers to an antigen binding protein or a fragment thereof.
  • the term “antibody” includes, but is not limited to, full-length antibodies, antibody fragments, chimeric antibodies, human antibodies, and humanized antibodies.
  • Antibody diabodies including bivalent diabodies and bispecific diabodies, trispecific mAbs bispecific T- cell engagers (BiTEs), bispecific and trispecific killer cell engagers (BiKEs and TRiKEs), and dual-affinity re-targeting antibodies (DARTs) and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134. Antibodies also include, but are not limited to, camelid antibodies and single domain antibodies (e.g., nanobodies). In certain embodiments, recombinant DNA techniques are used to produce antibodies. [0067] In some embodiments, the therapeutic antibody comprises at least one heavy chain and at least one light chain.
  • heavy chain refers to a polypeptide comprising sufficient heavy chain variable region sequence to confer antigen specificity either alone or in combination with a light chain.
  • light chain refers to a polypeptide comprising sufficient light chain variable region sequence to confer antigen specificity either alone or in combination with a heavy chain.
  • heavy and light chains are expressed as separate proteins and are joined by endogenous B cell processing to form functional antibodies.
  • the therapeutic antibody is expressed as a single protein.
  • the antibody is expressed as a single-chain fragment variable (scFv) that contains the antigen-binding domains of a whole antibody and which can be expressed as a single protein sequence.
  • the single protein e.g., scFv will comprise the variable light and variable heavy chain regions of an antibody connected by a flexible linker sequence, such as a glycine and serine-based linker sequence, for example having a length of 10-100 amino acids, for example about 15-20 amino acids.
  • the therapeutic antibody is expressed as a Fab, for example, comprising a single protein comprising an antibody light chain linked to an antibody heavy chain.
  • the single protein e.g., expressed as LC-L-HC or HC-L-LC will comprise the light and heavy chains of an antibody connected by a flexible linker sequence, such as a glycine and serine-based linker sequence, for example having a length of 10-100 amino acids, for example about 15-20 amino acids.
  • the antibodies are produced as the product of two protein sequences, a heavy chain gene (IgH) and a kappa (Ig or lambda (Ig , which chains are expressed and processed by the in-vivo machinery of the recipient organism to create functional antibodies.
  • a heavy chain gene IgH
  • a kappa Ig or lambda
  • Ig which chains are expressed and processed by the in-vivo machinery of the recipient organism to create functional antibodies.
  • Techniques for antibody production such as engineering B cells for antibody expression, are known in the art. (See, e.g. Moffett, Howell F., et al. "B cells engineered to express pathogen-specific antibodies protect against infection.” Science immunology 4.35 (2019): eaax0644; Hartweger, Harald, et al.
  • the scope of the invention encompasses the expression of an antibody, including Fab, scFv, and other versions of an antibody, or other antigen-binding variants of an antibody.
  • the antibody comprises an antibody, including Fab, scFv or other antigen binding fragment of an antibody selected from the group consisting of: Abciximab, Adalimumab, , Aducanumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Ansuvimab, Atezolizumab, Atoltivimab, Maftivimab, Odesivimab, Avelumab, Axatilimab, Basiliximab, Belantamab, Belimumab, Benralizumab, Bevacizumab, Bezlotoxumab, Bimekizumab, Blinatumomab, Brentuximab, Brodalumab, Brolucizumab, Burosumab, Camrelizumab, Canakinumab, Caplacizumab, Casirivimab, Imdevimab, Catumaxo
  • antigen e.g., human immunoglobulin, human immunoglobulin, and human immunoglobulin, human immunoglobulin, and human immunoglobulin, and human immunoglobulin, human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulin, and human immunoglobulfin, antigen, antigen, antigen, antigen, and target molecules.
  • target molecules include, but are not limited to, polypeptides, nucleic acids, and polysaccharides.
  • treating refers to any one of the following: ameliorating one or more symptoms of a disease or condition (e.g., HIV); preventing the manifestation of such symptoms before they occur; slowing down or completely preventing the progression of the disease or condition (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.); enhancing the onset of a remission period; slowing down the irreversible damage caused in the progressive-chronic stage of the disease or condition (both in the primary and secondary stages); delaying the onset of said progressive stage; or any combination thereof.
  • a disease or condition e.g., HIV
  • preventing the manifestation of such symptoms before they occur slowing down or completely preventing the progression of the disease or condition (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.); enhancing the onset of a remission period; slowing down the irreversible damage caused in the progressive-chronic stage of the disease or condition (both in
  • the terms “subject”, “individual” or “patient” refer, interchangeably, to a warm-blooded animal such as a mammal. In particular embodiments, the term refers to a human. A subject may have, be suspected of having, or be predisposed to a lysosomal storage disorder as described herein.
  • the term also includes livestock, pet animals, or animals kept for study, including horses, cows, sheep, poultry, pigs, cats, dogs, zoo animals, goats, primates (e.g., chimpanzee), and rodents.
  • a “subject in need thereof” refers to a subject that has one or more symptoms of a disease or condition (e.g., HIV), that has received a diagnosis of a disease or condition, that is suspected of having or being predisposed to an disease or condition, or that is thought to potentially benefit from increased expression of a therapeutic antibody as described herein.
  • An “effective amount” refers to an amount of a compound or composition, as disclosed herein effective to achieve a particular biological, therapeutic, or prophylactic result. Such results include, without limitation, the treatment of a disease or condition disclosed herein as determined by any means suitable in the art.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences.
  • “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a therapeutic antibody can have an increased stability, assembly, or activity as described herein.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y.
  • amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.
  • Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated.
  • this definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.
  • sequence comparison algorithm typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol.215: 403-410.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)).
  • CRISPR/Cas system refers to a class of bacterial systems for defense against foreign nucleic acids. CRISPR-Cas systems are found in a wide range of bacterial and archaeal organisms.
  • CRISPR-Cas systems fall into two classes with six types, I, II, III, IV, V, and VI as well as many sub-types, with Class 1 including types I and III CRISPR systems, and Class 2 including types II, IV, V and VI; Class 1 subtypes include subtypes I-A to I-F, for example. See, e.g., Fonfara et al., Nature 532, 7600 (2016); Zetsche et al., Cell 163, 759-771 (2015); Adli et al. (2018).
  • Endogenous CRISPR-Cas systems include a CRISPR locus containing repeat clusters separated by non-repeating spacer sequences that correspond to sequences from viruses and other mobile genetic elements, and Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage.
  • Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage.
  • these activities are effected by multiple Cas proteins, with Cas3 providing the endonuclease activity, whereas in class 2 systems they are all carried out by a single Cas, Cas9.
  • a “homologous repair template” refers to a polynucleotide sequence that can be used to repair a double stranded break (DSB) in the DNA, e.g., a CRISPR/Cas9-mediated break at a target locus (e.g., a CCR5 locus) as induced using the herein-described methods and compositions.
  • the homologous repair template comprises homology to the genomic sequence surrounding the DSB, i.e., comprising CCR5 homology arms of the disclosure.
  • two distinct homologous regions are present on the template, with each region comprising at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides or more of homology with the corresponding genomic sequence.
  • the templates comprise two homology arms comprising about 500 nucleotides of homology extending from either site of the sgRNA target site.
  • the repair template can be present in any form, e.g., on a plasmid that is introduced into the cell, as a free floating doubled stranded DNA template (e.g., a template that is liberated from a plasmid in the cell), or as single stranded DNA.
  • the template is present within a viral vector, e.g., an adeno-associated viral vector such as AAV6.
  • the templates comprise an expression cassette comprising a sequence encoding a therapeutic antibody (e.g., anti-HIV antibody), operably linked to a promoter, such that the expression cassette is integrated into the genome at the target locus (e.g., a CCR5 locus) and the therapeutic antibody is expressed.
  • a therapeutic antibody e.g., anti-HIV antibody
  • HR refers to insertion of a nucleotide sequence during repair of double-strand breaks in DNA via homology-directed repair mechanisms.
  • This process uses a “donor template” or “homologous repair template” with homology to nucleotide sequence in the region of the break as a template for repairing a double-strand break.
  • the presence of a double-stranded break facilitates integration of the donor sequence.
  • the donor sequence may be physically integrated or used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence.
  • This process is used by a number of different gene editing platforms that create the double-strand break, such as meganucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 gene editing systems.
  • HR involves double- stranded breaks induced by CRISPR-Cas9.
  • the present disclosure provides methods and compositions for integrating and expressing transgenes encoding therapeutic antibodies, e.g., anti-HIV antibodies, into a safe harbor locus or an immunoglobulin-associated locus in cells from a subject in need.
  • the cells are hematopoietic stem and progenitor cells (HSPCs) or primary B cells.
  • HSPCs hematopoietic stem and progenitor cells
  • the cells can be modified using the methods described herein and then reintroduced into the subject, wherein the expression of the therapeutic antibody in the modified cells in vivo can treat the subject in need.
  • the present disclosure is based in part on the identification of CRISPR guide sequences that specifically direct the cleavage of the target locus, e.g., a CCR5 locus, by RNA- guided nucleases such as Cas9.
  • the methods involve the introduction of ribonucleoproteins (RNPs) comprising an sgRNA targeting specific locus and Cas9, as well as a template DNA molecule comprising homology arms flanking the transgene encoding the therapeutic antibody.
  • RNPs ribonucleoproteins
  • sgRNAs [0089]
  • the single guide RNAs (sgRNAs) used in the present disclosure target a specific locus wherein the integration of transgenes allows robust expression of the therapeutic antibodies but without affecting cell physiology.
  • the locus is a safe harbor locus.
  • the locus is an immunoglobulin-associated locus.
  • the sgRNAs used in the present disclosure interact with a site-directed nuclease such as Cas9 and specifically bind to or hybridize to a target nucleic acid within the genome of a cell, such that the sgRNA and the site-directed nuclease co-localize to the target nucleic acid in the genome of the cell.
  • the sgRNAs as used herein comprise a targeting sequence comprising homology (or complementarity) to a target DNA sequence at a selected locus, and a constant region that mediates binding to Cas9 or another RNA-guided nuclease.
  • the sgRNA can target any sequence within the selected locus adjacent to a PAM sequence.
  • the sgRNA targets a sequence within a safe harbor locus, such as within a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In other instances, the sgRNA targets a sequence within an immunoglobulin-associated locus, such as an IgH locus, an an . [0091] In some embodiments, the sgRNA targets at a CCR5 locus. In particular embodiments, the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 1.
  • the sgRNA comprises the sequence shown as SEQ ID NO: 4, or a sequence having, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 4, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in SEQ ID NO: 4.
  • the sgRNA targets at an AAVS1 locus.
  • the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 39.
  • the sgRNA comprises the sequence shown as SEQ ID NO: 40, or a sequence having, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 40, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in SEQ ID NO: 40.
  • the sgRNA targets at an immunoglobulin-associated locus, such as an IgH locus, an an .
  • the sgRNA targets at an IgH locus.
  • the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 27, 31, or 35.
  • the sgRNA comprises the sequence shown as SEQ ID NO: 40, or a sequence having, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to any one of SEQ ID NOs: 28, 32, and 36, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in any one of SEQ ID NOs: 28, 32, and 36.
  • the targeting sequence of the sgRNAs may be, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or 15-25, 18-22, or 19-21 nucleotides in length, and shares homology with a targeted genomic sequence, in particular at a position adjacent to a CRISPR PAM sequence.
  • the sgDNA targeting sequence is designed to be homologous to the target DNA, i.e., to share the same sequence with the non-bound strand of the DNA template or to be complementary to the strand of the template DNA that is bound by the sgRNA.
  • the homology or complementarity of the targeting sequence can be perfect (i.e., sharing 100% homology or 100% complementarity to the target DNA sequence) or the targeting sequence can be substantially homologous (i.e., having less than 100% homology or complementarity, e.g., with 1-4 mismatches with the target DNA sequence).
  • Each sgRNA also includes a constant region that interacts with or binds to the site- directed nuclease, e.g., Cas9.
  • the constant region of an sgRNA can be from about 70 to 250 nucleotides in length, or about 75-100 nucleotides in length, 75-85 nucleotides in length, or about 80-90 nucleotides in length, or 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length.
  • the overall length of the sgRNA can be, e.g., from about 80-300 nucleotides in length, or about 80-150 nucleotides in length, or about 80-120 nucleotides in length, or about 90-110 nucleotides in length, or, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length.
  • the sgRNAs comprise one or more modified nucleotides.
  • the polynucleotide sequences of the sgRNAs may also comprise RNA analogs, derivatives, or combinations thereof.
  • the probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates).
  • the sgRNAs comprise 3’ phosphorothiate internucleotide linkages, 2’-O-methyl- 3’-phosphoacetate modifications, 2’-fluoro-pyrimidines, S-constrained ethyl sugar modifications, or others, at one or more nucleotides.
  • the sgRNAs - O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides see, e.g., Hendel et al. (2015) Nat. Biotech.
  • the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47.
  • the sgRNAs can be obtained in any of a number of ways.
  • primers can be synthesized in the laboratory using an oligo synthesizer, e.g., as sold by Applied Biosystems, Biolytic Lab Performance, Sierra Biosystems, or others.
  • RNA-guided nucleases Any CRISPR/Cas nuclease can be used in the method, i.e., a CRISPR/Cas nuclease capable of interacting with a guide RNA and cleaving the DNA at the target site as defined by the guide RNA.
  • the nuclease is Cas9 or Cpf1.
  • the nuclease is Cas9.
  • the Cas9 or other nuclease used in the present methods can be from any source, so long that it is capable of binding to an sgRNA of the disclosure and being guided to and cleaving the specific sequence targeted by the targeting sequence of the sgRNA.
  • Cas9 is from Streptococcus pyogenes.
  • CRISPR/Cas or CRISPR/Cpf1 systems that target and cleave DNA at the selected locus.
  • the selected locus is a safe harbor locus, such as a CCR5 locus, an AAVS1 locus, or a ROSA26 locus.
  • the selected locus is an immunoglobulin-associated locus, such as an IgH locus, an or an .
  • An exemplary CRISPR/Cas system comprises (a) a Cas (e.g., Cas9) or Cpf1 polypeptide or a nucleic acid encoding said polypeptide, and (b) an sgRNA that hybridizes specifically to the selected locus, or a nucleic acid encoding said guide RNA.
  • the nuclease systems described herein further comprises a donor template as described herein.
  • the CRISPR/Cas system comprises an RNP comprising an sgRNA targeting a specific locus and a Cas protein such as Cas9.
  • the Cas9 is a high fidelity (HiFi) Cas9.
  • HiFi high fidelity
  • CRISPR/Cas9 platform which is a type II CRISPR/Cas system
  • alternative systems exist including type I CRISPR/Cas systems, type III CRISPR/Cas systems, and type V CRISPR/Cas systems.
  • CRISPR/Cas9 systems including Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9) and Neisseria cinerea Cas9 (NcCas9) to name a few.
  • Alternatives to the Cas system include the Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) systems.
  • any of the above CRISPR systems may be used to induce a single or double stranded break at a CCR5 locus to carry out the methods disclosed herein.
  • Introducing the sgRNA and Cas protein into cells [0102]
  • the sgRNA and nuclease can be introduced into a cell using any suitable method, e.g., by introducing one or more polynucleotides encoding the sgRNA and the nuclease into the cell, e.g., using a vector such as a viral vector or delivered as naked DNA or RNA, such that the sgRNA and nuclease are expressed in the cell.
  • the sgRNA and nuclease are assembled into ribonucleoproteins (RNPs) prior to delivery to the cells, and the RNPs are introduced into the cell by, e.g., electroporation.
  • RNPs are complexes of RNA and RNA-binding proteins.
  • the RNPs comprise the RNA- binding nuclease (e.g., Cas9) assembled with the guide RNA (e.g., sgRNA), such that the RNPs are capable of binding to the target DNA (through the gRNA component of the RNP) and cleaving it (via the protein nuclease component of the RNP).
  • an RNP for use in the present methods can comprise any of the herein-described guide RNAs and any of the herein-described RNA-guided nucleases.
  • Animal cells, mammalian cells, preferably human cells, modified ex vivo, in vitro, or in vivo are contemplated. Also included are cells of other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, rats; birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • the cell is an embryonic stem cell, a stem cell, a progenitor cell, a pluripotent stem cell, an induced pluripotent stem (iPS) cell, a somatic stem cell, a differentiated cell, a mesenchymal stem cell or a mesenchymal stromal cell, a neural stem cell, a hematopoietic stem cell or a hematopoietic progenitor cell, an adipose stem cell, a keratinocyte, a skeletal stem cell, a muscle stem cell, a fibroblast, an NK cell, a B-cell, a T cell, or a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the cells are hematopoietic stem and progenitor cells (HSPCs), e.g., cord blood-derived (CB) or adult peripheral blood-derived (PB) HSPCs, or neuronal stem cells.
  • HSPCs hematopoietic stem and progenitor cells
  • CB cord blood-derived
  • PB peripheral blood-derived
  • the cells are primary B cells.
  • the cells to be modified are preferably derived from the subject’s own cells.
  • the mammalian cells are autologous cells from the subject to be treated with the modified cells.
  • the cells are allogeneic, i.e., isolated from an HLA-matched or HLA-compatible, or otherwise suitable, donor.
  • cells are harvested from the subject and modified according to the methods disclosed herein, which can include selecting certain cell types, optionally expanding the cells and optionally culturing the cells, and which can additionally include selecting cells that contain the transgene integrated into the selected locus (e.g., the CCR5 locus).
  • the cells are induced to undergo differentiation, e.g., into macrophages or monocytes, using methods known in the art and as described herein.
  • such modified, selected, and/or differentiated cells are then reintroduced into the subject.
  • nuclease systems to produce the modified host cells described herein, comprising introducing into the cell (a) an RNP of the disclosure that targets and cleaves DNA at the selected locus (e.g., the CCR5 locus), and (b) a homologous donor template or vector as described herein.
  • Each component can be introduced into the cell directly or can be expressed in the cell by introducing a nucleic acid encoding the components of said one or more nuclease systems.
  • Such methods will target integration of the transgene encoding the therapeutic antibody to the selected locus (e.g., the CCR5 locus) in a host cell ex vivo.
  • Such methods can further comprise (a) introducing a donor template or vector into the cell, optionally after expanding said cells, or optionally before expanding said cells, and (b) optionally culturing the cell.
  • the disclosure herein contemplates a method of producing a modified mammalian host cell, the method comprising introducing into a mammalian cell: (a) an RNP comprising a Cas nuclease such as Cas9 and an sgRNA specific to the selected locus (e.g., the CCR5 locus), and (b) a homologous donor template or vector as described herein.
  • the nuclease can produce one or more single stranded breaks within the selected locus (e.g., the CCR5 locus), or a double stranded break within the selected locus (e.g., the CCR5 locus).
  • the selected locus e.g., the CCR5 locus
  • the methods can further comprise (c) selecting cells that contain the transgene integrated into the selected locus (e.g., the CCR5 locus).
  • transgenes including large transgenes, capable of expressing functional proteins, including enzymes, cytokines, antibodies, and cell surface receptors are known in the art.
  • functional proteins including enzymes, cytokines, antibodies, and cell surface receptors.
  • transgene to be integrated is typically present within a homologous repair template, or homologous donor template.
  • the transgene can be any transgene encoding a therapeutic antibody has a beneficial effect in subjects in need.
  • the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. In particular embodiments, the transgene encodes a therapeutic antibody against HIV infection. [0113] In some embodiments, the transgene is flanked in the template by homology regions of the selected locus. In some instances, the selected locus is a safe harbor locus, such as a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In other instances, the selected locus is an immunoglobulin-associated locus, such as an IgH locus, an an .
  • the transgene is flanked in the template by homology regions of a CCR5 locus.
  • an exemplary template can comprise, in linear order: a first CCR5 homology region, a promoter, a coding sequence for a therapeutic antibody, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH-PolyA), and a second CCR5 homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site.
  • bGH-PolyA bovine growth hormone polyadenylation sequence
  • one of the homology regions comprises the sequence of SEQ ID NO: 2
  • the other homology region comprises the sequence of SEQ ID NO: 3, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 2 and/or SEQ ID NO: 3.
  • the transgene is flanked in the template by homology regions of an AAVS1 locus.
  • an exemplary template can comprise, in linear order: a first AAVS1 homology region, a promoter, a coding sequence for a therapeutic antibody, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH- PolyA), and a second AAVS1 homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site.
  • a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH- PolyA)
  • bGH- PolyA bovine growth hormone polyadenylation sequence
  • an exemplary template can comprise, in linear order: a first IgH homology region, a promoter, a coding sequence for a therapeutic antibody, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH- PolyA), and a second IgH homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site.
  • a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH- PolyA)
  • bGH- PolyA bovine growth hormone polyadenylation sequence
  • one of the homology regions comprises the sequence of SEQ ID NO: 28, and the other homology region comprises the sequence of SEQ ID NO: 29, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 28 and/or SEQ ID NO: 29.
  • one of the homology regions comprises the sequence of SEQ ID NO: 33
  • the other homology region comprises the sequence of SEQ ID NO: 34, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 33 and/or SEQ ID NO: 34.
  • one of the homology regions comprises the sequence of SEQ ID NO: 36
  • the other homology region comprises the sequence of SEQ ID NO: 37, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 36 and/or SEQ ID NO: 37.
  • the transgene is flanked within the polynucleotide or donor construct by sequences homologous to the target genomic sequence, i.e., CCR5.
  • the transgene is flanked by sequences in a CCR5 locus surrounding the site of cleavage as defined by sgRNA.
  • the transgene is flanked on one side by a sequence comprising SEQ ID NO: 2 or a fragment thereof, and on the other side by a sequence comprising SEQ ID NO: 3 or a fragment thereof.
  • the homology regions can be of any size, e.g., 50-2000, 100-1500 bp, 300-900 bp, 400-600 bp, or about 50, 100, 200, 300, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more bp.
  • Any suitable method can be used to introduce the polynucleotide, or donor construct, into the cell.
  • the polynucleotide is introduced using a recombinant adeno-associated viral vector (rAAV).
  • rAAV a recombinant adeno-associated viral vector
  • the rAAV can be from serotype 1 (e.g., an rAAV1 vector), 2 (e.g., an rAAV2 vector), 3 (e.g., an rAAV3 vector), 4 (e.g., an rAAV4 vector), 5 (e.g., an rAAV5 vector), 6 (e.g., an rAAV6 vector), 7 (e.g., an rAAV7 vector), 8 (e.g., an rAAV8 vector), 9 (e.g., an rAAV9 vector), 10 (e.g., an rAAV10 vector), or 11 (e.g., an rAAV11 vector).
  • serotype 1 e.g., an rAAV1 vector
  • 2
  • the vector is an rAAV6 vector.
  • the donor template is single stranded, double stranded, a plasmid or a DNA fragment.
  • plasmids comprise elements necessary for replication, including a promoter and optionally a 3’ UTR.
  • vectors comprising (a) one or more nucleotide sequences homologous to the selected locus, and (b) a transgene encoding a therapeutic factor of the disclosure.
  • the vector can be a viral vector, such as a retroviral, lentiviral (both integration competent and integration defective lentiviral vectors), adenoviral, adeno-associated viral or herpes simplex viral vector.
  • Viral vectors may further comprise genes necessary for replication of the viral vector.
  • the targeting construct comprises: (1) a viral vector backbone, e.g. an AAV backbone, to generate virus; (2) arms of homology to the target site of at least 200 bp but ideally at least 400 bp on each side to assure high levels of reproducible targeting to the site (see, Porteus, Annual Review of Pharmacology and Toxicology, Vol.
  • transgene encoding a therapeutic antibody and capable of expressing the therapeutic antibody
  • expression control sequence operably linked to the transgene
  • optionally (5) an additional marker gene to allow for enrichment and/or monitoring of the modified host cells.
  • Any AAV known in the art can be used.
  • the primary AAV serotype is AAV6.
  • Suitable marker genes are known in the art and include Myc, HA, FLAG, GFP, truncated NGFR, truncated EGFR, truncated CD20, truncated CD19, as well as antibiotic resistance genes (e.g., pac (puromycin-N-acetyl transferase), aph (aminoglycoside phosphotransferase), or bsd (blasticidin S deaminase), providing resistance to puromycin, G418, and blasticidin, respectively).
  • antibiotic resistance genes e.g., pac (puromycin-N-acetyl transferase), aph (aminoglycoside phosphotransferase), or bsd (blasticidin S deaminase), providing resistance to puromycin, G418, and blasticidin, respectively).
  • the donor template or vector comprises a nucleotide sequence homologous to a fragment of the selected locus, wherein the nucleotide sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 or more consecutive nucleotides of the selected locus, e.g., a CCR5 locus.
  • the inserted construct can also include a safety switch, such as a standard suicide gene, (e.g., inducible Caspase 9) into the locus in circumstances where rapid removal of cells might be required due to acute toxicity.
  • a safety switch such as a standard suicide gene, (e.g., inducible Caspase 9) into the locus in circumstances where rapid removal of cells might be required due to acute toxicity.
  • the present disclosure provides a robust safety switch so that any engineered cell transplanted into a body can be eliminated, e.g., by removal of an auxotrophic factor. This is especially important if the engineered cell has transformed into a cancerous cell.
  • Non-limiting examples of safety switches include inducible Caspase 9 (iCaspase9), such as, e.g., those described in Martin RM, et al. Nat. Commun.
  • the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9 (iCaspase9).
  • the inducible Caspase 9 is a Caspase9-FKBP F36V .
  • Treatment with small-molecule AP20187 dimerizes the Caspase9-FKBP F36V protein leading to rapid, irreversible apoptosis of the cell.
  • the inserted construct includes a destabilization domain (DD), such as a FKBP12-derived destabilization domain.
  • DD destabilization domain
  • a destabilization domain (DD) refers to an amino acid sequence that is inherently unstable under physiological conditions and is efficiently targeted for protein degradation. When fused to a protein of interest (POI), the entire fusion product is rapidly degraded under untreated conditions. However, the DD-POI fusion protein can be stabilized upon the addition of a small molecule ligand (e.g., Shield1 ligand) which binds to and stabilizes the DD, allowing for quick, conditional regulation of the POI.
  • a small molecule ligand e.g., Shield1 ligand
  • the present disclosure provides a genetically modified cell comprising an expression cassette encoding a fusion protein, such as, e.g., a therapeutic antibody fused to a DD.
  • a fusion protein such as, e.g., a therapeutic antibody fused to a DD.
  • the DD is a FKBP12-derived destabilization domain.
  • the fusion protein comprising the therapeutic antibody and the DD is either undetectable or detected at a very low level in the cell under normal conditions.
  • a small molecule such as a Shield1 ligand
  • the fusion protein is rescued from proteasomal degradation, resulting in rapid accumulation of the fusion protein.
  • the small molecule stabilizer is a Shield1 ligand.
  • An exemplary structure of a Shield1 ligand is shown below: . 5.
  • the present disclosure provides methods and compositions for introducing and integrating a transgene encoding therapeutic antibody at a selected locus.
  • the selected locus is a safe harbor locus.
  • safe harbor loci known to exist within mammalian cells include, but are not limited to, a CCR5 locus, an AAVS1 locus, a ROSA26 locus, and a CLYBL locus.
  • the safe harbor locus is a human C-C chemokine receptor type 5 (CCR5) locus (chromosome 3 p21.31).
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome, whereupon the nuclease cleaves the CCR5 locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell.
  • HDR homology directed recombination
  • the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1.
  • the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof.
  • the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof.
  • the safe harbor locus is a human adeno-associated virus integration site 1 (AAVS1) locus (chromosome 19 q13.42).
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the AAVS1 locus, an RNA- guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the AAVS1 locus in the genome, whereupon the nuclease cleaves the AAVS1 locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved AAVS1 locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell.
  • HDR homology directed recombination
  • the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39.
  • the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof.
  • the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof.
  • the safe harbor locus is a human reverse orientation splice acceptor 26 (ROSA26) locus (chromosome 3 p25.3).
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the ROSA26 locus, an RNA- guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the ROSA26 locus in the genome, whereupon the nuclease cleaves the ROSA26 locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved ROSA26 locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell.
  • HDR homology directed recombination
  • the safe harbor locus is the human citrate lyase beta like (CLYBL) locus (chromosome 13 q32.3).
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CLYBL locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CLYBL locus in the genome, whereupon the nuclease cleaves the CLYBL locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR)
  • HDR homology directed recombination
  • Immunoglobulin-Associated Loci The methods and compositions disclosed herein can be also used to introduce and express a therapeutic antibody at an immunoglobulin-associated locus.
  • immunoglobulin-associated loci known to exist within mammalian cells include, but are not limited to, an IgH locus, an and an .
  • the immunoglobulin-associated locus is a human immunoglobulin heavy locus (IgH, chromosome 14 q32.33).
  • Examples of expressing antibody in a human immunoglobulin heavy locus include but not limited to, as those described in Moffett HF, et al. Science Immunology. 2019;4(35) and Nahmad AD, et al.
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting an IgH locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the IgH locus in the genome, whereupon the nuclease cleaves the IgH locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved IgH locus; and the integrated
  • HDR homology directed recombination
  • the target sequence of the sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 27, 31 and 35.
  • the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 28, 32 or 36.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 28, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29, or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof.
  • the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof.
  • the immunoglobulin-associated locus is the human immunoglobulin light locus chromosome 2 p11.2).
  • Examples of expressing antibody in a include but not limited to, as those described in Moffett HF, et al. Science Immunology. 2019;4(35) and Nahmad AD, et al. Nature Communications. 2020;11(1):5851, each of which is hereby incorporated by reference in its entirety.
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting an locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at locus in the genome, whereupon the nuclease cleaves locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell.
  • HDR homology directed recombination
  • the immunoglobulin-associated locus is the human immunoglobulin light locus chromosome 22 p11.2).
  • Examples of expressing antibody in a can include but not limited to as those described in Moffett HF, et al. Science Immunology. 2019;4(35) and Nahmad AD, et al. Nature Communications. 2020;11(1):5851, each of which is hereby incorporated by reference in its entirety.
  • a method of genetically modifying a cell from a subject comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting an locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at locus in the genome, whereupon the nuclease cleaves locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell.
  • HDR homology directed recombination
  • the system disclosed herein can be used to introduce and express any therapeutic antibody within the genomically modified cells in subjects in need.
  • the therapeutic antibody binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen.
  • the coding sequence encodes an anti-HIV antibody, an anti-CD4 antibody, an anti-TNF- antibody, an anti-PCSK9 antibody, or any other therapeutic antibody beneficial to the subjects in need.
  • Other potential therapeutic antibodies include, but not limit to, any antibodies binding to RSV, Ebola virus, SARS-CoV2, Angiopoietin-like 3, PD-1, PD-L1, C1S, etc.
  • the therapeutic antibody comprises an antibody selected from the group consisting of: Abciximab, Adalimumab, Aducanumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Ansuvimab, Atezolizumab, Atoltivimab, Maftivimab, Odesivimab, Avelumab, Axatilimab, Basiliximab, Belantamab, Belimumab, Benralizumab, Bevacizumab, Bezlotoxumab, Bimekizumab, Blinatumomab, Brentuximab, Brodalumab, Brolucizumab, Burosumab, Camrelizumab, Canakinumab, Caplacizumab, Casiriv
  • the transgene encodes a therapeutic antibody against HIV infection.
  • the genomically modified cell encodes an anti- therapeutic antibody (e.g., for autoimmune disease treatment).
  • the genomically modified cell encodes an anti-PCSK9 therapeutic antibody (e.g., for hypercholesterolemia treatment).
  • the antibody is a bNAb targeting an HIV virus.
  • the antibody is selected from the group consisting of 2F5, 4E10, F105, hNM01, KD-247 3BNC117, 10-1074, VRC01 VRC-HIVMAB080-00-AB (VRC01LS), VRC- HIVMAB075-00-AB (VRC07-523LS), VRC-HIVMAB095-00-AB (10E8VLS), PGT121, PGDM1400, PGT121.414.LS CAP256V2LS (VRC-HIVMAB0102-00-AB), VRC- HIVMAB091-00-AB (N6LS), 10E8.4/iMab, and 3BNC117.
  • the antibody is a bNAb targeting an influenza virus.
  • the antibody is selected from the group consisting of: C179 (4hlz), CR6261 (3gbn), F10 (3fku), CR8020 (3sdy), CR8043 (4nm8), FI6 (3ztn), CR9114 (4fqy), 39.29 (4kvn), MEDI8852 (5jw4), CT149 (4r8w), 56.a.09 (59k9), 31.b.09 (5k9o), 16.a.26 (5k9q), 16.g.07 (5kan), 31.a.83 (5kaq), C05 (4fqr), CR8059 (4fqk, stabilized variant of CR8071), 6F12, C179, CR6261, 9H10, CT-P27, VIS410, 39.29, 5A7, CR8033, CR8071, MHAA4549A, and 46B8.
  • the antibody is a bNAb targeting a SARS-CoV-2 virus.
  • the antibody is selected from the group consisting of bebtelovimab, etesevimab, Omi-42, AZD3152, cilgavimab, S304, IY- 2A, and EY6A.
  • the antibody is a bNAb targeting a tuburcolosis virus.
  • the antibody is selected from the group consisting of p4-36, p4-170, T1AM09 (aka AM009) and L1AM04.
  • the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain.
  • the at least one light chain and the at least one heavy chain are linked by a linker.
  • the linker can be a cleavable or uncleavable linker.
  • the linker comprises a sequence having 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to SEQ ID NO: 5.
  • the linker comprises a sequence having 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to SEQ ID NO: 6.
  • the therapeutic antibody comprises an amino acid sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18, e.g., a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of SEQ ID NOs: 11-18.
  • the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26., e.g., a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of SEQ ID NOs: 19-26.
  • the therapeutic antibody comprises an amino acid sequence of any one of SEQ ID NOs: 11-18.
  • the transgene comprises a nucleotide sequence of any one of SEQ ID NOs: 19-26.
  • Expression cassette [0140] In some embodiments, the transgene encoding a therapeutic antibody is present within an expression cassette when introduced into the cell.
  • the expression cassette comprises a coding sequence for the therapeutic antibody.
  • the coding sequence for the therapeutic antibody comprises at least one sequence encoding a light chain and at least one sequence encoding a heavy chain. In some embodiments, the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain.
  • the expression cassette comprises a coding sequence for the therapeutic antibody., operably linked to a promoter.
  • a promoter any promoter that can induce expression of the therapeutic antibody in the cell can be used, including endogenous and exogenous promoters, inducible promoters, constitutive promoters, cell-specific promoters, and others.
  • the promoter is a B-cell specific promoter.
  • the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof.
  • the EEK promoter comprises a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 7.
  • the B29 promoter comprises a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 8.
  • the IgH promoter comprises a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity SEQ ID NO: 9 or 10.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, -actin promoter and other constitutive promoters.
  • HPTR hypoxanthine phosphoribosyl transferase
  • adenosine deaminase adenosine deaminase
  • -actin promoter other constitutive promoters.
  • Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • promoters from the simian virus papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses
  • LTR long terminal repeats
  • thymidine kinase promoter of herpes simplex virus.
  • promoters including the CMV (cytomegalovirus) promoter/enhancer, EF1a (elongation - -a - globin), Ubiquitin C and PGK, all of which provide constitutively active, high-level gene expression in most cell types.
  • CMV cytomegalovirus
  • EF1a elongation - -a - globin
  • Ubiquitin C elongation - -a - globin
  • PGK elongation - -a - globin
  • Inducible promoters are activated in the presence of an inducing agent.
  • the metallothionein promoter is activated to increase transcription and translation in the presence of certain metal ions.
  • Other inducible promoters include alcohol-regulated, tetracycline-regulated, steroid-regulated, metal-regulated, nutrient-regulated promoters, and temperature-regulated promoters.
  • Tissue-specific and/or physiologically regulated expression can also be pursued by modifying mRNA stability and/or translation efficiency (post-transcriptional targeting) of the transgenes.
  • miRNA target recognition sites miRNA target recognition sites
  • the incorporation of miRNA target recognition sites (miRTs) into the expressed mRNA has been used to recruit the endogenous host cell machinery to block transgene expression (detargeting) in specific tissues or cell types.
  • miRNAs are noncoding RNAs, approximately 22 nucleotides, that are fully or partially region of particular mRNA, referred to as miRTs. Binding of a miRNA to its particular miRTs promotes translational attenuation/inactivation and/or degradation.
  • the expression cassette is optionally linked to one or more regulatory elements such as enhancers or post-transcriptional regulatory sequences such as a polyadenylation (polyA) fragment.
  • regulatory elements such as enhancers or post-transcriptional regulatory sequences such as a polyadenylation (polyA) fragment.
  • polyA polyadenylation
  • the expression cassette is further linked to an exogenous polyadenylation (polyA) fragment.
  • the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment.
  • the expression cassette further comprises a signal sequence encoding a signal peptide.
  • the signal sequence is present at the 5’ end of the coding sequence for the therapeutic antibody. In other instances, the signal sequence is present at the 3’ end of the coding sequence for the therapeutic antibody.
  • the signal peptide prompts to translocate the synthesized therapeutic antibody to the cellular membrane and/or secret out of the cell. In some embodiments, the signal peptide is cleaved from the therapeutic antibody after translocation.
  • the expression control sequence functions to express the therapeutic transgene following the same expression pattern as in normal individuals (physiological expression) (See Toscano et al., Gene Therapy (2011) 18, 117–127 (2011), incorporated herein by reference in its entirety for its references to promoters and regulatory sequences). 8.
  • a plurality of modified cells can be reintroduced into the subject, such that they can repopulate and differentiate into, e.g., macrophages or monocytes, and due to the expression of the therapeutic antibody, can improve one or more abnormalities or symptoms in the subject in need.
  • the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection.
  • the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject.
  • methods of treating an individual in need thereof comprising providing to the individual one-time therapy using the genome modification methods disclosed herein.
  • the method comprises reintroducing a modified host cell comprising a transgene encoding a therapeutic antibody integrated at the selected locus, back into the individual for the treatment.
  • the guide RNA displays off-target activity (e.g., > 0.1% indels) at less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 location.
  • the off-target activity occurs at less than 4, 3, 2, or 1 location. In particular embodiments, the off-target activity occurs at 1 or 0 locations when a HiFi Cas9 is used. [0151] In some embodiments, following introduction of the guide RNA, RNA-guided nuclease, and donor template, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of the targeted cells comprise an integrated transgene.
  • chimerism in the subject is at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%).
  • Cells [0152] Disclosed herein, in some embodiments, are genetically modified cells.
  • the genetically modified cell is a hematopoietic stem and progenitor cell (HSPC).
  • the genetically modified cell is a primary B cell.
  • an HSPC comprises a sgRNA, an RNA-guided nuclease (e.g., Cas9), and/or a homologous donor template as disclosed herein.
  • a B cell comprises a sgRNA, an RNA-guided nuclease (e.g., Cas9), and/or a homologous donor template as disclosed herein.
  • a genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody.
  • the selected locus is a safe harbor locus, including but not limited to, a CCR5 locus, an AAVS1 locus, and a ROSA26 locus. In other instances, the selected locus is an immunoglobulin-associated locus, including but not limited to, an IgH locus, an and an .
  • the genetically modified cell expresses a thereutic antibody. In some embodiments, the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. In some embodiments, the genetically modified cell further comprises a sequence encoding an inducible Caspase 9.
  • the genetically modified cell comprises a sequence encoding a fusion protein comprising an therapeutic antibody fused to a destabilization domain (DD).
  • DD destabilization domain
  • Pharmaceutical compositions Disclosed herein, in some embodiments, are methods, compositions and kits for use of the modified cells, including pharmaceutical compositions, therapeutic methods, and methods of administration. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any animals.
  • a pharmaceutical composition comprises a modified autologous host cell of the disclosure is provided. The modified autologous host cell is genetically engineered to comprise an integrated transgene encoding the therapeutic antibody at the selected locus.
  • the modified host cell of the disclosure herein may be formulated using one or more excipients to, e.g.: (1) increase stability; (2) alter the biodistribution (e.g., target the cell line to specific tissues or cell types); (3) alter the release profile of an encoded therapeutic factor.
  • a pharmaceutical composition comprises a plurality of HSPCs, a plurality of B cells, or a plurality of genetically modified cells as disclosed herein.
  • Formulations of the present disclosure can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • the composition may include between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5- 80%, or at least 80% (w/w) active ingredient.
  • Excipients include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R.
  • Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the modified host cells of the present disclosure included in the pharmaceutical compositions described above may be administered by any delivery route, systemic delivery or local delivery, which results in a therapeutically effective outcome.
  • the cells are transplanted intrafemorally or intrahepatically.
  • the composition may take the form of solid, semi- solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • the cell is reintroduced into the subject by systemic transplantation. In some embodiments, the cell is reintroduced into the subject by local transplantation.
  • the local transplantation is intrafemoral or intrahepatic.
  • a subject will undergo a conditioning regime before cell transplantation. For example, before hematopoietic stem cell transplantation, a subject may undergo myeloablative therapy, non-myeloablative therapy or reduced intensity conditioning to prevent rejection of the stem cell transplant even if the stem cell originated from the same subject.
  • the conditioning regime may involve administration of cytotoxic agents.
  • the conditioning regime may also include immunosuppression, antibodies, and irradiation.
  • conditioning needs to be used to create space in the brain for microglia derived from engineered hematopoietic stem cells (HSCs) to migrate in to deliver the protein of interest (as in recent gene therapy trials for ALD and MLD).
  • the conditioning regimen is also designed to create niche “space” to allow the transplanted cells to have a place in the body to engraft and proliferate.
  • the conditioning regimen creates niche space in the bone marrow for the transplanted HSCs to engraft. Without a conditioning regimen, the transplanted HSCs cannot engraft.
  • compositions including the modified host cell of the present disclosure are directed to methods of providing pharmaceutical compositions including the modified host cell of the present disclosure to target tissues of mammalian subjects, by contacting target tissues with pharmaceutical compositions including the modified host cell under conditions such that they are substantially retained in such target tissues.
  • pharmaceutical compositions including the modified host cell include one or more cell penetration agents, although “naked” formulations (such as without cell penetration agents or other agents) are also contemplated, with or without pharmaceutically acceptable excipients.
  • the present disclosure additionally provides methods of administering modified host cells in accordance with the disclosure to a subject in need thereof.
  • compositions including the modified host cell, and compositions of the present disclosure may be administered to a subject using any amount and any route of administration effective for preventing, treating, or managing the diseases or conditions.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the subject may be a human, a mammal, or an animal.
  • the specific therapeutically or prophylactically effective dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration; the duration of the treatment; drugs used in combination or coincidental with the specific modified host cell employed; and like factors well known in the medical arts.
  • modified host cell pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from, e.g., about 1 x 10 4 to 1 x 10 5 , 1 x 10 5 to 1 x 10 6 , 1 x 10 6 to 1 x 10 7 , or more modified cells to the subject, or any amount sufficient to obtain the desired therapeutic or prophylactic, effect.
  • the desired dosage of the modified host cells of the present disclosure may be administered one time or multiple times.
  • delivery of the modified host cell to a subject provides a therapeutic effect for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.
  • the modified host cells may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, either sequentially or concurrently. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • Use of a modified mammalian host cell according to the present disclosure for treatment of a lysosomal disease, disorder or condition is also encompassed by the disclosure.
  • kits comprising compositions or components of the disclosure, e.g., sgRNA, Cas9, RNPs, and/or homologous templates, as well as, optionally, reagents for, e.g., the introduction of the components into cells.
  • kits can also comprise one or more containers or vials, as well as instructions for using the compositions in order to modify cells and treat subjects according to the methods described herein. 9. Examples [0170] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1.
  • CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously.
  • Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.
  • each antibody was produced in the conventional manner from a separate cassette for the light and heavy chains (traditional antibody) and from a single cassette with a linker pairing the light and heavy chains (linker antibody) ( Figure 1A). Purified antibodies were applied to western blot analysis under reducing conditions and stained for IgG.
  • CRISPR-Cas9 editing with electroporation of ribonucleoprotein (RNP) and adeno-associated virus serotype 6 (AAV6) delivery of DNA donor templates for knock-in by homology directed repair (HDR).
  • RNP ribonucleoprotein
  • AAV6 adeno-associated virus serotype 6
  • HDR homology directed repair
  • CCR5 KO is achieved with a highly efficient single guide RNA (sgRNA) we previously demonstrated to induce KO INDELs (Figure 7A) that confer resistance to CCR5-tropic HIV-1 infection in primary CD4 + T cells. 50 Off-target analysis previously performed for this sgRNA showed it to be specific with no measurable off-target INDEL formation.
  • AAV6-delivered donor templates for each linker antibody contain homology flanking the CCR5 cut site and are driven by a previously defined B cell promoter, the EEK promoter ( Figure 2A), for strong expression in B cells as the professional antibody secreting cell type.
  • 45 Cord blood (CB) CD34 + HSPCs were edited with each antibody construct individually.
  • Knock-in analysis through in-out droplet digital PCR (ddPCR) determined that each antibody construct individually was incorporated in an average of 31% to 41% of alleles ( Figures 2B and 7B).
  • CFU colony forming unit
  • AZD7648 treatment did not impact total colony formation or the distribution of colony formation between the three major sub-types, CFU-GEMM (colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte), CFU-GM (colony forming unit-granulocyte and monocyte), and BFU-E (burst forming unit-erythroid) ( Figures 2G-H).
  • Single colonies were genotyped to determine the impact of AZD7648 on mono-allelic and bi-allelic knock-in frequency, as well as INDEL formation.
  • AZD7648 treatment resulted in a 2.4-fold increase in the proportion of bi-allelic knock-in events across colony sub-types (21% to 51%) ( Figure 2I).
  • Human chimerism was measured in bone marrow and spleen through flow cytometry analysis of human and mouse markers. Cells from each condition successfully engrafted in the bone marrow and showed migration to the spleen, a secondary lymphatic site, with similar human chimerism between each gene-edited condition ( Figures 3D-E). The observed decrease in total human engraftment due to RNP/AAV6-based editing was in line with previously published works.
  • Input cells carried a knock-in frequency averaging 50% with efficient knock-in of both 10-1074 and Ibalizumab constructs ( Figures 4A-B).
  • Human chimerism and lineage formation were analyzed in the bone marrow at 12 weeks post-transplantation.
  • Gene- edited cells engrafted at a lower frequency than mock edited cells, though this decrease is in line with those seen in the literature ( Figure 4C).
  • 50,59-62 As expected when human chimerism is low, gene-edited HSPCs showed a myeloid bias in reconstitution within the bone marrow ( Figure 4D).
  • Modifying a patient’s own cells removes the need for identifying a matched donor and circumvents the risks specific to allogeneic transplantation. Additionally, precise genetic engineering can allow for the delivery of multi-factored resistance to R5-tropic and X4-tropic HIV-1.
  • genetically engineered HSPCs Following transplantation, genetically engineered HSPCs have the potential to persist for a patient’s lifetime and provide a long-term source of therapeutic antibody secreting B cells.
  • Recent trials investigating direct injection of bNAbs for HIV-1 suppression show that repeated dosing is required to maintain efficacy, as viral titers rebound once the serum concentrations drop below a therapeutic threshold. 18,19 Additionally, AAV and B cell-mediated antibody delivery platforms rely on somatic cells and are unlikely to rival the longevity of HSCT. Therefore, the engraftment of genetically engineered HSPCs represents one of the most promising methods for a one-time therapy to combat HIV-1.
  • B cells are the professional antibody-producing cells of the hematopoietic system, we believe that expression from B cell progeny is a favorable strategy for sustained delivery of engineered antibodies.
  • our cassettes will act as passengers to normal B cell function and should therefore persist across B cell sub-types. This will ideally allow for our antibodies to reach a steady-state concentration in the bloodstream, providing an advantage over traditional antibody injection therapies that have peaks and troughs with each dosage. Additionally, expression of these antibodies from B cells directly may benefit from the inclusion of natural post-translational modifications that are not incorporated when expressed from other cell types.
  • CD34 + HSPC isolation and culture Human CD34 + HSPCs were isolated from cord blood by the Stanford Binns Program for Cord Blood Research and cultured as previously described. 80 Briefly, isolated mononuclear cells were positively selected for CD34 using the CD34+ Microbead kit Ultrapure (Miltenyi Biotec, San Diego, CA, USA, cat.: 130-100-453).
  • Cells were cultured at 1.5 ⁇ 10 5 –2.5 ⁇ 10 5 cells/mL in CellGenix® GMP Stem Cell Growth Medium (SCGM, CellGenix, Freiburg, Germany, cat.: 20802-0500) supplemented with a human cytokine (PeproTech, Rocky Hill, NJ, USA) cocktail: stem cell factor (100 ng/mL), thrombopoietin (100 ng/mL), Fms-like tyrosine kinase 3 ligand (100 ng/mL), interleukin 6 (100 ng/mL), streptomycin (20 mg/mL), and penicillin (20 U/mL), and 35 nM of UM171 (APExBIO, Houston, TX, USA, cat.: A89505).
  • SCGM CellGenix® GMP Stem Cell Growth Medium
  • PCG CellGenix® GMP Stem Cell Growth Medium
  • a human cytokine PeproTech, Rocky Hill, NJ, USA
  • stem cell factor 100 ng
  • the target sequence for the sgRNA are as follows: sg-CCR5, - ATGCACAGGGTGGAACAAGA- (SEQ ID NO:72) ; sg-CCR5-#2, 5’- GCAGCATAGTGAGCCCAGAA-3’ (SEQ ID NO:73).
  • HiFi Cas9 protein was purchased from IDT (cat.: 1081061) or Aldevron (Fargo, ND, USA, cat:. 9214). RNPs were complexed at a Cas9:sgRNA molar ratio of 1:2.5 at room temperature for 15-30 min.
  • CD34 + cells 2.5 ⁇ 10 5 -1x10 6 CD34 + cells were resuspended in P3 buffer (Lonza, Basel, Switzerland, cat.: V4XP-3032) with complexed RNPs and electroporated using the Lonza 4D Nucleofector and 4D- Nucleofector X Unit (program DZ-100). Electroporated cells were then plated at 2.5 ⁇ 10 5 cells/mL in the previously described cytokine-supplemented media. Immediately following electroporation, AAV6 was dispensed onto cells at 0.625 ⁇ 10 3 -2.5 ⁇ 10 3 vector genomes/cell as noted in figure legends.
  • AZD7648 (Selleck Chemicals, Houston, TX, cat.: S8843) for 24 hours, as previously described.
  • AZD7648 Selleck Chemicals, Houston, TX, cat.: S8843
  • 58 [0193] B cell isolation, culture, and genome editing.
  • Leukoreduction system (LRS) chambers were obtained from the Stanford Blood Center and primary human B cells were isolated by negative selection using the human B Cell Isolation Kit II (Miltenyi Biotec, cat: 130-091-151).
  • IMDM modified Dulbecco’s medium
  • bovine growth serum Cytiva, Marlborough, MA, USA, cat.: SH30541.03HI
  • penicillin-streptomycin -mercaptoethanol Sigma-Aldrich, St.
  • B cells were edited 3-5 days after isolation or thawing using the Lonza Nucleofector 4D (program EO-117) using 1 ⁇ 10 6 cells per well of a 16-well Nucleocuvette Strip (Lonza). Immediately following nucleofection, cells were incubated with AAV6 donor vector (2.5 ⁇ 10 4 for 3-4 hours and then replated at 1 ⁇ 10 6 cells/mL in complete B cell activation media. 82 [0194] Measurement of knock-in alleles by ddPCR. Cells were harvested 2-3 days post- electroporation and genomic DNA (gDNA) was harvested using QuickExtract DNA extraction solution (Biosearch Technologies, Hoddesdon, UK, cat.: QE09050).
  • ddPCR To quantify knock-in alleles via ddPCR, we employed CCR5 specific in-out PCR primers and a probe corresponding to the expected knock-in event (1:3.6 primer to probe ratio). 60 We also used an established genomic DNA reference (REF) at the CCRL2 locus. 60 The ddPCR reaction was prepared and underwent droplet generation following the manufacturer’s instructions with a Bio-Rad QX200 ddPCR machine (Bio-Rad, Hercules, CA, USA).
  • REF genomic DNA reference
  • Thermocycler settings were as follows: 95°C (10 min, 1°C/s ramp), 94°C (30 s, 1°C/s ramp), 60°C (30 s, 1°C/s ramp), 72°C (2 min, 1°C/s ramp), return to step 2 for 50 cycles, and 98°C (10 min, 1°C/s ramp). Analysis of droplet samples was then performed using the QX200 Droplet Digital PCR System (Bio-Rad).
  • Antibody constructs were expressed in Expi293F cells (Thermo Fisher Scientific, cat.: A14527) and grown in combined media (66% FreeStyle/33% Expi media, Thermo Fisher Scientific, cat.: 12338018 and A1435101, respectively) at 37°C and 8% CO2 with shaking at Antibody constructs were transfected using FectoPRO transfection reagent (Polyplus, Sébastien Brant, France, cat.: 101000014) with a transfection mixture of 10 mL of culture media, 50 of total DNA and 130 of FectoPro for each 90 mL of cell culture (for a total transfected cell culture volume of 100 mL or scaled down at the same ratios for a 75 mL total volume).
  • FectoPRO transfection reagent Polyplus, Sébastien Brant, France, cat.: 101000014
  • Heavy and light chain plasmids were used at a 1:1 ratio, while single plasmid linker antibody constructs were used alone.
  • Cells were transfected at a cell density 3-4 ⁇ 10 6 cells/mL and harvested post- transfection by spinning at >4200g for Cell culture supernatants were filtered through a 0.45- filter, combined with 1/10th volume of 10x phosphate-buffered saline (PBS) and purified using the ⁇ KTA pure fast performance liquid chromotography (FPLC, Cytiva) with a 5mL MabSelect PrismA column (Cytvia, cat.: 17549802) using wash steps with 1x PBS and elution with glycine (pH 2.8) into one-tenth volume of 1 M Tris (pH 8.0).
  • PBS phosphate-buffered saline
  • the eluted proteins were then concentrated using 50-kDa or 100-kDa cutoff centrifugal concentrators and further purified by size exclusion on the same ⁇ KTA FPLC with Superdex 200 Increase 10/300 GL column (Cytiva, cat.: 28-9909-44). Proteins were then further concentrated using 50-kDa or 100-kDa cutoff centrifugal concentrators. [0200] Immunophenotyping of B cells.
  • ELISA for Antibody Concentration Antigen specific ELISA was used to determine the concentration of each HIV-inhibiting antibody in B cell supernatant.
  • Anti-IgG Fc ELISA was used to detect human IgG in mouse serum. 10-1074, Ibalizumab, and total human IgG were detected using a previously described protocol.
  • Detection was performed with the 1-StepTM TMB Substrate Kit (Thermo Fisher Scientific, cat.: 34021) and quenched with 3M H 2 SO 4 . Plates were read at 450nm using a Molecular Devices SpectraMax M3 plate reader with SoftMax Pro software. Standard curves were created using purified versions of each HIV inhibiting antibody or purified human IgG/kappa from normal serum (Bethyl, Montgomery, TX, USA, cat.: P80-111). Results were analyzed using GraphPad Prism v10 software to calculate a standard curve using a 4-parameter or 5-parameter sigmoidal algorithm. The curve with a higher r-squared was selected and results were interpolated for each sample.
  • PGDM1400 and CAP256V2LS were detected with a modified protocol.
  • recombinant HIV-1 Env trimer BG505 SOSIP
  • EZ-LinkTM-Biotinylation Kit Thermo Fisher Scientific, cat.: 21435
  • ChonBlock Thermo Fisher Scientific, cat.: 50-152- 6971
  • TZM-bl Infection Assay for Determining IC 50 .
  • the TZM-bl HIV-1 pseudotype infection assay was adapted from previously described protocols.
  • 86,87 TZM-bl cells were obtained through the NIH AIDS Reagent Program (cat.: 8129) and cultured in DMEM with 10% BGS, and 1% penicillin-streptomycin (complete DMEM) at 37°C, 5% CO2, and ambient oxygen levels.
  • HIV-1 env pseudotyped lentivirus was produced as previously described using a pNL4- env plasmids provided by the NIH AIDS Reagents Program (cat.: 11100 and cat.: 12670, respectively). 88 Briefly, 5x10 3 cells were plated in black- walled, clear-bottom 96-well plates (Corning, Corning, NY, USA, cat.: 07-200-565) and incubated overnight. The next day, each antibody was incubated with HIV-1 pseudovirus for 1 - 5 -fold serial dilution in -dextran (Sigma-Aldrich, D9885). Culture media was aspirated from the TZM-bl cells and replaced with antibody-virus mixture.
  • Virus only (no antibodies added) and cells only (no virus or antibodies) wells were included for determining 100% and 0% infection readouts. Cells were incubated for 48 hours then measured for luciferase signal corresponding to infection. Briefly, cells were lysed with Reporter Lysis Buffer (Promega, Madison, WI, USA, cat.: E3971) and freeze-thawed at -80°C.
  • Reporter Lysis Buffer Promega, Madison, WI, USA, cat.: E3971
  • Lysed samples were read for relative luminescence units (RLU) using a Synergy H1 plate reader (BioTek, Winooski, VT, USA) that automatically injected luciferin solution consisting of the following: Firefly Luciferase Signal Enhancer -luciferin (Biosynth Chemistry & Biology, Staad, Switzerland, cat.: L8220).
  • RLU relative luminescence units
  • Percent infection was determined by normalizing RLU values to the average RLU of virus-only and cells-only wells using GraphPad Prism v10.
  • IC50 was calculated using a linear regression dose-response curve fit for inhibitor versus response (three parameters) based on the average of technical duplicate wells using GraphPad Prism v10.
  • TZM-bl Infection Assay with B cell supernatant. Six days post-editing, B cells were plated at 1 ⁇ 10 6 cells/mL and antibody was allowed to accumulate in supernatant for five days. Culture supernatant was collected following centrifugation to remove cells. A modified TZM-bl assay was performed as described above with the following changes. Culture supernatant was diluted in complete DMEM with two-fold serial dilutions. Then, 50uL of each dilution was mixed with 50uL of HIV-1 pseudovirus + complete DMEM for final dilutions of -dextran (Sigma-Aldrich, cat.: D9885).
  • CFU Colony forming unit
  • mice NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) and NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ (NBSGW) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in the Stanford University barrier facility. All experiments were completed under the Administrative Panel on Laboratory Animal Care (APLAC Protocol #25065).
  • CD34 + HSPC transplantation in to immunodeficient mice For the experiment in NSG mice, 6-8 week old mice were irradiated with 2Gy approximately 4 hours prior to transplantation.8.5 ⁇ 10 5 mock or gene-edited cells were transplanted into each mouse via retro- orbital injection. For the experiment in NSGBW mice, 6-8 week old mice received no conditioning and 5 ⁇ 10 5 mock or gene-edited cells were transplanted into each mouse via retro- orbital injection.
  • Assessment of human HSPC engraftment Human engraftment was assessed at 12- (NSGBW) or 16-weeks (NSG) post-transplantation. Mice were euthanized and bone marrow and spleen were harvested from recipient mice.
  • Mononuclear cells from bone marrow samples were isolated via Ficoll gradient centrifugation. Spleen samples were treated with RBC Lysis Buffer (IBI Scientific, Dubuque, IA, USA, cat.: IB47620) to eliminate mature red blood cells.
  • RBC Lysis Buffer IBI Scientific, Dubuque, IA, USA, cat.: IB47620
  • HSPCs hematopoietic stem and progenitor cells
  • GT-Ab gene- targeted antibodies
  • Figure 16 shows the levels of anti-PCSK9 antibodies in B cell culture supernatant of cells editing with anti-PCSK9 antibody expression cassette with fused destabilization domain with and without addition of small molecule stabilizer (1uM Shield-1). This ability to regulate antibody level may be essential to achieve both safety and efficacy of sustained cell- based delivery of monoclonal antibodies.
  • this platform represents a modular approach for treating chronic diseases currently reliant on repeated administration of therapeutic antibodies, offering the potential for a durable production of antibodies from a single treatment. 10.
  • Gaebler C., Nogueira, L., Stoffel, E., Oliveira, T.Y., Breton, G.L., Millard, K.G., Turroja, M., Butler, A., Ramos, V., Seaman, M.S., et al. (2022). Prolonged viral suppression with anti-HIV-1 antibody therapy. Nature. 10.1038/s41586-022-04597-1. Julg, B., Stephenson, K.E., Wagh, K., Tan, S.C., Zash, R., Walsh, S., Ansel, J., Kanjilal, D., Nkolola, J., Walker-Sperling, V.E.K., et al.
  • Antibody 10-1074 suppresses viremia in HIV-1-infected individuals. Nature Medicine 23, 185-191. 10.1038/nm.4268.
  • Lipid Nanoparticles Allow Efficient and Harmless Ex Vivo Gene Editing of Human Hematopoietic Cells. Blood. Xu, L., Lahiri, P., Skowronski, J., Bhatia, N., Lattanzi, A., and Porteus, M.H. (2023).
  • Antibody-mediated prevention of vaginal HIV transmission is dictated by IgG subclass in humanized mice. Science translational medicine 14, eabn9662. 85. Dam, K.-M.A., Mutia, P.S., and Bjorkman, P.J. (2022). Comparing methods for immobilizing HIV-1 SOSIPs in ELISAs that evaluate antibody binding. Scientific Reports 12, 11172. 86. Montefiori, D.C. (2009). Measuring HIV neutralization in a luciferase reporter gene assay. HIV protocols, 395-405. 87.
  • CATNAP a tool to compile, analyze and tally neutralizing antibody panels.
  • Embodiment 1 A method of genetically modifying a cell from a subject, the method comprising: introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting a selected locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein: the sgRNA binds to the nuclease and directs it to a target sequence at the selected locus in a genome, whereupon the nuclease cleaves the locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed
  • Embodiment 2 The method of Embodiment 1, wherein the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, the RNA- guided nuclease, and the homologous donor template.
  • Embodiment 3 The method of Embodiment 1 or 2, wherein the sgRNA comprises chemical modifications at one or more nucleotides.
  • Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the - O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides.
  • Embodiment 5. The method of Embodiment -O-methyl- - [0223] Embodiment 6.
  • Embodiment 7 The method of Embodiment 6, wherein the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus.
  • Embodiment 8 The method of Embodiment 7, wherein the safe harbor locus is the CCR5 locus and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1.
  • Embodiment 9 The method of Embodiment 8, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4.
  • Embodiment 8 or 9 wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof.
  • Embodiment 11 The method of any one of Embodiments 8 to 10, wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof.
  • Embodiment 12. The method of Embodiment 7, wherein the safe harbor locus is the AAVS1 locus, and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39.
  • Embodiment 13 The method of Embodiment 12, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40.
  • Embodiment 14 The method of Embodiment 12 or 13, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof.
  • Embodiment 15 The method of any one of Embodiments 12 to 14, wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof.
  • Embodiment 16 The method of any one of Embodiments 1 to 5, wherein the selected locus is an immunoglobulin-associated locus.
  • Embodiment 17 The method of Embodiment 16, wherein the immunoglobulin- associated locus is an IgH locus, an [0235] Embodiment 18.
  • Embodiment 17 wherein the immunoglobulin- associated locus is the IgH locus, and wherein the target sequence of the sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 27, 31 and 35.
  • Embodiment 19 The method of Embodiment 18, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 28, 32 or 36.
  • Embodiment 20 The method of Embodiment 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 28, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29, or a fragment thereof.
  • Embodiment 21 The method of Embodiment 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof.
  • Embodiment 22 The method of Embodiment 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof.
  • Embodiment 23 Embodiment 23.
  • Embodiment 24 The method of any one of Embodiments 1 to 23, wherein the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
  • Embodiment 25 The method of Embodiment 24, wherein the RNP is introduced into the cell by electroporation.
  • Embodiment 26 The method of any one of Embodiments 1 to 25, wherein the transgene is present within an expression cassette.
  • Embodiment 27 Embodiment 27.
  • Embodiment 26 wherein the expression cassette comprises a coding sequence for the therapeutic antibody, operably linked to a promoter, and an exogenous polyadenylation (polyA) fragment.
  • Embodiment 28 The method of Embodiment 27, wherein the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain.
  • Embodiment 29 The method of Embodiment 28, wherein the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain.
  • Embodiment 30 The method of any one of Embodiments 27 to 29, wherein the promoter is a B-cell specific promoter.
  • Embodiment 31 The method of Embodiment 30, wherein the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof.
  • Embodiment 32 The method of Embodiment 31, wherein the EEK promoter comprises a sequence having at least 80% identity to SEQ ID NO: 7.
  • Embodiment 33 The method of Embodiment 31, wherein the B29 promoter comprises a sequence having at least 80% identity to SEQ ID NO: 8.
  • Embodiment 34 The method of Embodiment 31, wherein the IgH promoter comprises a sequence having at least 80% identity to SEQ ID NO: 9 or 10.
  • Embodiment 35 The method of Embodiment 27, wherein the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment.
  • Embodiment 36 The method of any one of Embodiments 27 to 35, wherein the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic antibody.
  • Embodiment 37 The method of any one of Embodiments 1 to 36, wherein the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen.
  • Embodiment 38 Embodiment 38.
  • Embodiment 39 The method of any one of Embodiments 1 to 38, wherein the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain.
  • Embodiment 40 The method of Embodiment 39, wherein the at least one light chain and the at least one heavy chain are linked by a linker.
  • Embodiment 41 The method of Embodiment 40, wherein the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6.
  • Embodiment 42 the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6.
  • Embodiment 43 The method of any one of Embodiments 1 to 42, wherein the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26.
  • Embodiment 44 The method of any one of Embodiments 1 to 43, wherein the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector.
  • rAAV recombinant adeno-associated virus
  • Embodiment 44 wherein the recombinant adeno- associated virus is serotype 6 (rAAV6).
  • Embodiment 46 The method of any one of Embodiments 1 to 45, wherein the cell is a hematopoietic stem and progenitor cell (HSPC).
  • Embodiment 47 The method of any one of Embodiments 1 to 45, wherein the cell is a primary B cell.
  • Embodiment 48 The method of any one of Embodiments 1 to 47, wherein the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9 or a destabilization domain fused to the therapeutic antibody.
  • Embodiment 49 The method of Embodiment 48, wherein the inducible Caspase 9 is a Caspase 9-FKBPF36V.
  • Embodiment 50 A method of treating a subject in need thereof, comprising (i) genetically modifying a cell from the subject using the method of any one of Embodiments 1 to 49, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject.
  • Embodiment 51 The method of Embodiment 50, wherein the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection.
  • Embodiment 52 The method of Embodiment 50, wherein the cell is reintroduced into the subject by systemic transplantation.
  • Embodiment 53 The method of Embodiment 50, wherein the cell is reintroduced into the subject by local transplantation.
  • Embodiment 54 The method of Embodiment 53, wherein the local transplantation is intrafemoral or intrahepatic.
  • Embodiment 55 The method of any one of Embodiments 50 to 54, wherein the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject.
  • Embodiment 56 The method of any one of Embodiments 50 to 54, wherein the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject.
  • Embodiment 57 The sgRNA of Embodiment 56, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 4.
  • Embodiment 58 A sgRNA that specifically targets an AAVS1 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 39.
  • Embodiment 59 A sgRNA that specifically targets an AAVS1 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 39.
  • Embodiment 60 A sgRNA that specifically targets an IgH locus, wherein the target sequence of the sgRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27, 31, and 35.
  • Embodiment 61 The sgRNA of Embodiment 60, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 28, 32, and 36.
  • Embodiment 62 Embodiment 62.
  • Embodiment 63 The sgRNA of Embodiment 62, wherein the sgRNA comprises - O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides.
  • Embodiment 64 The sgRNA of Embodiment -O-methyl- -
  • Embodiment 65 The sgRNA of Embodiment 64, wherein the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47.
  • Embodiment 66 Embodiment 66.
  • a homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic antibody, operably linked to (b) a promoter and (c) a polyadenylation signal at the 3’ end of the coding sequence; (ii) a first homology region located to one side of the expression cassette within the donor template; and (iii) a second homology region located to the other side of the expression cassette within the donor template.
  • Embodiment 67 The donor template of Embodiment 66, wherein the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic.
  • Embodiment 68 Embodiment 68.
  • Embodiment 66 or 67 wherein the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain.
  • Embodiment 69 The donor template of Embodiment 68, wherein the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain.
  • Embodiment 70 The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof.
  • Embodiment 71 The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof.
  • Embodiment 72 The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 30 or a fragment thereof.
  • Embodiment 73 Embodiment 73.
  • Embodiment 75 The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34 or a fragment thereof.
  • Embodiment 74 The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 38 or a fragment thereof.
  • Embodiment 75 Embodiment 75.
  • Embodiment 77 wherein the at least one light chain and the at least one heavy chain are linked by a linker.
  • Embodiment 79 The donor template of Embodiment 78, wherein the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6.
  • Embodiment 80 The donor template of any one of Embodiments 66 to 79, wherein the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18.
  • Embodiment 81 The donor template of any one of Embodiments 66 to 80, wherein the coding sequence comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26.
  • Embodiment 82 An HSPC comprising the sgRNA of any one of Embodiments 56 to 64 and/or the homologous donor template of any one of Embodiments 66 to 81.
  • Embodiment 83 A B cell comprising the sgRNA of any one of Embodiments 56 to 64 and/or the homologous donor template of any one of Embodiments 66 to 81.
  • Embodiment 84 A genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody.
  • Embodiment 85 Embodiment 85.
  • Embodiment 84 The genetically modified cell of Embodiment 84, wherein the selected locus is a safe harbor locus.
  • Embodiment 86 The genetically modified cell of Embodiment 85, wherein the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus.
  • Embodiment 87 The genetically modified cell of Embodiment 84, wherein the selected locus is an immunoglobulin-associated locus.
  • Embodiment 88 The genetically modified cell of Embodiment 87, wherein the immunoglobulin- [0306] Embodiment 89.
  • Embodiment 90 The genetically modified cell of any one of Embodiments 84 to 89, wherein the cell was modified using the method of any one of Embodiments 1 to 49.
  • Embodiment 91 pharmaceutical composition comprising a plurality of HSPCs of Embodiment 82, a plurality of B cells of Embodiment 83, or a plurality of genetically modified cells of any one of Embodiments 84 to 90.

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Abstract

The present disclosure provides methods and compositions for disease (e.g., cancer) treatment in subjects in need through genetically modifying host cells by introducing and integrating a transgene encoding a therapeutic antibody at a selected locus of the genomic DNA.

Description

PATENT Attorney Docket No.079445-013410PC-1488491 Client Ref. No. S23-412 GENETIC ENGINEERING OF CELLS FOR SECRETION OF THERAPEUTIC ANTIBODIES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 63/561,249, filed March 4, 2024, the disclosure of which is herein incorporated by reference in its entirety for all purposes. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said .xml copy, created on February 25, 2025, is named 079445-1488491-013410PC, and is 101,440 bytes in size. BACKGROUND [0003] There are 39 million people worldwide living with HIV infection.1 While great advances in small-molecule antiretroviral therapy (ART) have resulted in combination drug regimens that can control viral loads, in 2022 alone there were over 600,000 AIDS-related deaths, underlining that HIV remains a global epidemic with substantial morbidity and mortality.1-3 This is due in part to the strict adherence necessary for ART to remain effective.4- 6 Current regimens call for lifetime daily dosing to suppress long-term viral reservoirs and prevent viral rebound.7,8 Because of this, significant research efforts have sought to identify novel treatment modalities that provide a more durable long-term cure. [0004] The discovery of highly potent HIV-1 inhibiting antibodies with relatively long half- lives has led to numerous clinical trials for sustained control of HIV-1.9,10 The first monoclonal antibody clinically approved to treat HIV-1, Ibalizumab, acts as a post-attachment inhibitor by binding HIV-1’s primary receptor on human cells, CD4.11-14 In addition to this, numerous extremely potent antibodies that act against diverse HIV-1 subtypes have been identified to bind and neutralize HIV-1 directly.15,16 These antibodies, known as broadly neutralizing antibodies (bNAbs), target highly conserved regions of the viral envelope to inhibit infection. Recent and ongoing trials are testing the efficacy of targeting multiple env epitopes simultaneously to prevent the generation of resistance mutations.9,10,17-19 These trials have shown that bNAbs can maintain viral suppression and prevent the formation of escape mutants so long as antibody titers remain above a therapeutic threshold. While the longer half-life of antibodies does allow for reduced frequency of dosing, lifelong repeated administration would still be required to maintain their efficacy. [0005] One promising method for sustained delivery of bNAbs is through AAV-mediated delivery of DNA antibody expression cassettes, known as vectored immunoprophylaxis.20-25 Trials in mice, macaques, and recently humans have demonstrated the potential of this strategy for sustained secretion of therapeutic anti-HIV-1 antibodies. However, pre-existing immunity against AAV vectors, low levels of antibody secretion, uncertainty regarding long-term expression, and seroconversion preventing re-dosing remain challenges limiting this approach.26,27 Another potential method for long-term maintenance of antibody expression is the direct editing of autologous B cells. B cells can be directly engineered for custom antibody expression from the B cell receptor locus, allowing for participation of the custom antibody in the humoral immune response.28-32 However, there is currently no clinical protocol for the engraftment of B cells in patients and it remains unclear how long they may persist in the body. [0006] Hematopoietic stem cell transplantation (HSCT) of CCR5 knockout (KO) cells is the only reported long-term functional cure for HIV-1 infection. Demonstrated first in the cases of the Berlin and London patients, allogeneic HSCT with cells from donors carrying the naturally occurring CCR5- 32 KO mutation resulted in reconstitution with cells that were resistant to their CCR5-tropic (R5-tropic) HIV-1.33,34 After treatment interruption of ART, each patient maintained viral suppression and was considered functionally cured of their infection. While this strategy has shown continued success, it is not widely available to the vast majority of patients.35 The rarity of identifying a CCR5 KO matched donor and the morbidities associated with allogeneic transplantation, such as graft versus host disease (GvHD), limit this treatment to only a small fraction of patients who require HSCT for an underlying malignancy.36-38 Moreover, HSCT with CCR5 KO cells is ineffective in patients carrying CXCR4-tropic (X4- tropic) HIV-1 strains that do not rely on CCR5 for cellular entry. This was demonstrated in the case of the Essen patient, where CCR5 KO HSCT followed by ART interruption resulted in rebound of X4-tropic virus.39 X4-tropic HIV-1 is estimated to be present in 18% to 52% of patients, highlighting the need for strategies to combat these strains.40,41 [0007] Autologous HSCT with genetically modified cells is a promising strategy to overcome the risk of GvHD and the need for rare donor cells. One recent trial demonstrated the feasibility of autologous transplantation of cells genetically modified for CCR5 KO in a patient with HIV-1.42 However, low editing rates resulted in a failure to prevent viral rebound, underscoring the importance of efficient modification to minimize viral replication in unedited cells. Other strategies have modified hematopoietic stem and progenitor cells (HSPCs) with CCR5 KO, lentiviral delivery of HIV-1 inhibiting proteins, neutralizing antibodies, and inhibitory RNAs against CCR5 and viral targets.42-48 Several of these strategies benefit from layering multiple methods of HIV-1 inhibition to control both R5-tropic and X4-tropic HIV-1, however, limited editing efficiency and the risk of malignancy resulting from insertional mutagenesis have hampered their application.49 Meanwhile, advances in precision editing with CRISPR-Cas9 have allowed for high-efficiency cellular engineering without the risks associated with lentiviral integration. Recently, we reported a CRISPR-Cas9-based strategy in HSPCs for simultaneous KO out of CCR5 with knock-in of expression cassettes for two HIV- 1 inhibiting proteins.50 While this work demonstrated the feasibility of high-efficiency editing to deliver multilayered genetic resistance to HIV-1, it relies on a cell-autonomous resistance scheme that leaves unedited cells vulnerable to infection. BRIEF SUMMARY [0008] In one aspect, the present disclosure provides a method of genetically modifying a cell from a subject. The method comprises introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting a selected locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the selected locus in a genome, whereupon the nuclease cleaves the locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. [0009] In some embodiments, the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, the RNA-guided nuclease, and the homologous donor template. In some embodiments, the sgRNA comprises chemical modifications at one or more nucleotides. In some embodiments, -O-methyl- - phosphorothioate (MS) modifications at one or more nucleotides. In some embodiments, the -O-methyl- -phosphorothioate (MS) modifications are present at the three terminal [0010] In some instances, the selected locus is a safe harbor locus. In some embodiments, the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In some embodiments, the safe harbor locus is a CCR5 locus and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof. In some embodiments, the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. In some embodiments, the safe harbor locus is an AAVS1 locus, and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof. In some embodiments, the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. [0011] In other instances, the selected locus is an immunoglobulin-associated locus. In some embodiments, the immunoglobulin-associated locus is an IgH locus, an an locus. In some embodiments, the immunoglobulin-associated locus is an IgH locus, and wherein the target sequence of the sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 27, 31 and 35. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 28, 32 or 36. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 28, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29, or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof. [0012] In some embodiments, the RNA-guided nuclease is a Cas9. In some embodiments, the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP). In some embodiments, the RNP is introduced into the cell by electroporation. [0013] In some embodiments, the transgene is present within an expression cassette. In some embodiments, the expression cassette comprises a coding sequence for the therapeutic antibody, operably linked to a promoter, and an exogenous polyadenylation (polyA) fragment. In some embodiments, the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. In some embodiments, the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain. [0014] In some embodiments, the promoter is a B-cell specific promoter. In some embodiments, the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof. In some embodiments, the EEK promoter comprises a sequence having at least 80% identity to SEQ ID NO: 7. In some embodiments, the B29 promoter comprises a sequence having at least 80% identity to SEQ ID NO: 8. In some embodiments, the IgH promoter comprises a sequence having at least 80% identity to SEQ ID NO: 9 or 10. [0015] In some embodiments, the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment. In some embodiments, the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic antibody. [0016] In some embodiments, the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. In some embodiments, the transgene encodes a therapeutic antibody against HIV infection. In some embodiments, the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain. In some embodiments, the at least one light chain and the at least one heavy chain are linked by a linker. In some embodiments, the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6. [0017] In some embodiments, the therapeutic antibody comprises an amino acid sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. In some embodiments, the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26. In some embodiments, the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the recombinant adeno-associated virus is serotype 6 (rAAV6). In some embodiments, the cell is a hematopoietic stem and progenitor cell (HSPC). In some embodiments, the cell is a primary B cell. [0018] In some embodiments, the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9. In some embodiments, the inducible Caspase 9 is a Caspase 9-FKBPF36V. In some embodiments, the transgene encodes a fusion protein comprising the therapeutic antibody fused with a destabilization domain. In some embodiments, the destabilization domain is a FKBP12-derived destabilization domain. [0019] In another aspect, the present disclosure provides a method of treating a subject in need thereof, comprising (i) genetically modifying a cell from the subject using the method described herein, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject. In some embodiments, the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection. In some embodiments, the cell is reintroduced into the subject by systemic transplantation. In some embodiments, the cell is reintroduced into the subject by local transplantation. In some embodiments, the local transplantation is intrafemoral or intrahepatic. In some embodiments, the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject. [0020] In another aspect, the present disclosure provides a sgRNA that specifically targets a CCR5 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 4. [0021] In another aspect, the present disclosure provides a sgRNA that specifically targets an AAVS1 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 39. In some embodiments, the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 40. [0022] In another aspect, the present disclosure provides a sgRNA that specifically targets an IgH locus, wherein the target sequence of the sgRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27, 31, and 35. In some embodiments, the sgRNA comprises a nucleotide sequence having 80% or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 28, 32, and 36. In some embodiments, the sgRNA comprises chemical modifications at one or more nucleotides. In some embodiments, the -O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides. In some embodiments, -O-methyl- -phosphorothioate (MS) modifications In some embodiments, the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47. [0023] In another aspect, the present disclosure provides a homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic antibody, operably linked to (b) a promoter and (c) a polyadenylation signal at the 3’ end of the coding sequence; (ii) a first homology region located to one side of the expression cassette within the donor template; and (iii) a second homology region located to the other side of the expression cassette within the donor template. [0024] In some embodiments, the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic. In some embodiments, the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. In some embodiments, the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain. [0025] In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 30 or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34 or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 38 or a fragment thereof. [0026] In some embodiments, the expression cassette encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. In some embodiments, the expression cassette encodes a therapeutic antibody against HIV infection. In some embodiments, the therapeutic antibody encoded by the expression cassette comprises at least one light chain and at least one heavy chain. In some embodiments, the at least one light chain and the at least one heavy chain are linked by a linker. In some embodiments, the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6. In some embodiments, the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. In some embodiments, the coding sequence comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 19- 26. [0027] In another aspect, the present disclosure provides a HSPC comprising the sgRNA, and/or the homologous donor template described herein. In another aspect, the present disclosure provides a B cell comprising the sgRNA and/or the homologous donor template described herein. [0028] In another aspect, the present disclosure provides a genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody. In some embodiments, the selected locus is a safe harbor locus. In some embodiments, the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In some embodiments, the selected locus is an immunoglobulin-associated locus. In some embodiments, the immunoglobulin-associated locus is an IgH locus, an an In some embodiments, the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11- 18. In some embodiments, the cell was modified using the method described herein. [0029] In another aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of HSPCs, a plurality of B cells, or a plurality of genetically modified cells described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0030] Figure 1: HIV Inhibiting Antibodies Maintain Function with a Peptide Linker. A) Diagram of antibody expression with and without a peptide linker. B) SDS-PAGE western blot of purified traditional and linker antibodies. C) IC50 of traditional and linker antibodies against a panel of HIV-1 pseudoviruses measured in vitro with TZM-bl infection assay (IC50 was calculated from technical duplicate infections across serial dilutions of each antibody). Pseudoviruses not shown on a graph were not inhibited within the tested antibody [0031] Figure 2: Efficient targeted integration of antibody expression cassettes at the CCR5 locus in HSPCs. A) Schematic of gene editing strategy to deliver linker antibody expression cassettes to the CCR5 locus. B) Allelic integration frequency in CB CD34+ HSPCs targeted with AAV6 cassettes for linker antibodies as indicated. Each AAV6 was used at a multiplicity of infection (MOI) of 1250. Values represent independent biologic donors with n = 6 for mock and 10-1074 conditions and n = 3 for Ibalizumab, PGDM1400, CAP256V2LS, and 10-1074+Ibalizumab conditions. C) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel 2B (n=3). D) Distribution frequency of WT, INDEL, and KI alleles in CB CD34+ HSPCs targeted at CCR5 E) Percent of knock-in alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in D (n=3). F) Distribution frequency of WT, INDEL, and KI alleles in CB CD34+ HSPCs used for the CFU assay described in G, H, and I. Cell were targeted at CCR5 G) Number of BFU-E, CFU-GM, and CFU-GEMM colonies formed per 500 cells plated in the CFU assay (n = 2, with technical duplicates wells for each donor). H) Relative frequency of BFU-E, CFU-GM, and CFU-GEMM colonies formed within the CFU assay (n=2, with technical duplicate wells for each donor). I) Frequency of genotypes from single-cell colonies within the “10- (n represents the total number of colonies genotyped across both biological donors). All replicates represent independent biological donors unless otherwise noted. All dots represent independent biologic donors. All bars represent mean and error bars represent standard deviation (SD). [0032] Figure 3: Antibody edited HSPCs maintain engraftment capacity and multilineage reconstitution in vivo. A) Diagram for editing and engraftment of CB CD34+ HSPCs in NSG mice to characterize engraftment capacity and multilineage potential. B) Distribution frequency of WT, INDEL, and KI alleles in CB CD34+ HSPCs prior to transplantation in NSG mice. Cells were targeted at CCR5 with an AAV6 MOI of 1250 for each antibody construct (n=1 pooled sample from 5 donors). C) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel b (n = 1). D) Percent human cell chimerism in the bone marrow at 16 weeks post-transplantation (n = 5 for mock, 10-1074, and 10-1074+Ibalizumab, n = 4 for Ibalizumab). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant; P = 0.3287; *P = 0.0496; **P = 0.0082). E) Percent human cell chimerism in the spleen at 16 weeks post- transplantation (n = 4 for mock, 10-1074, and 10-1074+Ibalizumab, n = 3 for Ibalizumab). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant, P = 0.0806 for Mock vs 10-1074, P = 0.1363 for Mock vs Ibalizumab; *P = 0.0216). . F) Percent of human cells in the bone marrow (n are the same as in D) or G) spleen (n are the same as in E) that are CD19+ (B cell lineage), CD33+ (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited HSPCs (10-1074, blue; Ibalizumab, purple; 10-1074 and Linker Ibalizumab, red). H) Percent of human alleles from the bone marrow or spleen with knock-in of the indicated antibody constructs (n are the same as in d and e for bone marrow and spleen, respectively). I) Percent of human alleles with knock- in from the bulk bone marrow (as shown in panel H) or in positively selected bone marrow CD19+ cells (n are the same as in D). Lines connect dots representing measurements from the same mice. Two-tailed Mann-Whitney test (*P = 0.0135). J) Percent of human alleles from the bone marrow with an INDEL at CCR5 (n are the same as in D). This analysis does not include alleles with KI. All bars represent mean. All dots represent individual mice engrafted with mock HSPCs or HSPCs edited with AAV6 and RNP for the antibody construct(s) indicated. [0033] Figure 4: HSPCs with high frequency knock-in maintain edited alleles following engraftment in vivo. A) Distribution frequency of WT, INDEL, and KI alleles in CB CD34+ HSPCs prior to transplantation in NBSGW mice. Cells were targeted at CCR5 with an AAV6 independent biologic donors; data also shown in Figure 2F). B) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel 4A (n=2). C) Percent human cell chimerism in the bone marrow 12 weeks post-transplantation, Two-tailed Mann-Whitney test (**P = 0.0040, n = 4 for mock and n = 8 for 10- 1074+Ibalizumab). D) Percent of human cells in the bone marrow that are CD19+ (B cell lineage), CD33+ (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited (red) HSPCs (n are the same as in C). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant; P = 0.1650 for CD33+ comparison, P = 0.3492 for other comparison; *P = 0.0474). E) Percent of human alleles with knock-in from the bulk bone marrow or in positively selected bone marrow CD19+ cells. Lines connect dots representing measurements from the same mice (n are the same as in c). Two- tailed Mann-Whitney test (ns, p>0.05). F) Percent of human alleles from the bone marrow with an INDEL at CCR5 (n are the same as in c). This analysis does not include alleles with KI. All bars represent mean and all error bars represent SD. All dots represent individual mice engrafted with mock HSPCs or HSPCs edited with AAV6 and RNP. [0034] Figure 5: Antibody Engineered B Cells Secrete Functional Linker Antibodies. A) Allelic integration frequency in adult peripheral blood CD19+ B Cells targeted with AAV6 cassettes for linker antibodies as indicated. Each AAV6 was used at an MOI of 25,000. Dots represent independent biologic donors, n = 9 for mock and 10-1074, n = 5 for Ibalizumab and 10-1074+Ibalizumab, n = 3 for PGDM1400 and CAP256V2LS). B) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel 5A (n=5). C) Measured IC50 for each antibody against TRO11 or CNE55 pseudoviruses as determined by TZM-bl assay in Figure 1C (NN, not-neutralizing). D) Inhibition of infection with TRO11 or E) CNE55 HIV-1 pseudovirus by culture supernatant from B cells engineered to express linker antibodies as indicated. Percent infection for each dose of supernatant from gene targeted B cells is normalized to infection at each dose of supernatant from mock B cells from the same donor (n=3 biological donors, data points and error bars represent mean with standard deviation of technical duplicate infections). [0035] Figure 6: Inhibition activity of traditional and linker antibodies against a global panel of HIV-1 pseudoviruses. A) Expected and measured IC50 of each antibody. Expected IC50 was derived from CATNAP unless otherwise noted. 89, 90 Traditional and linker antibody IC50 was determined as described in Figure 1C (data also shown in Figure 1C). [0036] Figure 7: CCR5 INDEL profile and construct specific ddPCR for integration analysis. A) Representative INDELs formed in CB CD34+ HSPCs following gene editing with CCR5 RNP. Image generated with Synthego ICE Analysis. The sequences shown from top to bottom are SEQ ID NO: 66-71, 67 and 67. B) Diagram of ddPCR primer design for measuring bulk and construct specific integration at the CCR5 locus. Bulk integration is based on the BGH-polyA signal that all antibody cassettes carry. Construct specific integration is based on IgG1 (10-1074) or IgG4 (Ibalizumab). C) Percent of KI alleles as measured by ddPCR using bulk integration or construct specific integration primers across dilutions of CB CD34+ HSPCs targeted with an IgG1 antibody construct (10-1074). D) Percent of KI alleles as measured by ddPCR using bulk integration or construct specific integration primers across dilutions of CB CD34+ HSPCs targeted with an IgG4 antibody construct (Ibalizumab, n = 1). E) Efficiency of construct specific ddPCR primers relative to bulk integration primers. Measurement efficiency is determined by dividing the construct specific integration measurement by the bulk integration measurement at the indicated dilutions of targeted cells. [0037] Figure 8: Bulk CB CD34+ HSPCs edited with three antibody cassettes simultaneously. A) Allelic integration frequency at CCR5 in CB CD34+ HSPCs targeted with AAV6 cassettes for linker antibodies as indicated (n=1 biological donor). B) Expected amplicon size for in-out PCR with primers designed to amplify each antibody individually when integrated in the CCR5 locus. C) Gel electrophoresis analysis of amplicons from in-out PCR for integration of each antibody construct. Genomic DNA from CB CD34+ HSPCs targeted as indicated for each lane was amplified simultaneously with all three sets of in-out PCR primers described in panel S3B. [0038] Figure 9: Maintenance of knockout and knock-in in xenografted NSG and NBSGW mice. A) Distribution frequency of WT, INDEL, and KI human alleles in the bone marrow of NSG mice at 16 weeks post-transplantation (Figure 3). Each number represents an individual mouse transplanted with cells targeted as indicated. B) Distribution frequency of WT, INDEL, and KI human alleles in the bone marrow of NBSGW mice at 12 weeks post- transplantation (Figure 4). Each number represents an individual mouse transplanted with cells targeted as indicated. C) Percent of KI alleles from the bulk bone marrow integrated with 10- 1074 or Ibalizumab within NBSGW mice receiving “10-1074+Ibalizumab” targeted cells (n=5). Bars represent mean and errors bars represent SD. [0039] Figure 10: Human IgG in xenografted NSG and NBSGW mice. A) Serum concentration of human IgG in NSG mice 16 weeks post-engraftment with CB CD34+ HSPCs targeted as indicated and described in Figure 3. B) Serum concentration of human IgG in NBSGW mice 12 weeks post-engraftment with CB CD34+ HSPCs targeted as indicated and described in Figure 4 (n = 4 for mock, n = 8 for 10-1074+Ibalizumab). All dots represent individual mice engrafted with mock HSPCs or HSPCs edited with AAV6 and RNP for the linker antibody construct(s) indicated. All lines represent mean. [0040] Figure 11: The EEK promoter is highly active in human B Cells. A) Schematic for integration of cassettes with the EEK promoter (left) or the ubiquitous human UBC promoter (right) driving expression of GFP (EEK-GFP and UBC-GFP). UBC-GFP is integrated using a different CCR5 sgRNA (sg-CCR5-#2) that cuts downstream within exon 2. B) Percent GFP+ cells from adult peripheral blood CD19+ B Cells targeted with AAV6 cassettes as indicated (n=1 biological donor). Each AAV6 was used at an MOI of 25,000. C) Median fluorescence intensity of GFP+ B cells targeted as indicated (n = 1). Each AAV6 was used at an MOI of 25,000. [0041] Figure 12: B cell subsets are not perturbed by expression of exogenous antibodies. A) Representative gating strategy for classification of B cell subsets. Gating derived from Velounias and Tull, 2022.91 B) Percent CD19+ positive cells from adult peripheral blood B Cells targeted with AAV6 cassettes as indicated (n=1 biological donor). Each AAV6 was used at an MOI of 25,000. “EEK-10-1074” represents integration of linker 10-1074 corresponding Figure 2A. Days are days post-editing. C) Distribution of cells within the CD19+ gate that are phenotypically plasmablasts (CD19+ CD27+ CD38+) or general B cells (CD19+ CD27+ CD38-, CD19+ CD27- CD38-, or CD19+ CD27- CD38+). D) Distribution of cells within the B cell gate that are phenotypically memory B cells (CD19+ CD27+ IgD-), marginal zone B cells (CD19+ CD27+ IgD+), Double negative B cells (CD19+ CD27- IgD-), or Naïve/Transitional B cells (CD19+ CD27- IgD-). E) Distribution of cells within the memory B cell gate that are phenotypically unswitched memory B cells (CD19+ CD27+ IgD- IgM+) or class switched memory B cells (CD19+ CD27+ IgD- IgM-). [0042] Figure 13: Engineered B cells secrete linker antibodies. Concentration of each antibody as determined by antigen specific ELISA from B cell supernatant from cells targeted with AAV6 cassettes as indicated (n=3 biological donors). Each AAV6 was used at an MOI of 25,000. B cells were plated at 1x106 cells per mL and supernatant was collected after 5 days. [0043] Figure 14: Targeted integration frequency of gene-targeted antibody (GT-Ab). A) Schematic of GT-Ab integration into CD34+ HSPCs. B) Targeted integration frequency of GT-Ab CD34+ HSPCs on day 3 post-editing measured by droplet digital PCR. C) Schematic of antibody secretion from GT-Ab B cells. D) Targeted integration frequency of GT-Ab B cells on day 3 post-editing measured by droplet digital PCR. E) Concentration of therapeutic antibodies in B cell culture supernatant 5 days after re-plating measured by ELISA. [0044] Figure 15: Expression of gene-targeted antibodies (GT-Abs) in the engineered cells. A) Concentration of therapeutic anti-PCSK9 antibody in the serum of mice 4-16 weeks post-transplantation measured by ELISA. B) Concentration of therapeutic anti- antibody in the serum of mice 4-16 weeks post-transplantation measured by ELISA. C) Total edited alleles in the spleen (SP) and peripheral blood (PB) of mice 16 weeks post-transplantation as measured by droplet digital PCR. D) Edited alleles at week 16 in the bulk spleen population of donor cells calculated by multiplying the total editing frequency measured by droplet digital PCR by the donor chimerism measured by flow cytometry. Edited alleles at week 16 in the B220+ B cells sorted from the spleens of mice measured by droplet digital PCR. E) Representative flow cytometry plots of the spleens of transplanted mice at week 16. Gated on B220+CD43-CD24+ and showing IgM and IgD expressing cells. [0045] Figure 16: Concentration of anti-PCSK9 antibodies in B cell culture supernatant measured by ELISA 5 days after re-plating of cells editing with anti-PCSK9 antibody expression cassette with fused destabilization domain with and without addition of small molecule stabilizer (1uM Shield-1). DETAILED DESCRIPTION 1. Introduction [0046] The present disclosure provides methods and compositions for disease (e.g., cancer) treatment in subjects in need through the introduction and integration at a locus of transgenes encoding therapeutic antibodies. The methods involve the introduction of single guide RNAs (sgRNAs) and RNA-guided nucleases (e.g., Cas9) into cells from the subject, as well as the introduction of homologous donor templates for transgene integration at a target locus. The transgenes encoding therapeutic antibodies can be introduced and integrate in a safe harbor locus (e.g., CCR5) or an immunoglobulin-associated locus (e.g., IgH). In particular embodiments, the RNP complexes, e.g., comprising CCR5 sgRNA and Cas9 protein, are delivered to cells via electroporation, followed by the transduction of the homologous donor template using an AAV6 viral vector. The homologous templates for repair are constructed to have arms of homology centered on the cut site within the target locus, located on either side of the coding sequence for a therapeutic antibody of interest, under the control of a designated promoter. Transcription is terminated using an exogenous polyadenylation signal. Depending on the promoter, the system can achieve, e.g., supraphysiological expression and/or cell- specific expression. This system can be used to modify any human cell, and in particular embodiments HSPCs are used. 2. General [0047] Practicing this disclosure utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this disclosure include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed.2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). [0048] For nucleic acids, sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and/or RNA can be given in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences. [0049] Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983). 3. Definitions [0050] As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0051] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth. [0052] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to “about X” specifically indicates at least the values X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” [0053] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). [0054] The term “gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The term “transgene” refers to an exogenous gene which does not naturally occur in a wild-type cell or organism but is typically introduced into the cell by molecular biological technique. [0055] The term “locus” refers to a specific location of a gene, DNA sequence, polypeptide- encoding sequence, or position on a chromosome of the genome of an organism. For example, a “CCR5 locus” may refer to the specific location of a C-C chemokine receptor type 5 (CCR5 or CD195) gene, CCR5 DNA sequence, CCR5-encoding sequence, or CCR5 position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. A “locus” may comprise a regulatory element of a gene, including, for example, an The term “safe harbor locus” refers to a locus in the genome which allows for expression of an inserted transgene without the risk of affecting surrounding endogenous genes. Examples of safe harbor loci known to exist within mammalian cells include but not limited to a CCR5 locus, an AAVS1 locus, a ROSA26 locus, and a CLYBL locus. The term “immunoglobulin- associated locus” refers to a locus in the genome which includes an immunoglobulin (Ig) gene and its regulatory elements for expressing the immunoglobulin protein. Examples of Immunoglobulin-associated loci known to exist within mammalian cells include but not limited to an IgH locus, an and an [0056] A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be an endogenous or exogenous promoter. An “endogenous promoter” refers to a promoter that occurs naturally within a cell or tissue. An “exogenous promoter” refers to a promoter that are not normally present in a cell or tissue but introduced into the cell by molecular biological technique. [0057] The promoter can also be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013/176772, herein incorporated by reference in its entirety for all purposes. [0058] A constitutive promoter is one that is active in all tissues or particular tissues at all developing stages. Examples of constitutive promoters include the human cytomegalovirus immediate early (hCMV), mouse cytomegalovirus immediate early (mCMV), human elongation factor 1 alpha (hEF1a), mouse elongation factor 1 alpha (mEF1a), mouse phosphoglycerate kinase (PGK), chicken beta actin hybrid (CAG or CBh), SV40 early, and beta 2 tubulin promoters. [0059] Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter). [0060] Tissue-specific promoters can be, for example, neuron-specific promoters, glia- specific promoters, muscle cell-specific promoters, heart cell-specific promoters, kidney cell- specific promoters, bone cell-specific promoters, endothelial cell-specific promoters, or immune cell-specific promoters (e.g., a B cell promoter or a T cell promoter). [0061] Developmentally regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell. [0062] An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism). [0063] As used herein, a first polynucleotide or polypeptide is "heterologous" to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence). [0064] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. [0065] The terms “expression” and “expressed” refer to the production of a transcriptional and/or translational product, e.g., of a therapeutic antibody and/or a nucleic acid sequence encoding a therapeutic antibody. In some embodiments, the term refers to the production of a transcriptional and/or translational product encoded by a polynucleotide or a portion thereof. The level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. [0066] The term “therapeutic antibody” relates to any antibody preparation which is intended for use in a subject (e.g., a human being) that results in the alleviation and/or a decrease in the progression of a disease in vivo. The term “antibody” refers to an antigen binding protein or a fragment thereof. The term “antibody” includes, but is not limited to, full-length antibodies, antibody fragments, chimeric antibodies, human antibodies, and humanized antibodies. Antibody diabodies, including bivalent diabodies and bispecific diabodies, trispecific mAbs bispecific T- cell engagers (BiTEs), bispecific and trispecific killer cell engagers (BiKEs and TRiKEs), and dual-affinity re-targeting antibodies (DARTs) and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134. Antibodies also include, but are not limited to, camelid antibodies and single domain antibodies (e.g., nanobodies). In certain embodiments, recombinant DNA techniques are used to produce antibodies. [0067] In some embodiments, the therapeutic antibody comprises at least one heavy chain and at least one light chain. As used herein, the term “heavy chain” refers to a polypeptide comprising sufficient heavy chain variable region sequence to confer antigen specificity either alone or in combination with a light chain. As used herein, the term “light chain” refers to a polypeptide comprising sufficient light chain variable region sequence to confer antigen specificity either alone or in combination with a heavy chain. [0068] In one implementation, heavy and light chains are expressed as separate proteins and are joined by endogenous B cell processing to form functional antibodies. In another implementation, the therapeutic antibody is expressed as a single protein. In some embodiments, the antibody is expressed as a single-chain fragment variable (scFv) that contains the antigen-binding domains of a whole antibody and which can be expressed as a single protein sequence. The single protein, e.g., scFv will comprise the variable light and variable heavy chain regions of an antibody connected by a flexible linker sequence, such as a glycine and serine-based linker sequence, for example having a length of 10-100 amino acids, for example about 15-20 amino acids. In some embodiments, the therapeutic antibody is expressed as a Fab, for example, comprising a single protein comprising an antibody light chain linked to an antibody heavy chain. The single protein, e.g., expressed as LC-L-HC or HC-L-LC will comprise the light and heavy chains of an antibody connected by a flexible linker sequence, such as a glycine and serine-based linker sequence, for example having a length of 10-100 amino acids, for example about 15-20 amino acids. In other embodiments, the antibodies are produced as the product of two protein sequences, a heavy chain gene (IgH) and a kappa (Ig or lambda (Ig , which chains are expressed and processed by the in-vivo machinery of the recipient organism to create functional antibodies. Techniques for antibody production, such as engineering B cells for antibody expression, are known in the art. (See, e.g. Moffett, Howell F., et al. "B cells engineered to express pathogen-specific antibodies protect against infection." Science immunology 4.35 (2019): eaax0644; Hartweger, Harald, et al. "HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells." Journal of Experimental Medicine 216.6 (2019): 1301-1310; Nahmad, Alessio D., et al. "Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion." Nature communications 11.1 (2020): 5851; Hung, King L., et al. "Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells." Molecular Therapy 26.2 (2018): 456-467; and Rogers GL, Huang C, et al. “Reprogramming human B cells with custom heavy chain antibodies. bioRxiv” [Preprint]. 2023 Jun 30:2023.06.28.546944. doi: 10.1101/2023.06.28.546944; each of which is hereby incorporated by reference in its entirety.) [0069] The scope of the invention encompasses the expression of an antibody, including Fab, scFv, and other versions of an antibody, or other antigen-binding variants of an antibody. In some embodiments, the antibody comprises an antibody, including Fab, scFv or other antigen binding fragment of an antibody selected from the group consisting of: Abciximab, Adalimumab, , Aducanumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Ansuvimab, Atezolizumab, Atoltivimab, Maftivimab, Odesivimab, Avelumab, Axatilimab, Basiliximab, Belantamab, Belimumab, Benralizumab, Bevacizumab, Bezlotoxumab, Bimekizumab, Blinatumomab, Brentuximab, Brodalumab, Brolucizumab, Burosumab, Camrelizumab, Canakinumab, Caplacizumab, Casirivimab, Imdevimab, Catumaxomab, Cemiplimab, Certolizumab, Cetuximab, Concizumab, Cosibelimab, Crizanlizumab, Crovalimab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarlimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Elranatamab, Emapalumab, Emicizumab, Enfortumab, Envafolimab, Epcoritamab, Eptinezumab, Erenumab, Evinacumab, Evolocumab, Faricimab, Fremanezumab, Frovocimab (LY 3015014), Galcanezumab, Garadacimab, Gemtuzumab, Glofitamab, Golimumab, Guselkumab, Ibalizumab, Ibritumomab, Idarucizumab, Inebilizumab, Infliximab, Inotuzumab, Ipilimumab, Isatuximab, Ixekizumab, Lanadelumab, Lebrikizumab, Lecanemab, Linvoseltamab, Loncastuximab, Margetuximab, Marstacimab, Mepolizumab, Mirikizumab, Mirvetuximab, Mogamulizumab, Mosunetuzumab, Moxetumomab, Narsoplimab, Natalizumab, Naxitamab, Necitumumab, Nemolizumab, Nirsevimab, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocrelizumab, Odronextamab, Ofatumumab, Olaratumab, Omalizumab, Ozoralizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab, Polatuzumab, Pozelimab, Ramucirumab, Ranibizumab, Ravulizumab, Raxibacumab, Regdanvimab, Relatlimab, Reslizumab, Retifanlimab, Risankizumab, Rituximab, Romosozumab, Rozanolixizumab, Sacituzumab, Sarilumab, Satralizumab, Secukinumab, Serplulimab, Siltuximab, Sintilimab, Sotrovimab, Spesolimab, Sugemalimab, Sutimlimab, Tafasitamab, Talquetamab, Tarlatamab, Tebentafusp, Teclistamab, Teplizumab, Teprotumumab, Tezepelumab, Tildrakizumab, Tislelizumab, Tisotumab, Tixagevimab, cilgavimab, Tocilizumab, Toripalimab, Tositumomab, Tralokinumab, Trastuzumab, Tremelimumab, Ublituximab, Ustekinumab, Vedolizumab, and Zolbetuximab. [0070] The terms “antigen,” “target molecule,” and “target” are used interchangeably to refer to molecules that are bound by an antibody. Exemplary antigens or target molecules include, but are not limited to, polypeptides, nucleic acids, and polysaccharides. [0071] The term “treating” or “treatment” refers to any one of the following: ameliorating one or more symptoms of a disease or condition (e.g., HIV); preventing the manifestation of such symptoms before they occur; slowing down or completely preventing the progression of the disease or condition (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.); enhancing the onset of a remission period; slowing down the irreversible damage caused in the progressive-chronic stage of the disease or condition (both in the primary and secondary stages); delaying the onset of said progressive stage; or any combination thereof. [0072] As used herein, the terms “subject”, “individual” or “patient” refer, interchangeably, to a warm-blooded animal such as a mammal. In particular embodiments, the term refers to a human. A subject may have, be suspected of having, or be predisposed to a lysosomal storage disorder as described herein. The term also includes livestock, pet animals, or animals kept for study, including horses, cows, sheep, poultry, pigs, cats, dogs, zoo animals, goats, primates (e.g., chimpanzee), and rodents. A “subject in need thereof” refers to a subject that has one or more symptoms of a disease or condition (e.g., HIV), that has received a diagnosis of a disease or condition, that is suspected of having or being predisposed to an disease or condition, or that is thought to potentially benefit from increased expression of a therapeutic antibody as described herein. [0073] An “effective amount” refers to an amount of a compound or composition, as disclosed herein effective to achieve a particular biological, therapeutic, or prophylactic result. Such results include, without limitation, the treatment of a disease or condition disclosed herein as determined by any means suitable in the art. [0074] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. [0075] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a therapeutic antibody can have an increased stability, assembly, or activity as described herein. [0076] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y. (1984)). [0077] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. [0078] In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence. [0079] As used in herein, the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length. [0080] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used. [0081] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. [0082] An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol.215: 403-410. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). [0083] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. 4. CRISPR/Cas system [0084] The “CRISPR/Cas” system refers to a class of bacterial systems for defense against foreign nucleic acids. CRISPR-Cas systems are found in a wide range of bacterial and archaeal organisms. CRISPR-Cas systems fall into two classes with six types, I, II, III, IV, V, and VI as well as many sub-types, with Class 1 including types I and III CRISPR systems, and Class 2 including types II, IV, V and VI; Class 1 subtypes include subtypes I-A to I-F, for example. See, e.g., Fonfara et al., Nature 532, 7600 (2016); Zetsche et al., Cell 163, 759-771 (2015); Adli et al. (2018). Endogenous CRISPR-Cas systems include a CRISPR locus containing repeat clusters separated by non-repeating spacer sequences that correspond to sequences from viruses and other mobile genetic elements, and Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage. In class 1 systems these activities are effected by multiple Cas proteins, with Cas3 providing the endonuclease activity, whereas in class 2 systems they are all carried out by a single Cas, Cas9. [0085] A “homologous repair template” refers to a polynucleotide sequence that can be used to repair a double stranded break (DSB) in the DNA, e.g., a CRISPR/Cas9-mediated break at a target locus (e.g., a CCR5 locus) as induced using the herein-described methods and compositions. The homologous repair template comprises homology to the genomic sequence surrounding the DSB, i.e., comprising CCR5 homology arms of the disclosure. In some embodiments, two distinct homologous regions are present on the template, with each region comprising at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides or more of homology with the corresponding genomic sequence. In particular embodiments, the templates comprise two homology arms comprising about 500 nucleotides of homology extending from either site of the sgRNA target site. The repair template can be present in any form, e.g., on a plasmid that is introduced into the cell, as a free floating doubled stranded DNA template (e.g., a template that is liberated from a plasmid in the cell), or as single stranded DNA. In particular embodiments of the present disclosure, the template is present within a viral vector, e.g., an adeno-associated viral vector such as AAV6. In particular embodiments, the templates comprise an expression cassette comprising a sequence encoding a therapeutic antibody (e.g., anti-HIV antibody), operably linked to a promoter, such that the expression cassette is integrated into the genome at the target locus (e.g., a CCR5 locus) and the therapeutic antibody is expressed. [0086] As used herein, “homologous recombination” or “HR” refers to insertion of a nucleotide sequence during repair of double-strand breaks in DNA via homology-directed repair mechanisms. This process uses a “donor template” or “homologous repair template” with homology to nucleotide sequence in the region of the break as a template for repairing a double-strand break. The presence of a double-stranded break facilitates integration of the donor sequence. The donor sequence may be physically integrated or used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence. This process is used by a number of different gene editing platforms that create the double-strand break, such as meganucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 gene editing systems. In particular embodiments of the present disclosure, HR involves double- stranded breaks induced by CRISPR-Cas9. [0087] The present disclosure provides methods and compositions for integrating and expressing transgenes encoding therapeutic antibodies, e.g., anti-HIV antibodies, into a safe harbor locus or an immunoglobulin-associated locus in cells from a subject in need. In particular embodiments, the cells are hematopoietic stem and progenitor cells (HSPCs) or primary B cells. The cells can be modified using the methods described herein and then reintroduced into the subject, wherein the expression of the therapeutic antibody in the modified cells in vivo can treat the subject in need. [0088] The present disclosure is based in part on the identification of CRISPR guide sequences that specifically direct the cleavage of the target locus, e.g., a CCR5 locus, by RNA- guided nucleases such as Cas9. In particular embodiments, the methods involve the introduction of ribonucleoproteins (RNPs) comprising an sgRNA targeting specific locus and Cas9, as well as a template DNA molecule comprising homology arms flanking the transgene encoding the therapeutic antibody. Using the present methods, high rates of targeted integration at the target locus and expression of the therapeutic antibodies can be achieved, with the result that the transplantation and long-term engraftment of the modified cells can lead to a treatment and a full recovery from a disease or condition (e.g., HIV). sgRNAs [0089] The single guide RNAs (sgRNAs) used in the present disclosure target a specific locus wherein the integration of transgenes allows robust expression of the therapeutic antibodies but without affecting cell physiology. In some embodiments, the locus is a safe harbor locus. In some embodiments, the locus is an immunoglobulin-associated locus. [0090] The sgRNAs used in the present disclosure interact with a site-directed nuclease such as Cas9 and specifically bind to or hybridize to a target nucleic acid within the genome of a cell, such that the sgRNA and the site-directed nuclease co-localize to the target nucleic acid in the genome of the cell. The sgRNAs as used herein comprise a targeting sequence comprising homology (or complementarity) to a target DNA sequence at a selected locus, and a constant region that mediates binding to Cas9 or another RNA-guided nuclease. The sgRNA can target any sequence within the selected locus adjacent to a PAM sequence. In some instances, the sgRNA targets a sequence within a safe harbor locus, such as within a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In other instances, the sgRNA targets a sequence within an immunoglobulin-associated locus, such as an IgH locus, an an . [0091] In some embodiments, the sgRNA targets at a CCR5 locus. In particular embodiments, the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 1. In particular embodiments, the sgRNA comprises the sequence shown as SEQ ID NO: 4, or a sequence having, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 4, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in SEQ ID NO: 4. [0092] In some embodiments, the sgRNA targets at an AAVS1 locus. In particular embodiments, the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 39. In particular embodiments, the sgRNA comprises the sequence shown as SEQ ID NO: 40, or a sequence having, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 40, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in SEQ ID NO: 40. [0093] In some embodiments, the sgRNA targets at an immunoglobulin-associated locus, such as an IgH locus, an an . In some embodiments, the sgRNA targets at an IgH locus. In particular embodiments, the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 27, 31, or 35. In particular embodiments, the sgRNA comprises the sequence shown as SEQ ID NO: 40, or a sequence having, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to any one of SEQ ID NOs: 28, 32, and 36, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in any one of SEQ ID NOs: 28, 32, and 36. [0094] The targeting sequence of the sgRNAs may be, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or 15-25, 18-22, or 19-21 nucleotides in length, and shares homology with a targeted genomic sequence, in particular at a position adjacent to a CRISPR PAM sequence. The sgDNA targeting sequence is designed to be homologous to the target DNA, i.e., to share the same sequence with the non-bound strand of the DNA template or to be complementary to the strand of the template DNA that is bound by the sgRNA. The homology or complementarity of the targeting sequence can be perfect (i.e., sharing 100% homology or 100% complementarity to the target DNA sequence) or the targeting sequence can be substantially homologous (i.e., having less than 100% homology or complementarity, e.g., with 1-4 mismatches with the target DNA sequence). [0095] Each sgRNA also includes a constant region that interacts with or binds to the site- directed nuclease, e.g., Cas9. In the nucleic acid constructs provided herein, the constant region of an sgRNA can be from about 70 to 250 nucleotides in length, or about 75-100 nucleotides in length, 75-85 nucleotides in length, or about 80-90 nucleotides in length, or 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length. The overall length of the sgRNA can be, e.g., from about 80-300 nucleotides in length, or about 80-150 nucleotides in length, or about 80-120 nucleotides in length, or about 90-110 nucleotides in length, or, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length. [0096] It will be appreciated that it is also possible to use two-piece gRNAs (cr:tracrRNAs) in the present methods, i.e., with separate crRNA and tracrRNA molecules in which the target sequence is defined by the crispr RNA (crRNA), and the tracrRNA provides a binding scaffold for the Cas nuclease. [0097] In some embodiments, the sgRNAs comprise one or more modified nucleotides. For example, the polynucleotide sequences of the sgRNAs may also comprise RNA analogs, derivatives, or combinations thereof. For example, the probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates). In some embodiments, the sgRNAs comprise 3’ phosphorothiate internucleotide linkages, 2’-O-methyl- 3’-phosphoacetate modifications, 2’-fluoro-pyrimidines, S-constrained ethyl sugar modifications, or others, at one or more nucleotides. In some embodiments, the sgRNAs -O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides (see, e.g., Hendel et al. (2015) Nat. Biotech. 33(9):985-989, the entire disclosure of which is herein incorporated by reference). In some -O-methyl- -phosphorothioate In particular embodiments, the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47. [0098] The sgRNAs can be obtained in any of a number of ways. For sgRNAs, primers can be synthesized in the laboratory using an oligo synthesizer, e.g., as sold by Applied Biosystems, Biolytic Lab Performance, Sierra Biosystems, or others. Alternatively, primers and probes with any desired sequence and/or modification can be readily ordered from any of a large number of suppliers, e.g., ThermoFisher, Biolytic, IDT, Sigma-Aldritch, GeneScript, etc. RNA-guided nucleases [0099] Any CRISPR/Cas nuclease can be used in the method, i.e., a CRISPR/Cas nuclease capable of interacting with a guide RNA and cleaving the DNA at the target site as defined by the guide RNA. In some embodiments, the nuclease is Cas9 or Cpf1. In particular embodiments, the nuclease is Cas9. The Cas9 or other nuclease used in the present methods can be from any source, so long that it is capable of binding to an sgRNA of the disclosure and being guided to and cleaving the specific sequence targeted by the targeting sequence of the sgRNA. In particular embodiments, Cas9 is from Streptococcus pyogenes. [0100] Also disclosed herein are CRISPR/Cas or CRISPR/Cpf1 systems that target and cleave DNA at the selected locus. In some embodiments, the selected locus is a safe harbor locus, such as a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In some embodiments, the selected locus is an immunoglobulin-associated locus, such as an IgH locus, an or an . An exemplary CRISPR/Cas system comprises (a) a Cas (e.g., Cas9) or Cpf1 polypeptide or a nucleic acid encoding said polypeptide, and (b) an sgRNA that hybridizes specifically to the selected locus, or a nucleic acid encoding said guide RNA. In some instances, the nuclease systems described herein, further comprises a donor template as described herein. In particular embodiments, the CRISPR/Cas system comprises an RNP comprising an sgRNA targeting a specific locus and a Cas protein such as Cas9. In some embodiments, the Cas9 is a high fidelity (HiFi) Cas9. [0101] In addition to the CRISPR/Cas9 platform (which is a type II CRISPR/Cas system), alternative systems exist including type I CRISPR/Cas systems, type III CRISPR/Cas systems, and type V CRISPR/Cas systems. Various CRISPR/Cas9 systems have been disclosed, including Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9) and Neisseria cinerea Cas9 (NcCas9) to name a few. Alternatives to the Cas system include the Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) systems. Any of the above CRISPR systems may be used to induce a single or double stranded break at a CCR5 locus to carry out the methods disclosed herein. Introducing the sgRNA and Cas protein into cells [0102] The sgRNA and nuclease can be introduced into a cell using any suitable method, e.g., by introducing one or more polynucleotides encoding the sgRNA and the nuclease into the cell, e.g., using a vector such as a viral vector or delivered as naked DNA or RNA, such that the sgRNA and nuclease are expressed in the cell. In particular embodiments, the sgRNA and nuclease are assembled into ribonucleoproteins (RNPs) prior to delivery to the cells, and the RNPs are introduced into the cell by, e.g., electroporation. RNPs are complexes of RNA and RNA-binding proteins. In the context of the present methods, the RNPs comprise the RNA- binding nuclease (e.g., Cas9) assembled with the guide RNA (e.g., sgRNA), such that the RNPs are capable of binding to the target DNA (through the gRNA component of the RNP) and cleaving it (via the protein nuclease component of the RNP). As used herein, an RNP for use in the present methods can comprise any of the herein-described guide RNAs and any of the herein-described RNA-guided nucleases. [0103] Animal cells, mammalian cells, preferably human cells, modified ex vivo, in vitro, or in vivo are contemplated. Also included are cells of other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, rats; birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys. [0104] In some embodiments, the cell is an embryonic stem cell, a stem cell, a progenitor cell, a pluripotent stem cell, an induced pluripotent stem (iPS) cell, a somatic stem cell, a differentiated cell, a mesenchymal stem cell or a mesenchymal stromal cell, a neural stem cell, a hematopoietic stem cell or a hematopoietic progenitor cell, an adipose stem cell, a keratinocyte, a skeletal stem cell, a muscle stem cell, a fibroblast, an NK cell, a B-cell, a T cell, or a peripheral blood mononuclear cell (PBMC). In particular embodiments, the cells are hematopoietic stem and progenitor cells (HSPCs), e.g., cord blood-derived (CB) or adult peripheral blood-derived (PB) HSPCs, or neuronal stem cells. In some embodiments, the cells are primary B cells. [0105] To avoid immune rejection of the modified cells when administered to a subject, the cells to be modified are preferably derived from the subject’s own cells. Thus, preferably the mammalian cells are autologous cells from the subject to be treated with the modified cells. In some embodiments, however, the cells are allogeneic, i.e., isolated from an HLA-matched or HLA-compatible, or otherwise suitable, donor. [0106] In some embodiments, cells are harvested from the subject and modified according to the methods disclosed herein, which can include selecting certain cell types, optionally expanding the cells and optionally culturing the cells, and which can additionally include selecting cells that contain the transgene integrated into the selected locus (e.g., the CCR5 locus). In some embodiments, the cells are induced to undergo differentiation, e.g., into macrophages or monocytes, using methods known in the art and as described herein. In some embodiments, such modified, selected, and/or differentiated cells are then reintroduced into the subject. [0107] Further disclosed herein are methods of using said nuclease systems to produce the modified host cells described herein, comprising introducing into the cell (a) an RNP of the disclosure that targets and cleaves DNA at the selected locus (e.g., the CCR5 locus), and (b) a homologous donor template or vector as described herein. Each component can be introduced into the cell directly or can be expressed in the cell by introducing a nucleic acid encoding the components of said one or more nuclease systems. [0108] Such methods will target integration of the transgene encoding the therapeutic antibody to the selected locus (e.g., the CCR5 locus) in a host cell ex vivo. Such methods can further comprise (a) introducing a donor template or vector into the cell, optionally after expanding said cells, or optionally before expanding said cells, and (b) optionally culturing the cell. [0109] In some embodiments, the disclosure herein contemplates a method of producing a modified mammalian host cell, the method comprising introducing into a mammalian cell: (a) an RNP comprising a Cas nuclease such as Cas9 and an sgRNA specific to the selected locus (e.g., the CCR5 locus), and (b) a homologous donor template or vector as described herein. [0110] In any of these methods, the nuclease can produce one or more single stranded breaks within the selected locus (e.g., the CCR5 locus), or a double stranded break within the selected locus (e.g., the CCR5 locus). In these methods, the selected locus (e.g., the CCR5 locus) is modified by homologous recombination with said donor template or vector to result in insertion of the transgene into the locus. The methods can further comprise (c) selecting cells that contain the transgene integrated into the selected locus (e.g., the CCR5 locus). [0111] Techniques for insertion of transgenes, including large transgenes, capable of expressing functional proteins, including enzymes, cytokines, antibodies, and cell surface receptors are known in the art. (See, e.g. Bak and Porteus, Cell Rep. 2017 Jul 18; 20(3): 750– 756 (integration of EGFR); Kanojia et al., Stem Cells. 2015 Oct;33(10):2985-94 (expression of anti-Her2 antibody); Eyquem et al., Nature. 2017 Mar 2;543(7643):113-117 (site-specific integration of a CAR); O’Connell et al., 2010 PLoS ONE 5(8): e12009 (expression of human IL-7); Tuszynski et al., Nat Med. 2005 May;11(5):551-5 (expression of NGF in fibroblasts); Sessa et al., Lancet. 2016 Jul 30;388(10043):476-87 (expression of arylsulfatase A in ex vivo gene therapy to treat MLD); Rocca et al., Science Translational Medicine 25 Oct 2017: Vol.9, Issue 413, eaaj2347 (expression of frataxin); Bak and Porteus, Cell Reports, Vol. 20, Issue 3, 18 July 2017, Pages 750-756 (integrating large transgene cassettes into a single locus), Dever et al., Nature 17 November 2016: 539, 384-389 (adding tNGFR into hematopoietic stem cells (HSC) and HSPCs to select and enrich for modified cells); each of which is hereby incorporated by reference in its entirety.) Homologous donor templates [0112] The transgene to be integrated is typically present within a homologous repair template, or homologous donor template. The transgene can be any transgene encoding a therapeutic antibody has a beneficial effect in subjects in need. In some embodiments, the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. In particular embodiments, the transgene encodes a therapeutic antibody against HIV infection. [0113] In some embodiments, the transgene is flanked in the template by homology regions of the selected locus. In some instances, the selected locus is a safe harbor locus, such as a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. In other instances, the selected locus is an immunoglobulin-associated locus, such as an IgH locus, an an . [0114] In some embodiments, the transgene is flanked in the template by homology regions of a CCR5 locus. In such embodiments, an exemplary template can comprise, in linear order: a first CCR5 homology region, a promoter, a coding sequence for a therapeutic antibody, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH-PolyA), and a second CCR5 homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site. In particular embodiments, one of the homology regions comprises the sequence of SEQ ID NO: 2, and the other homology region comprises the sequence of SEQ ID NO: 3, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 2 and/or SEQ ID NO: 3. [0115] In some embodiments, the transgene is flanked in the template by homology regions of an AAVS1 locus. In such embodiments, an exemplary template can comprise, in linear order: a first AAVS1 homology region, a promoter, a coding sequence for a therapeutic antibody, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH- PolyA), and a second AAVS1 homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site. In particular embodiments, one of the homology regions comprises the sequence of SEQ ID NO: 41, and the other homology region comprises the sequence of SEQ ID NO: 42, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 41 and/or SEQ ID NO: 42. [0116] In some embodiments, the transgene is flanked in the template by homology regions of an immunoglobulin-associated locus. In particular embodiments, the immunoglobulin- associated locus is an IgH locus. In such embodiments, an exemplary template can comprise, in linear order: a first IgH homology region, a promoter, a coding sequence for a therapeutic antibody, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH- PolyA), and a second IgH homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site. In some instances, one of the homology regions comprises the sequence of SEQ ID NO: 28, and the other homology region comprises the sequence of SEQ ID NO: 29, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 28 and/or SEQ ID NO: 29. In other instances, one of the homology regions comprises the sequence of SEQ ID NO: 33, and the other homology region comprises the sequence of SEQ ID NO: 34, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 33 and/or SEQ ID NO: 34. In yet other instances, one of the homology regions comprises the sequence of SEQ ID NO: 36, and the other homology region comprises the sequence of SEQ ID NO: 37, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 36 and/or SEQ ID NO: 37. [0117] To facilitate homologous recombination, the transgene is flanked within the polynucleotide or donor construct by sequences homologous to the target genomic sequence, i.e., CCR5. In one particular embodiment, the transgene is flanked by sequences in a CCR5 locus surrounding the site of cleavage as defined by sgRNA. In such embodiment, the transgene is flanked on one side by a sequence comprising SEQ ID NO: 2 or a fragment thereof, and on the other side by a sequence comprising SEQ ID NO: 3 or a fragment thereof. The homology regions can be of any size, e.g., 50-2000, 100-1500 bp, 300-900 bp, 400-600 bp, or about 50, 100, 200, 300, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more bp. [0118] Any suitable method can be used to introduce the polynucleotide, or donor construct, into the cell. In particular embodiments, the polynucleotide is introduced using a recombinant adeno-associated viral vector (rAAV). For example, the rAAV can be from serotype 1 (e.g., an rAAV1 vector), 2 (e.g., an rAAV2 vector), 3 (e.g., an rAAV3 vector), 4 (e.g., an rAAV4 vector), 5 (e.g., an rAAV5 vector), 6 (e.g., an rAAV6 vector), 7 (e.g., an rAAV7 vector), 8 (e.g., an rAAV8 vector), 9 (e.g., an rAAV9 vector), 10 (e.g., an rAAV10 vector), or 11 (e.g., an rAAV11 vector). In particular embodiments, the vector is an rAAV6 vector. In some instances, the donor template is single stranded, double stranded, a plasmid or a DNA fragment. In some instances, plasmids comprise elements necessary for replication, including a promoter and optionally a 3’ UTR. [0119] Further disclosed herein are vectors comprising (a) one or more nucleotide sequences homologous to the selected locus, and (b) a transgene encoding a therapeutic factor of the disclosure. The vector can be a viral vector, such as a retroviral, lentiviral (both integration competent and integration defective lentiviral vectors), adenoviral, adeno-associated viral or herpes simplex viral vector. Viral vectors may further comprise genes necessary for replication of the viral vector. [0120] In some embodiments, the targeting construct comprises: (1) a viral vector backbone, e.g. an AAV backbone, to generate virus; (2) arms of homology to the target site of at least 200 bp but ideally at least 400 bp on each side to assure high levels of reproducible targeting to the site (see, Porteus, Annual Review of Pharmacology and Toxicology, Vol. 56:163-190 (2016); which is hereby incorporated by reference in its entirety); (3) a transgene encoding a therapeutic antibody and capable of expressing the therapeutic antibody; (4) an expression control sequence operably linked to the transgene; and optionally (5) an additional marker gene to allow for enrichment and/or monitoring of the modified host cells. Any AAV known in the art can be used. In some embodiments the primary AAV serotype is AAV6. [0121] Suitable marker genes are known in the art and include Myc, HA, FLAG, GFP, truncated NGFR, truncated EGFR, truncated CD20, truncated CD19, as well as antibiotic resistance genes (e.g., pac (puromycin-N-acetyl transferase), aph (aminoglycoside phosphotransferase), or bsd (blasticidin S deaminase), providing resistance to puromycin, G418, and blasticidin, respectively). [0122] In any of the preceding embodiments, the donor template or vector comprises a nucleotide sequence homologous to a fragment of the selected locus, wherein the nucleotide sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 or more consecutive nucleotides of the selected locus, e.g., a CCR5 locus. Safety switch [0123] In some embodiments, the inserted construct can also include a safety switch, such as a standard suicide gene, (e.g., inducible Caspase 9) into the locus in circumstances where rapid removal of cells might be required due to acute toxicity. The present disclosure provides a robust safety switch so that any engineered cell transplanted into a body can be eliminated, e.g., by removal of an auxotrophic factor. This is especially important if the engineered cell has transformed into a cancerous cell. Non-limiting examples of safety switches include inducible Caspase 9 (iCaspase9), such as, e.g., those described in Martin RM, et al. Nat. Commun. 2020;11(1):2713, and Straathof KC, et al. Blood. 2005;105(11):4247-4254. [0124] As disclosed herein, in some embodiments, the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9 (iCaspase9). In some embodiments, the inducible Caspase 9 is a Caspase9-FKBPF36V. Treatment with small-molecule AP20187 dimerizes the Caspase9-FKBPF36V protein leading to rapid, irreversible apoptosis of the cell. Destabilization Domain (DD) [0125] In some embodiments, the inserted construct includes a destabilization domain (DD), such as a FKBP12-derived destabilization domain. A destabilization domain (DD) refers to an amino acid sequence that is inherently unstable under physiological conditions and is efficiently targeted for protein degradation. When fused to a protein of interest (POI), the entire fusion product is rapidly degraded under untreated conditions. However, the DD-POI fusion protein can be stabilized upon the addition of a small molecule ligand (e.g., Shield1 ligand) which binds to and stabilizes the DD, allowing for quick, conditional regulation of the POI. [0126] The present disclosure provides a genetically modified cell comprising an expression cassette encoding a fusion protein, such as, e.g., a therapeutic antibody fused to a DD. In some embodiments, the DD is a FKBP12-derived destabilization domain. The fusion protein comprising the therapeutic antibody and the DD is either undetectable or detected at a very low level in the cell under normal conditions. However, upon treatment with a small molecule, such as a Shield1 ligand, the fusion protein is rescued from proteasomal degradation, resulting in rapid accumulation of the fusion protein. In some embodiments, the small molecule stabilizer is a Shield1 ligand. An exemplary structure of a Shield1 ligand is shown below: . 5. Safe Harbor Loci [0127] The present disclosure provides methods and compositions for introducing and integrating a transgene encoding therapeutic antibody at a selected locus. In some embodiments, the selected locus is a safe harbor locus. Examples of safe harbor loci known to exist within mammalian cells include, but are not limited to, a CCR5 locus, an AAVS1 locus, a ROSA26 locus, and a CLYBL locus. [0128] In some embodiments, the safe harbor locus is a human C-C chemokine receptor type 5 (CCR5) locus (chromosome 3 p21.31). In such embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome, whereupon the nuclease cleaves the CCR5 locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. In some embodiments, the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof. In some embodiments, the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. [0129] In some embodiments, the safe harbor locus is a human adeno-associated virus integration site 1 (AAVS1) locus (chromosome 19 q13.42). In such embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the AAVS1 locus, an RNA- guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the AAVS1 locus in the genome, whereupon the nuclease cleaves the AAVS1 locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved AAVS1 locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. In some embodiments, the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof. In some embodiments, the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. [0130] In some embodiments, the safe harbor locus is a human reverse orientation splice acceptor 26 (ROSA26) locus (chromosome 3 p25.3). In such embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the ROSA26 locus, an RNA- guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the ROSA26 locus in the genome, whereupon the nuclease cleaves the ROSA26 locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved ROSA26 locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. [0131] In some embodiments, the safe harbor locus is the human citrate lyase beta like (CLYBL) locus (chromosome 13 q32.3). In such embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CLYBL locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the CLYBL locus in the genome, whereupon the nuclease cleaves the CLYBL locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CLYBL locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. 6. Immunoglobulin-Associated Loci [0132] The methods and compositions disclosed herein can be also used to introduce and express a therapeutic antibody at an immunoglobulin-associated locus. Examples of immunoglobulin-associated loci known to exist within mammalian cells include, but are not limited to, an IgH locus, an and an . [0133] In some embodiments, the immunoglobulin-associated locus is a human immunoglobulin heavy locus (IgH, chromosome 14 q32.33). Examples of expressing antibody in a human immunoglobulin heavy locus, include but not limited to, as those described in Moffett HF, et al. Science Immunology. 2019;4(35) and Nahmad AD, et al. Nature Communications, 2020;11(1):5851, each of which is hereby incorporated by reference in its entirety. In some embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting an IgH locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at the IgH locus in the genome, whereupon the nuclease cleaves the IgH locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved IgH locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. In some embodiments, the target sequence of the sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 27, 31 and 35. In some embodiments, the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 28, 32 or 36. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 28, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29, or a fragment thereof. In some embodiments, the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof. the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof. [0134] In some embodiments, the immunoglobulin-associated locus is the human immunoglobulin light locus chromosome 2 p11.2). Examples of expressing antibody in a , include but not limited to, as those described in Moffett HF, et al. Science Immunology. 2019;4(35) and Nahmad AD, et al. Nature Communications. 2020;11(1):5851, each of which is hereby incorporated by reference in its entirety. In some embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting an locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at locus in the genome, whereupon the nuclease cleaves locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. [0135] In some embodiments, the immunoglobulin-associated locus is the human immunoglobulin light locus chromosome 22 p11.2). Examples of expressing antibody in a can include but not limited to as those described in Moffett HF, et al. Science Immunology. 2019;4(35) and Nahmad AD, et al. Nature Communications. 2020;11(1):5851, each of which is hereby incorporated by reference in its entirety. In some embodiments, a method of genetically modifying a cell from a subject is provided, comprising introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting an locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein the sgRNA binds to the nuclease and directs it to a target sequence at locus in the genome, whereupon the nuclease cleaves locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. 7. Therapeutic Antibody [0136] The system disclosed herein can be used to introduce and express any therapeutic antibody within the genomically modified cells in subjects in need. In some embodiments, the therapeutic antibody binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. In some embodiments, the coding sequence encodes an anti-HIV antibody, an anti-CD4 antibody, an anti-TNF- antibody, an anti-PCSK9 antibody, or any other therapeutic antibody beneficial to the subjects in need. Other potential therapeutic antibodies include, but not limit to, any antibodies binding to RSV, Ebola virus, SARS-CoV2, Angiopoietin-like 3, PD-1, PD-L1, C1S, etc. Therapeutic antibodies suitable for the present disclosure can be referenced from https://www.antibodysociety.org/resources/approved- antibodies/. In some embodiments, the therapeutic antibody comprises an antibody selected from the group consisting of: Abciximab, Adalimumab, Aducanumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Ansuvimab, Atezolizumab, Atoltivimab, Maftivimab, Odesivimab, Avelumab, Axatilimab, Basiliximab, Belantamab, Belimumab, Benralizumab, Bevacizumab, Bezlotoxumab, Bimekizumab, Blinatumomab, Brentuximab, Brodalumab, Brolucizumab, Burosumab, Camrelizumab, Canakinumab, Caplacizumab, Casirivimab, Imdevimab, Catumaxomab, Cemiplimab, Certolizumab, Cetuximab, Concizumab, Cosibelimab, Crizanlizumab, Crovalimab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarlimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Elranatamab, Emapalumab, Emicizumab, Enfortumab, Envafolimab, Epcoritamab, Eptinezumab, Erenumab, Evinacumab, Evolocumab, Faricimab, Fremanezumab, Frovocimab (LY 3015014), Galcanezumab, Garadacimab, Gemtuzumab, Glofitamab, Golimumab, Guselkumab, Ibalizumab, Ibritumomab, Idarucizumab, Inebilizumab, Infliximab, Inotuzumab, Ipilimumab, Isatuximab, Ixekizumab, Lanadelumab, Lebrikizumab, Lecanemab, Linvoseltamab, Loncastuximab, Margetuximab, Marstacimab, Mepolizumab, Mirikizumab, Mirvetuximab, Mogamulizumab, Mosunetuzumab, Moxetumomab, Narsoplimab, Natalizumab, Naxitamab, Necitumumab, Nemolizumab, Nirsevimab, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocrelizumab, Odronextamab, Ofatumumab, Olaratumab, Omalizumab, Ozoralizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab, Polatuzumab, Pozelimab, Ramucirumab, Ranibizumab, Ravulizumab, Raxibacumab, Regdanvimab, Relatlimab, Reslizumab, Retifanlimab, Risankizumab, Rituximab, Romosozumab, Rozanolixizumab, Sacituzumab, Sarilumab, Satralizumab, Secukinumab, Serplulimab, Siltuximab, Sintilimab, Sotrovimab, Spesolimab, Sugemalimab, Sutimlimab, Tafasitamab, Talquetamab, Tarlatamab, Tebentafusp, Teclistamab, Teplizumab, Teprotumumab, Tezepelumab, Tildrakizumab, Tislelizumab, Tisotumab, Tixagevimab, cilgavimab, Tocilizumab, Toripalimab, Tositumomab, Tralokinumab, Trastuzumab, Tremelimumab, Ublituximab, Ustekinumab, Vedolizumab, and Zolbetuximab. In particular embodiments, the transgene encodes a therapeutic antibody against HIV infection. In some embodiments, the genomically modified cell encodes an anti- therapeutic antibody (e.g., for autoimmune disease treatment). In some embodiments, the genomically modified cell encodes an anti-PCSK9 therapeutic antibody (e.g., for hypercholesterolemia treatment). [0137] In some embodiments, the antibody is a bNAb targeting an HIV virus. In some embodiments, the antibody is selected from the group consisting of 2F5, 4E10, F105, hNM01, KD-247 3BNC117, 10-1074, VRC01 VRC-HIVMAB080-00-AB (VRC01LS), VRC- HIVMAB075-00-AB (VRC07-523LS), VRC-HIVMAB095-00-AB (10E8VLS), PGT121, PGDM1400, PGT121.414.LS CAP256V2LS (VRC-HIVMAB0102-00-AB), VRC- HIVMAB091-00-AB (N6LS), 10E8.4/iMab, and 3BNC117. In some embodiments, the antibody is a bNAb targeting an influenza virus. In some embodiments, the antibody is selected from the group consisting of: C179 (4hlz), CR6261 (3gbn), F10 (3fku), CR8020 (3sdy), CR8043 (4nm8), FI6 (3ztn), CR9114 (4fqy), 39.29 (4kvn), MEDI8852 (5jw4), CT149 (4r8w), 56.a.09 (59k9), 31.b.09 (5k9o), 16.a.26 (5k9q), 16.g.07 (5kan), 31.a.83 (5kaq), C05 (4fqr), CR8059 (4fqk, stabilized variant of CR8071), 6F12, C179, CR6261, 9H10, CT-P27, VIS410, 39.29, 5A7, CR8033, CR8071, MHAA4549A, and 46B8. In some embodiments, the antibody is a bNAb targeting a SARS-CoV-2 virus. In some embodiments, the antibody is selected from the group consisting of bebtelovimab, etesevimab, Omi-42, AZD3152, cilgavimab, S304, IY- 2A, and EY6A. In some embodiments, the antibody is a bNAb targeting a tuburcolosis virus. In some embodiments, the antibody is selected from the group consisting of p4-36, p4-170, T1AM09 (aka AM009) and L1AM04. [0138] In some embodiments, the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain. In some embodiments, the at least one light chain and the at least one heavy chain are linked by a linker. The linker can be a cleavable or uncleavable linker. In some embodiments, the linker comprises a sequence having 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to SEQ ID NO: 5. In some embodiments, the linker comprises a sequence having 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to SEQ ID NO: 6. [0139] In some embodiments, the therapeutic antibody comprises an amino acid sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18, e.g., a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of SEQ ID NOs: 11-18. In such embodiments, the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26., e.g., a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of SEQ ID NOs: 19-26. In particular embodiments, the therapeutic antibody comprises an amino acid sequence of any one of SEQ ID NOs: 11-18. In particular embodiments, the transgene comprises a nucleotide sequence of any one of SEQ ID NOs: 19-26. Expression cassette [0140] In some embodiments, the transgene encoding a therapeutic antibody is present within an expression cassette when introduced into the cell. In some embodiments, the expression cassette comprises a coding sequence for the therapeutic antibody. In some embodiments, the coding sequence for the therapeutic antibody comprises at least one sequence encoding a light chain and at least one sequence encoding a heavy chain. In some embodiments, the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain. [0141] In some embodiments, the expression cassette comprises a coding sequence for the therapeutic antibody., operably linked to a promoter. As disclosed herein, any promoter that can induce expression of the therapeutic antibody in the cell can be used, including endogenous and exogenous promoters, inducible promoters, constitutive promoters, cell-specific promoters, and others. In some embodiments, the promoter is a B-cell specific promoter. In some embodiments, the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof. In some embodiments, the EEK promoter comprises a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 7. In some embodiments, the B29 promoter comprises a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 8. In some embodiments, the IgH promoter comprises a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity SEQ ID NO: 9 or 10. [0142] Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, -actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Commonly used promoters including the CMV (cytomegalovirus) promoter/enhancer, EF1a (elongation - -a - globin), Ubiquitin C and PGK, all of which provide constitutively active, high-level gene expression in most cell types. Other constitutive promoters are known to those of ordinary skill in the art. [0143] Inducible promoters are activated in the presence of an inducing agent. For example, the metallothionein promoter is activated to increase transcription and translation in the presence of certain metal ions. Other inducible promoters include alcohol-regulated, tetracycline-regulated, steroid-regulated, metal-regulated, nutrient-regulated promoters, and temperature-regulated promoters. [0144] Tissue-specific and/or physiologically regulated expression can also be pursued by modifying mRNA stability and/or translation efficiency (post-transcriptional targeting) of the transgenes. Alternatively, the incorporation of miRNA target recognition sites (miRTs) into the expressed mRNA has been used to recruit the endogenous host cell machinery to block transgene expression (detargeting) in specific tissues or cell types. miRNAs are noncoding RNAs, approximately 22 nucleotides, that are fully or partially region of particular mRNA, referred to as miRTs. Binding of a miRNA to its particular miRTs promotes translational attenuation/inactivation and/or degradation. Regulation of expression through miRNAs is described in Geisler and Fechner, World J Exp Med. 2016 May 20, 6(2): 37–54; Brown and Naldini, Nat Rev Genet. 2009 Aug, 10(8):578-85; Gentner and Naldini, Tissue Antigens. 2012 Nov, 80(5):393-403. [0145] In some instances, in addition to the promoter, the expression cassette is optionally linked to one or more regulatory elements such as enhancers or post-transcriptional regulatory sequences such as a polyadenylation (polyA) fragment. For example, one can include regulatory sequences (microRNA (miRNA) target sites) in the RNA to avoid expression in certain tissues (post-transcriptional targeting). In some embodiments, the expression cassette is further linked to an exogenous polyadenylation (polyA) fragment. In particular embodiments, the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment. [0146] In some embodiments, the expression cassette further comprises a signal sequence encoding a signal peptide. In some instances, the signal sequence is present at the 5’ end of the coding sequence for the therapeutic antibody. In other instances, the signal sequence is present at the 3’ end of the coding sequence for the therapeutic antibody. The signal peptide prompts to translocate the synthesized therapeutic antibody to the cellular membrane and/or secret out of the cell. In some embodiments, the signal peptide is cleaved from the therapeutic antibody after translocation. [0147] In some instances, the expression control sequence functions to express the therapeutic transgene following the same expression pattern as in normal individuals (physiological expression) (See Toscano et al., Gene Therapy (2011) 18, 117–127 (2011), incorporated herein by reference in its entirety for its references to promoters and regulatory sequences). 8. Methods of Treatment [0148] Following the integration of the transgene into the genome of the cell, e.g., HSPC, and confirming expression of the encoded therapeutic antibody, a plurality of modified cells can be reintroduced into the subject, such that they can repopulate and differentiate into, e.g., macrophages or monocytes, and due to the expression of the therapeutic antibody, can improve one or more abnormalities or symptoms in the subject in need. In some embodiments, the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection. In some embodiments, the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject. [0149] Disclosed herein, in some embodiments, are methods of treating an individual in need thereof, the method comprising providing to the individual one-time therapy using the genome modification methods disclosed herein. In some instances, the method comprises reintroducing a modified host cell comprising a transgene encoding a therapeutic antibody integrated at the selected locus, back into the individual for the treatment. [0150] In some embodiments, the guide RNA displays off-target activity (e.g., > 0.1% indels) at less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 location. In particular embodiments, the off-target activity occurs at less than 4, 3, 2, or 1 location. In particular embodiments, the off-target activity occurs at 1 or 0 locations when a HiFi Cas9 is used. [0151] In some embodiments, following introduction of the guide RNA, RNA-guided nuclease, and donor template, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of the targeted cells comprise an integrated transgene. In some embodiments, following transplantation of the genetically modified cells, chimerism in the subject is at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%). Cells [0152] Disclosed herein, in some embodiments, are genetically modified cells. In some embodiments, the genetically modified cell is a hematopoietic stem and progenitor cell (HSPC). In some embodiments, the genetically modified cell isa primary B cell. [0153] In some embodiments, an HSPC comprises a sgRNA, an RNA-guided nuclease (e.g., Cas9), and/or a homologous donor template as disclosed herein. In some embodiments, a B cell comprises a sgRNA, an RNA-guided nuclease (e.g., Cas9), and/or a homologous donor template as disclosed herein. [0154] In some embodiments, a genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody. In some instances, the selected locus is a safe harbor locus, including but not limited to, a CCR5 locus, an AAVS1 locus, and a ROSA26 locus. In other instances, the selected locus is an immunoglobulin-associated locus, including but not limited to, an IgH locus, an and an . In some embodiments, the genetically modified cell expresses a thereutic antibody. In some embodiments, the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. In some embodiments, the genetically modified cell further comprises a sequence encoding an inducible Caspase 9. In some embodiments, the genetically modified cell comprises a sequence encoding a fusion protein comprising an therapeutic antibody fused to a destabilization domain (DD). Pharmaceutical compositions [0155] Disclosed herein, in some embodiments, are methods, compositions and kits for use of the modified cells, including pharmaceutical compositions, therapeutic methods, and methods of administration. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any animals. [0156] In some embodiments, a pharmaceutical composition comprises a modified autologous host cell of the disclosure is provided. The modified autologous host cell is genetically engineered to comprise an integrated transgene encoding the therapeutic antibody at the selected locus. The modified host cell of the disclosure herein may be formulated using one or more excipients to, e.g.: (1) increase stability; (2) alter the biodistribution (e.g., target the cell line to specific tissues or cell types); (3) alter the release profile of an encoded therapeutic factor. In some embodiments, a pharmaceutical composition comprises a plurality of HSPCs, a plurality of B cells, or a plurality of genetically modified cells as disclosed herein. [0157] Formulations of the present disclosure can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof. Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions including at least one active ingredient (e.g., a modified host cell) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutical compositions of the present disclosure may be sterile. [0158] Relative amounts of the active ingredient (e.g., the modified host cell), a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may include between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may include between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5- 80%, or at least 80% (w/w) active ingredient. [0159] Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. [0160] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof. [0161] Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. [0162] The modified host cells of the present disclosure included in the pharmaceutical compositions described above may be administered by any delivery route, systemic delivery or local delivery, which results in a therapeutically effective outcome. These include, but are not limited to, enteral, gastroenteral, epidural, oral, transdermal, intracerebral, intracerebroventricular, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intra- arterial, intramuscular, intracardiac, intraosseous, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavernous), interstitial, intra-abdominal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrathecal, intratubular, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, soft tissue, and topical. In particular embodiments, the cells are transplanted intrafemorally or intrahepatically. In certain embodiments, the composition may take the form of solid, semi- solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. In some embodiments, the cell is reintroduced into the subject by systemic transplantation. In some embodiments, the cell is reintroduced into the subject by local transplantation. In some embodiments, the local transplantation is intrafemoral or intrahepatic. [0163] In some embodiments, a subject will undergo a conditioning regime before cell transplantation. For example, before hematopoietic stem cell transplantation, a subject may undergo myeloablative therapy, non-myeloablative therapy or reduced intensity conditioning to prevent rejection of the stem cell transplant even if the stem cell originated from the same subject. The conditioning regime may involve administration of cytotoxic agents. The conditioning regime may also include immunosuppression, antibodies, and irradiation. Other possible conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318(5854) Science 1296-9 (2007); Palchaudari et al., 34(7) Nature Biotechnology 738- 745 (2016); Chhabra et al., 10:8(351) Science Translational Medicine 351ra105 (2016)) and CAR T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2018); each of which is hereby incorporated by reference in its entirety). For example, conditioning needs to be used to create space in the brain for microglia derived from engineered hematopoietic stem cells (HSCs) to migrate in to deliver the protein of interest (as in recent gene therapy trials for ALD and MLD). The conditioning regimen is also designed to create niche “space” to allow the transplanted cells to have a place in the body to engraft and proliferate. In HSC transplantation, for example, the conditioning regimen creates niche space in the bone marrow for the transplanted HSCs to engraft. Without a conditioning regimen, the transplanted HSCs cannot engraft. [0164] Certain aspects of the present disclosure are directed to methods of providing pharmaceutical compositions including the modified host cell of the present disclosure to target tissues of mammalian subjects, by contacting target tissues with pharmaceutical compositions including the modified host cell under conditions such that they are substantially retained in such target tissues. In some embodiments, pharmaceutical compositions including the modified host cell include one or more cell penetration agents, although “naked” formulations (such as without cell penetration agents or other agents) are also contemplated, with or without pharmaceutically acceptable excipients. [0165] The present disclosure additionally provides methods of administering modified host cells in accordance with the disclosure to a subject in need thereof. The pharmaceutical compositions including the modified host cell, and compositions of the present disclosure may be administered to a subject using any amount and any route of administration effective for preventing, treating, or managing the diseases or conditions. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. The specific therapeutically or prophylactically effective dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration; the duration of the treatment; drugs used in combination or coincidental with the specific modified host cell employed; and like factors well known in the medical arts. [0166] In certain embodiments, modified host cell pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from, e.g., about 1 x 104 to 1 x 105, 1 x 105 to 1 x 106, 1 x 106 to 1 x 107, or more modified cells to the subject, or any amount sufficient to obtain the desired therapeutic or prophylactic, effect. The desired dosage of the modified host cells of the present disclosure may be administered one time or multiple times. In some embodiments, delivery of the modified host cell to a subject provides a therapeutic effect for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. [0167] The modified host cells may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, either sequentially or concurrently. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. [0168] Use of a modified mammalian host cell according to the present disclosure for treatment of a lysosomal disease, disorder or condition is also encompassed by the disclosure. [0169] The present disclosure also contemplates kits comprising compositions or components of the disclosure, e.g., sgRNA, Cas9, RNPs, and/or homologous templates, as well as, optionally, reagents for, e.g., the introduction of the components into cells. The kits can also comprise one or more containers or vials, as well as instructions for using the compositions in order to modify cells and treat subjects according to the methods described herein. 9. Examples [0170] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1. Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies Abstract: [0171] Autologous transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is extremely limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously. Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation. Introduction: [0172] Here, we seek to combine the success of CCR5 KO HSCT with the potency of sustained antibody therapy through a simultaneous knockout knock-in gene editing strategy in HSPCs. Our system is designed to deliver broad resistance against CCR5-tropic HIV-1 through CCR5 KO and to include additional layers of non-cell-autonomous protection through the secretion of multiple HIV-1 inhibiting antibodies that act broadly against both R5- and X4- tropic viruses. To combat the formation of escape mutants, we designed our system for use with multiple well-established antibodies targeting diverse epitopes. In this study, we demonstrate that HSPCs can be efficiently edited at the CCR5 locus for knock-in of multiple HIV-1 inhibiting antibody expression cassettes. Importantly, these engineered HSPCs maintain their engraftment capacity and multilineage potential following transplantation in immunodeficient mice. Moreover, we show that primary human B cells carrying the antibody expression cassettes secrete neutralizing levels of antibodies in vitro. Overall, this work establishes a strategy for autologous transplantation of HSPCs modified for long-term secretion of therapeutic antibodies from B cell progeny. Results: HIV-1 inhibiting antibodies maintain function with a peptide linker. [0173] Recent trials investigating direct injection of anti-HIV-1 antibodies to control disease highlight the need to use multiple antibodies targeting different epitopes in combination to prevent viral escape.17-19 To this end, we utilize well-established antibodies including the clinically approved antibody Ibalizumab and several bNAbs that target different highly conserved regions of the HIV-1 envelope: 10-1074 (V3 Loop), PGDM1400 (V1/2 Loop), CAP256V2LS (V1/2 Loop), 3BNC117 (CD4 binding site), and 1-18 (CD4 binding site).51-55 [0174] While traditional production of these IgG antibodies involves transfection of cell culture with two separate plasmids encoding the IgG heavy and light chain, the delivery system demonstrated in this example encompasses the delivery of the antibody components as a single transcript for knock-in to the target locus, in this example, the CCR5 locus. We employed a linker system to physically pair the therapeutic antibody heavy and light chain upon knock-in to mitigate the risk for mispairing and the potential formation of deleterious antibody products (Figure 1A).56 [0175] To determine if the addition of the linker impacts function, each antibody was produced in the conventional manner from a separate cassette for the light and heavy chains (traditional antibody) and from a single cassette with a linker pairing the light and heavy chains (linker antibody) (Figure 1A). Purified antibodies were applied to western blot analysis under reducing conditions and stained for IgG. Traditional antibodies were detected at the expected mass for a heavy chain alone at approximately 50 kDa, while linker antibodies formed a band with an expected higher molecular weight at 80kDa, corresponding to the increased mass of the linker and light chain paired with the heavy chain (Figure 1B). Each antibody was then tested against a panel of HIV-1 pseudoviruses representing the global diversity of HIV-1 in a TZM-bl infection assay to ensure neutralization potency was preserved with the added linker.57 The traditional IgG and linker versions of 10-1074, Ibalizumab, PGDM1400, and CAP256V2LS show comparable efficacy as demonstrated by measured half-maximal inhibitory concentration (IC50) for each pseudovirus (Figure 1C and 6). Two antibodies, 3BNC117 and 1-18, demonstrated a marked reduction in neutralization efficacy when expressed with a peptide linker. Interestingly, these two antibodies both target the CD4 binding site on HIV-1, indicating that further optimization may be needed to employ a linker for this subset of antibodies. Overall, these results demonstrate that therapeutic antibodies can maintain function when expressed from a single transcript with a peptide linker physically pairing the light and heavy chains. Simultaneous CCR5 KO and Antibody Cassette Knock-in in CD34+ HSPCs. [0176] The modification of CD34+ HSPCs is a promising strategy to deliver long-term HIV- 1 treatment through hematopoietic reconstitution with resistant cells. However, high-efficiency editing is needed to mitigate the potential for escape mutants to form in non-edited cells and to deliver therapeutically relevant antibody levels. To this end, we employ CRISPR-Cas9 editing with electroporation of ribonucleoprotein (RNP) and adeno-associated virus serotype 6 (AAV6) delivery of DNA donor templates for knock-in by homology directed repair (HDR). CCR5 KO is achieved with a highly efficient single guide RNA (sgRNA) we previously demonstrated to induce KO INDELs (Figure 7A) that confer resistance to CCR5-tropic HIV-1 infection in primary CD4+ T cells.50 Off-target analysis previously performed for this sgRNA showed it to be specific with no measurable off-target INDEL formation.50 AAV6-delivered donor templates for each linker antibody contain homology flanking the CCR5 cut site and are driven by a previously defined B cell promoter, the EEK promoter (Figure 2A), for strong expression in B cells as the professional antibody secreting cell type.45 Cord blood (CB) CD34+ HSPCs were edited with each antibody construct individually. We also edited cells with 10- 1074 and Ibalizumab constructs in combination as proof of concept for simultaneous delivery of multiple antibodies. Knock-in analysis through in-out droplet digital PCR (ddPCR) determined that each antibody construct individually was incorporated in an average of 31% to 41% of alleles (Figures 2B and 7B). When used in combination, 10-1074 and Ibalizumab knock-in was detected in up to 44% of alleles. We designed a construct-specific ddPCR assay to determine the prevalence of each antibody within the bulk cell population and found slightly higher knock-in of the Ibalizumab cassette compared to 10-1074 (Figures 2C and 7B-E). While we focus on the use of 10-1074 and Ibalizumab together, further combinations can be made through editing with any two or three antibody cassettes simultaneously in CD34+ HSPCs (Figure 8). Small molecule inhibition of NHEJ improves knock-in efficiency of antibody constructs. [0177] Inhibition of NHEJ by inhibiting DNA-PKcs with the small molecule AZD7648 has recently been shown to improve allelic knoco-in frequency in RNP/AAV6 HDR-based editing.58 Therefore, we tested the use of AZD7648 treatment with our constructs to increase editing frequency and reduce the multiplicity of infection (MOI) of AAV6 used for targeted knock-in. We found that AZD7648 treatment allowed us to reduce the AAV6 used by half while maintaining a high frequency of knock-in and CCR5 KO resulting from either knock-in or INDEL formation (Figure 2D). NHEJ inhibitor treatment did not impact the relative prevalence of 10-1074 and Ibalizumab in cells edited with both constructs (Figure 2E). [0178] To analyze the impact of AZD7648 treatment on differentiation capacity and cell health, we performed colony forming unit (CFU) assay analysis that measures myelo-erythroid differentiation potential in vitro. Two donors of CB CD34+ cells were treated with or without AZD7648 and received electroporation only (mock) or gene editing using a low MOI of both 10-1074 and Ibalizumab AAV6 together. As expected, we found that AZD7648 treatment increased knock-in frequency in the bulk cell population (Figure 2F). AZD7648 treatment did not impact total colony formation or the distribution of colony formation between the three major sub-types, CFU-GEMM (colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte), CFU-GM (colony forming unit-granulocyte and monocyte), and BFU-E (burst forming unit-erythroid) (Figures 2G-H). Single colonies were genotyped to determine the impact of AZD7648 on mono-allelic and bi-allelic knock-in frequency, as well as INDEL formation. We found that AZD7648 treatment resulted in a 2.4-fold increase in the proportion of bi-allelic knock-in events across colony sub-types (21% to 51%) (Figure 2I). Indeed, just 9% of colonies maintained a wild-type (WT) allele following treatment with AZD7648. These results demonstrate that we can achieve efficient CCR5 KO and knock-in of our antibody expression cassettes in CD34+ HSPCs, with further improved knock-in frequencies through the use of small molecule inhibition of NHEJ. Engineered HSPCs maintain engraftment capacity and multilineage reconstitution in immunodeficient mice. [0179] Genetically engineered HSPCs must maintain the ability to engraft and reconstitute the hematopoietic lineages in order to remain a viable therapeutic modality for long-term HIV- 1 treatment. We therefore edited CB CD34+ cells with 10-1074, Ibalizumab, or both 10-1074 and Ibalizumab together (at higher AAV6 MOI and without AZD7648) and transplanted them in sub-lethally irradiated immunodeficient NSG mice (Figure 3A). Prior to transplantation, we determined that each knock-in condition had similar frequencies of knock-in (33-38%) and CCR5 KO INDEL formation (52-56%), with a slight prevalence of Ibalizumab knock-in in the combination edited cells (Figures 3B-C). Engraftment, multilineage reconstitution, and editing frequencies of transplanted cells were measured at 16 weeks post-transplantation. Human chimerism was measured in bone marrow and spleen through flow cytometry analysis of human and mouse markers. Cells from each condition successfully engrafted in the bone marrow and showed migration to the spleen, a secondary lymphatic site, with similar human chimerism between each gene-edited condition (Figures 3D-E). The observed decrease in total human engraftment due to RNP/AAV6-based editing was in line with previously published works.50,59-62 Hematopoietic lineage distribution was consistent between conditions, with each editing condition producing similar frequencies of CD19+ B lineage cells and CD33+ myeloid lineage cells in both the bone marrow and spleen, indicating that our editing strategy does not introduce reconstitution bias (Figures 3F-G). Knock-in alleles were present at the endpoint in both the bone marrow and spleen, though with a lower knock-in frequency than the bulk cells at the time of engraftment (Figures 3H). Importantly, isolated CD19+ cells maintained knock- in alleles, indicating that the introduction of exogenous antibody cassettes does not inhibit B cell formation in vivo (Figure 3I). CCR5 KO alleles were also maintained in all conditions receiving RNP (Figures 3J and 9A). These analyses demonstrate that our engineered HSPCs maintain their ability to engraft and reconstitute the hematopoietic lineages, providing promise for the development of an autologous transplant therapy. However, the relatively low frequency of integrated alleles in the cells at the engraftment endpoint warranted further optimization. The results shown in Figure 2 demonstrated that an approach to optimization was to incorporate DNA-PKcs inhibition while lowering the MOI of AAV6. High-efficiency editing in CD34+ HSPCs allows for maintenance of integrated alleles following engraftment in vivo. [0180] Achieving a high frequency of integrated antibody cassettes in engrafted cells will be important for future studies determining the minimum editing threshold needed to achieve clinically relevant concentrations of each antibody. To this end, we employed AZD7648 treatment to improve the initial knock-in frequency in the CD34+ HSPCs cells prior to transplantation. Additionally, the use of AZD7684 allowed us to employ reduced AAV6 doses to potentially improve cell health and engraftment of edited cells, as reducing AAV6 is known to reduce p21 response in HSPCs.63 Two donors of CB CD34+ cells were edited for knock-in of both 10-1074 and Ibalizumab and transplanted into non-conditioned NBSGW immunodeficient mice. Input cells carried a knock-in frequency averaging 50% with efficient knock-in of both 10-1074 and Ibalizumab constructs (Figures 4A-B). Human chimerism and lineage formation were analyzed in the bone marrow at 12 weeks post-transplantation. Gene- edited cells engrafted at a lower frequency than mock edited cells, though this decrease is in line with those seen in the literature (Figure 4C).50,59-62 As expected when human chimerism is low, gene-edited HSPCs showed a myeloid bias in reconstitution within the bone marrow (Figure 4D).50 Importantly, we found the allelic knock-in frequency in the endpoint engrafted cells to remain above 50% (52-80%) in all but one mouse (11%) (Figure 4E). Knock-in frequency was also maintained in the CD19+ B cell lineage population within the bone marrow, with consistent frequencies of both 10-1074 and Ibalizumab constructs, further demonstrating that the knock-in of our constructs does not impact early B cell formation (Figures 4E and 9B- C). Finally, CCR5 KO INDELs were also maintained in non-integrated alleles (Figure 4F). These results demonstrate that AZD7648 can be used to increase the frequency of edited alleles within the long-term engrafted cell population. Such an improvement provides a favorable editing profile for introducing both CCR5 KO and long-term expression from our therapeutic antibody cassettes. [0181] While we were able to achieve efficient engraftment of gene-edited cells, this model limits our ability to effectively analyze the in vivo capacity of our system to secrete antibodies. It is well known that human HSPCs transplanted into NSG background mice do not efficiently mature into highly differentiated human B cells.64,65 This is highlighted by our results and others showing that even mice engrafted with high human chimerism do not produce relevant levels of human IgG (Figure 10).65-67 While both humans and immunocompetent mice are expected to produce over 6 mg/mL of IgG, we observed less than 300ng/mL (>4 logs lower) , 3 logs lower). Therefore, other models beyond xenotransplantation in immunodeficient mice are needed to assess the functional secretion of antibodies within our system. Engineered Human B cells secrete functional levels of each HIV-1 inhibiting antibody. [0182] Due to limitations in the humanized mouse model, we were unable to directly demonstrate if B cells derived from engineered HSPCs produce therapeutically relevant concentrations of linker antibodies in vivo. To bridge this gap, we sought to directly edit adult peripheral blood CD19+ B cells at the CCR5 locus with the same RNP/AAV6 guide RNA and antibody constructs used in HSPCs. We first confirmed the function of the EEK promoter in adult B cells through knock-in of constructs driving GFP expression through either the EEK promoter or the strong ubiquitous ubiquitin C (UBC) promoter. Flow cytometry analysis for median fluorescent intensity (MFI) of GFP+ cells was higher when driven by the EEK promoter, demonstrating its strong activity in B cells (Figure 11). Next, we edited B cells with each linker antibody construct individually or with the 10-1074 and Ibalizumab constructs together. As with CB CD34+ HSPCS, we achieved highly efficient knock-in into the CCR5 locus at frequencies up to 43% of alleles (Figure 5A), with 10-1074 and Ibalizumab constructs integrating at a similar frequency when used in combination (Figure 5B). Additionally, the knock-in of an exogenous antibody cassette did not obviously impact any B cell subset within the in vitro culture system (Figure 12). [0183] We next demonstrated that the knock-in of the EEK-driven linker antibody constructs resulted in the secretion of functional antibodies. B cells were edited, allowed to recover for six days, and then plated at a density of 1 million cells per mL of media for five days. Culture supernatant was collected, and antigen-specific ELISA analysis was used to confirm the presence of each antibody (Figure 13). Serial dilutions of cell culture supernatant were then tested for neutralization potency in the TZM-bl infection assay against either TRO11 or CNE55 HIV-1 pseudoviruses for which we empirically determined the expected IC50 for each antibody in Figure 1 (Figure 5C). Supernatant from B cells edited with 10-1074 and Ibalizumab cassettes individually inhibited infection, with near complete inhibition at a 1:2 media dilution (Figure 5D). As expected from the known IC50 values, PGDM1400 supernatant showed a reduced inhibition capacity and CAP256V2LS supernatant did not inhibit infection with TRO11. Supernatant from B cells expressing both 10-1074 and Ibalizumab effectively inhibited TRO11 infection with a similar potency to that of cells edited for each antibody alone. When tested against CNE55, we found that supernatant from B cells expressing 10-1074 did not inhibit infection, while supernatant from B cells expressing Ibalizumab, PGDM1400, and CAP256V2LS each effectively inhibited infection corresponding with the known IC50 of each antibody (Figure 5E). Importantly, while cells edited te express 10-1074 alone failed to inhibit infection, cells edited to express both 10-1074 and Ibalizumab together effectively inhibited CNE55, highlighting the importance of employing multiple antibodies to overcome deficiencies of any single antibody. These results demonstrate that B cells carrying linker antibody expression cassettes are capable of effectively secreting inhibitory concentrations of antibodies individually and in combination, thus highlighting the significance of a multi- epitope targeting strategy for efficacy against diverse HIV-1 strains. Discussion: [0184] The transplantation of HSPCs that generate HIV-resistant progeny represents a promising strategy to achieve a functional cure for HIV-1. However, the currently established method for allogeneic transplantation of CCR5 KO HSPCs is severely limited due to the rarity of available donor cells, the risk for GvHD, and a lack of efficacy against X4-tropic virus. Transplantation of genetically engineered autologous HSPCs is a treatment modality with the potential to address these limitations. Modifying a patient’s own cells removes the need for identifying a matched donor and circumvents the risks specific to allogeneic transplantation. Additionally, precise genetic engineering can allow for the delivery of multi-factored resistance to R5-tropic and X4-tropic HIV-1. To this end, we have established a highly efficient genome editing strategy to combine the known benefits of CCR5 KO with antibody therapy in a manner applicable towards autologous HSPC transplantation. [0185] Here we demonstrate the simultaneous knockout of CCR5 and knock-in of antibody expression cassettes in human HSPCs for secretion of HIV-1 inhibiting antibodies in B cell progeny of edited cells. Following transplantation, genetically engineered HSPCs have the potential to persist for a patient’s lifetime and provide a long-term source of therapeutic antibody secreting B cells. Recent trials investigating direct injection of bNAbs for HIV-1 suppression show that repeated dosing is required to maintain efficacy, as viral titers rebound once the serum concentrations drop below a therapeutic threshold.18,19 Additionally, AAV and B cell-mediated antibody delivery platforms rely on somatic cells and are unlikely to rival the longevity of HSCT. Therefore, the engraftment of genetically engineered HSPCs represents one of the most promising methods for a one-time therapy to combat HIV-1. To this end, we show that RNP/AAV6 editing of HSPCs delivers a favorable profile of modified alleles, achieving a knock-in frequency of up to 50% of alleles and KO frequency of up to 90% of alleles at CCR5 (Figure 2). Importantly, engineered HSPCs maintain their ability to engraft and reconstitute the hematopoietic lineages in immunodeficient mice (Figure 3), and small molecule inhibition of NHEJ can be used to address the decrease in knock-in alleles normally seen following engraftment (Figure 4). The development of this system for highly efficient CCR5 KO and knock-in of antibody expression cassettes is a favorable first step towards developing an autologous transplantation therapy. [0186] Recently, the transplantation of gene edited autologous HSPCs has displayed clinical success with the approval of Casgevy for the treatment of sickle cell disease and beta- thalassemia.68-70 Of these, Casgevy uses RNP editing for INDEL generation in HSPCs, acting as a proof of concept for RNP-edited HSCT. Now, efforts are underway to expand the therapeutic potential of CRISPR-Cas9 editing to include knock-in of large therapeutic cassettes using AAV6/RNP mediated editing. However, it is regularly shown that AAV6 treatment negatively impacts HSPCs, as demonstrated through reduced colony formation in CFU assays and decreased chimerism following xenotransplantation.71 We and others are exploring methods to alleviate toxicity associated with AAV6 in HSPCs to improve the long-term engraftment of engineered cells. For one, the use of small molecule NHEJ inhibitors allows for reduced doses of AAV6 and RNP to achieve the same editing frequencies, potentially decreasing toxicity through a reduction in p53 activation.58,63,72 Additionally, inhibition of the cell’s DNA damage response through transient inhibition of 53BP1 with peptides or siRNA has been shown to reduce toxicity in HSPCs.72,73 Finally, it has been demonstrated that simply increasing the number of transplanted cells can compensate for AAV6 associated toxicity in xenograft mouse models.61 While more work is needed to refine optimal editing conditions and improve engraftment of AAV6/RNP-edited HSPCs, the data presented here represent an important proof of concept study for the efficacy of a long-term antibody delivery platform for treatment of HIV and other diseases treatable by recombinant antibody delivery. [0187] Engraftment in immunodeficient mice is the gold-standard for functional analysis of genetically engineered human HSPCs, however, the model fails to recapitulate normal B cell development and maturation. While our engineered HSPCs do engraft and maintain edited alleles in CD19+ progeny, we are not able to assess functional antibody expression due to a lack of mature antibody secreting B cells in vivo. To address the our system’s ability to drive therapeutic antibody expression, we edited adult peripheral blood B cells with the same constructs used in HSPCs. These modified B cells were capable of secreting inhibiting levels of each tested antibody into the culture supernatant (Figure 5). It is well known that multiple antibodies should be employed simultaneously to combat viral diversity and escape. We therefore designed our system for use with multiple antibodies concurrently and show that linker 10-1074 and linker Ibalizumab could be expressed together from one bulk population of B cells (Figures 5 and 13). The modular design of our cassettes permits antibodies to be readily switched out, allowing for optimization of antibody pairings and potential customization based on the viral diversity of various patient populations across regions. [0188] Because B cells are the professional antibody-producing cells of the hematopoietic system, we believe that expression from B cell progeny is a favorable strategy for sustained delivery of engineered antibodies. By expressing from the CCR5 locus, our cassettes will act as passengers to normal B cell function and should therefore persist across B cell sub-types. This will ideally allow for our antibodies to reach a steady-state concentration in the bloodstream, providing an advantage over traditional antibody injection therapies that have peaks and troughs with each dosage. Additionally, expression of these antibodies from B cells directly may benefit from the inclusion of natural post-translational modifications that are not incorporated when expressed from other cell types.74 Carrying these modifications may decrease immune recognition of HIV-1 inhibiting antibodies and reduce the potential for anti- drug antibody formation, an issue encountered by both vectored immunoprophylaxis and direct injection trials.24,27,75,76 Additionally, endogenous glycosylation and sialylation have the potential to improve FC-mediated clearance of HIV-1 infected cells.77,78 Moving forward, it will be important to investigate the impact of post-translational modifications on antibodies expressed from B cells. [0189] Overall, this work serves as a platform strategy for the lifetime secretion of desired therapeutic antibodies. Beyond its usefulness for HIV-1 resistance, knock-in into the CCR5 safe harbor site allows for broad applications across chronic diseases. Future work will test alternate antibodies for use within our system as a new delivery modality for current treatment regimens that require long-term dosing of monoclonal antibodies. Methods: [0190] rAAV6 vector design, production, and purification. The sequence of each antibody construct was cloned from a gblock Gene Fragment (Integrated DNA Technologies, IDT, San Jose, CA, USA). Restriction enzyme digest or Gibson Assembly (New England Biolabs, Ipswich, MA, USA) was used to clone each antibody sequence plus 400 base pair (bp) homology arms on each side of the construct into an adeno-associated virus, serotype 6 (AAV6) vector plasmid derived from the pAAV-MCS plasmid (Agilent Technologies, Santa Clara, CA, USA). Experiments were performed with rAAV6 vectors produced and purified by SignaGen Laboratories (Frederick, MD, USA) and Packgene Biotech (Houston, TX, USA). All viral titers were determined using droplet digital PCR (ddPCR) to measure the number of vector genomes as previously described.79 [0191] CD34+ HSPC isolation and culture. Human CD34+ HSPCs were isolated from cord blood by the Stanford Binns Program for Cord Blood Research and cultured as previously described.80 Briefly, isolated mononuclear cells were positively selected for CD34 using the CD34+ Microbead kit Ultrapure (Miltenyi Biotec, San Diego, CA, USA, cat.: 130-100-453). Cells were cultured at 1.5×105–2.5×105 cells/mL in CellGenix® GMP Stem Cell Growth Medium (SCGM, CellGenix, Freiburg, Germany, cat.: 20802-0500) supplemented with a human cytokine (PeproTech, Rocky Hill, NJ, USA) cocktail: stem cell factor (100 ng/mL), thrombopoietin (100 ng/mL), Fms-like tyrosine kinase 3 ligand (100 ng/mL), interleukin 6 (100 ng/mL), streptomycin (20 mg/mL), and penicillin (20 U/mL), and 35 nM of UM171 (APExBIO, Houston, TX, USA, cat.: A89505). Cells were cultured in a 37°C hypoxic incubator with 5% CO2 and 5% O2. Cells were cultured for 3 days prior to editing. [0192] Genome editing of HSPCs. HPLC-purified synthetic chemically modified sgRNAs were purchased from TriLink Biotechnologies (San Diego, CA, USA). Chemical -O-methyl- -phosphorothioate at the three terminal ird, and fourth bases from the 3’ end as described previously.81 The target sequence for the sgRNA are as follows: sg-CCR5, - ATGCACAGGGTGGAACAAGA- (SEQ ID NO:72) ; sg-CCR5-#2, 5’- GCAGCATAGTGAGCCCAGAA-3’ (SEQ ID NO:73). HiFi Cas9 protein was purchased from IDT (cat.: 1081061) or Aldevron (Fargo, ND, USA, cat:. 9214). RNPs were complexed at a Cas9:sgRNA molar ratio of 1:2.5 at room temperature for 15-30 min. 2.5×105-1x106 CD34+ cells were resuspended in P3 buffer (Lonza, Basel, Switzerland, cat.: V4XP-3032) with complexed RNPs and electroporated using the Lonza 4D Nucleofector and 4D- Nucleofector X Unit (program DZ-100). Electroporated cells were then plated at 2.5×105 cells/mL in the previously described cytokine-supplemented media. Immediately following electroporation, AAV6 was dispensed onto cells at 0.625×103-2.5×103 vector genomes/cell as noted in figure legends. For some editing experiments, in addition to the steps described -PKcs inhibitor, AZD7648 (Selleck Chemicals, Houston, TX, cat.: S8843) for 24 hours, as previously described.58 [0193] B cell isolation, culture, and genome editing. Leukoreduction system (LRS) chambers were obtained from the Stanford Blood Center and primary human B cells were isolated by negative selection using the human B Cell Isolation Kit II (Miltenyi Biotec, cat: 130-091-151). Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific, Waltham, MA, USA cat.: 12440053) supplemented with 10% bovine growth serum (Cytiva, Marlborough, MA, USA, cat.: SH30541.03HI), 1% penicillin-streptomycin -mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA, cat.: M3148), 50 ng/mL of IL-2 (Peprotech, cat.: 200-02), 50 ng/mL of IL-10 (Peprotech, cat.: 200-10), 10 ng/mL of IL-15 (Peprotech, cat.: 200-15), 100 ng/mL of recombinant human MEGACD40L (Enzo Life Sciences, Farmingdale, NY, USA, cat.: ALX-522-110-C010), and -2006-1) at a density of 1×106 cells/mL, as described previously.28 B cells were cultured at 37°C, 5% CO2, and ambient oxygen levels. Genome editing was performed as described above for HSPCs with the following modifications. B cells were edited 3-5 days after isolation or thawing using the Lonza Nucleofector 4D (program EO-117) using 1×106 cells per well of a 16-well Nucleocuvette Strip (Lonza). Immediately following nucleofection, cells were incubated with AAV6 donor vector (2.5×104 for 3-4 hours and then replated at 1×106 cells/mL in complete B cell activation media.82 [0194] Measurement of knock-in alleles by ddPCR. Cells were harvested 2-3 days post- electroporation and genomic DNA (gDNA) was harvested using QuickExtract DNA extraction solution (Biosearch Technologies, Hoddesdon, UK, cat.: QE09050). To quantify knock-in alleles via ddPCR, we employed CCR5 specific in-out PCR primers and a probe corresponding to the expected knock-in event (1:3.6 primer to probe ratio).60 We also used an established genomic DNA reference (REF) at the CCRL2 locus.60 The ddPCR reaction was prepared and underwent droplet generation following the manufacturer’s instructions with a Bio-Rad QX200 ddPCR machine (Bio-Rad, Hercules, CA, USA). Thermocycler settings were as follows: 95°C (10 min, 1°C/s ramp), 94°C (30 s, 1°C/s ramp), 60°C (30 s, 1°C/s ramp), 72°C (2 min, 1°C/s ramp), return to step 2 for 50 cycles, and 98°C (10 min, 1°C/s ramp). Analysis of droplet samples was then performed using the QX200 Droplet Digital PCR System (Bio-Rad). We and probes were used in the ddPCR reaction: CCR5-BGH: Forward Primer (FP): 5’-GGGAGGATTGGGAAGACA-3’ (SEQ ID NO: 48) Reverse Primer (RP): 5’-AGGTGTTCAGGAGAAGGACA-3’ (SEQ ID NO: 49) Probe: 5’-6-FAM/AGCAGGCATGCTGGGGATGCGGTGG/3IABkFQ-3’ (SEQ ID NO: 50) CCR5-IgG1: FP: 5’-CCTGAGCCCCGGAAAATAG-3’ (SEQ ID NO: 51) RP: 5’-AGGTGTTCAGGAGAAGGACA-3’ (SEQ ID NO: 52) Probe: 5’-6-FAM/AGCAGGCATGCTGGGGATGCGGTGG/3IABkFQ-3’ (SEQ ID NO: 53) CCR5-IgG4: FP: 5’-CCTCTCCCTGTCTCTGGGTA-3’ (SEQ ID NO: 54) RP: 5’-AGGTGTTCAGGAGAAGGACA-3’ (SEQ ID NO: 55) Probe: 5’-6-FAM/AGCAGGCATGCTGGGGATGCGGTGG/3IABkFQ-3’ (SEQ ID NO: 56) CCRL2: FP: 5’- GCTGTATGAATCCAGGTCC-3’, (SEQ ID NO: 57) RP: 5’- CCTCCTGGCTGAGAAAAAG -3’ (SEQ ID NO: 58) Probe: 5’- HEX/TGTTTCCTC/ZEN/CAGGATAAGGCAGCTGT/3IABkFQ -3’ (SEQ ID NO: 59) [0195] INDEL analysis using ICE software. Two- or three-days following editing, cells were collected and genomic DNA was extracted using QuickExtract DNA extraction solution (Biosearch Technologies, cat.: QE09050). The following primer sequences were used to amplify the CCR5 locus around the sgRNA cut site as previously described:50 FP: 5’-CAGGGAAGCTAGCAGCAAACC-3’ (SEQ ID NO: 60) RP: 5’-AGACGCAAACACAGCCACC-3’ (SEQ ID NO: 61) [0196] PCR products were run on a 1% agarose gel and purified using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, cat.: FERK0692). Sanger sequencing of respective PCR samples was performed using the reverse primer. Sequencing files were then used as input for INDEL frequency analysis relative to a mock, unedited sample using the online ICE CRISPR analysis tool (Synthego, Redwood City, CA, USA).83 [0197] PCR and Gel Electrophoresis for Knock-in. Two- or three-days following editing, cells were collected and genomic DNA was extracted using QuickExtract DNA extraction solution (Biosearch Technologies, cat.: QE09050). In-out PCR was performed with 3 forward primers and one reverse primer to selectively amplify knock-in events of the Ibalizumab, 10- 1074, and PGDM1400 antibody cassettes. Primers used were as follows: Ibalizumab FP: 5’-ACAGTCCTCAGGACTCTACTCC-3’ (SEQ ID NO: 62) 10-1074 FP: 5’-TATGGCGTGGTGAGCTTTGG-3’ (SEQ ID NO: 63) PGDM1400 FP: 5’-CTGGGACCTCCGTAAAGGTCT-3’ (SEQ ID NO: 64) CCR5 RP: 5’-AGACGCAAACACAGCCACC-3’ (SEQ ID NO: 65) [0198] PCR products were run on a 1% agarose gel and imaged using a Bio-Rad ChemiDoc XRS+ gel imager. [0199] Antibody production and purification. Full length heavy and light chains for each antibody were cloned by restriction enzyme digest or Gibson Assembly (New England Biolabs) into pCMVR either individually or with a linker. Antibody constructs were expressed in Expi293F cells (Thermo Fisher Scientific, cat.: A14527) and grown in combined media (66% FreeStyle/33% Expi media, Thermo Fisher Scientific, cat.: 12338018 and A1435101, respectively) at 37°C and 8% CO2 with shaking at Antibody constructs were transfected using FectoPRO transfection reagent (Polyplus, Sébastien Brant, France, cat.: 101000014) with a transfection mixture of 10 mL of culture media, 50 of total DNA and 130 of FectoPro for each 90 mL of cell culture (for a total transfected cell culture volume of 100 mL or scaled down at the same ratios for a 75 mL total volume). Heavy and light chain plasmids were used at a 1:1 ratio, while single plasmid linker antibody constructs were used alone. Cells were transfected at a cell density 3-4×106 cells/mL and harvested post- transfection by spinning at >4200g for Cell culture supernatants were filtered through a 0.45- filter, combined with 1/10th volume of 10x phosphate-buffered saline (PBS) and purified using the ÄKTA pure fast performance liquid chromotography (FPLC, Cytiva) with a 5mL MabSelect PrismA column (Cytvia, cat.: 17549802) using wash steps with 1x PBS and elution with glycine (pH 2.8) into one-tenth volume of 1 M Tris (pH 8.0). The eluted proteins were then concentrated using 50-kDa or 100-kDa cutoff centrifugal concentrators and further purified by size exclusion on the same ÄKTA FPLC with Superdex 200 Increase 10/300 GL column (Cytiva, cat.: 28-9909-44). Proteins were then further concentrated using 50-kDa or 100-kDa cutoff centrifugal concentrators. [0200] Immunophenotyping of B cells. All samples were blocked for non-specific binding with human FcR Blocking Reagent (Miltenyi Biotec, cat.: 130-059-901) for 10 min at room temperature and then stained for 30 min at 4°C with the following antibody cocktail: anti- human CD19 (Becton, Dickinson and Company, BD, Franklin Lakes, NJ, USA, cat.: 562440), anti-human CD20 (BioLegend, San Diego, CA, USA, cat.: 302326), anti-human CD27 (BioLegend, cat.: 302832), anti-human CD38 (BD, cat.: 555462), anti-human IgD (BioLegend, cat.: 348232), and anti-human IgM (BD, cat.: 563903). Samples were analyzed on a FACSAria II SORP (BD). Data was subsequently analyzed using FlowJo (v.10.9.0). [0201] ELISA for Antibody Concentration. Antigen specific ELISA was used to determine the concentration of each HIV-inhibiting antibody in B cell supernatant. Anti-IgG Fc ELISA was used to detect human IgG in mouse serum. 10-1074, Ibalizumab, and total human IgG were detected using a previously described protocol.84 Briefly, Nunc MaxiSorp 96 Well Plates (Thermo Fisher Scientific, cat.: 44-2404- recombinant HIVJR-CSF gp120 (for detecting 10-1074; Immune Technology, Tarrytown, NY, USA, cat.: IT-001- mbinant rhesus monkey CD4 protein (for detecting Ibalizumab; Abcam, Cambridge, UK, cat.: ab208305), or 1:100 diluted goat anti-human IgG- Fc (for detecting total human IgG; Fortis Life Sciences, Waltham, MA, USA, cat.: A80-104A) in 0.05 M carbonate-bicarbonate buffer solution (Sigma-Aldrich, cat.: C3041) for 1 hour at room temperature or overnight at 4°C. Plates were then blocked in tris-buffered saline (TBS) with 1% bovine serum albumin (BSA; Miltenyi Biotech, cat.: 130-091-376) for 30 minutes. Media and serum samples were diluted in TBS with 1% BSA and 0.05% Tween-20 and incubated on the plate for 4 hours at room temperature or overnight at 4°C. Plates were then incubated with horseradish peroxidase (HRP)–conjugated goat anti-human IgG-Fc antibody (Fortis Life Sciences, cat.: A80-104A) at a 1:2500 dilution. Detection was performed with the 1-Step™ TMB Substrate Kit (Thermo Fisher Scientific, cat.: 34021) and quenched with 3M H2SO4. Plates were read at 450nm using a Molecular Devices SpectraMax M3 plate reader with SoftMax Pro software. Standard curves were created using purified versions of each HIV inhibiting antibody or purified human IgG/kappa from normal serum (Bethyl, Montgomery, TX, USA, cat.: P80-111). Results were analyzed using GraphPad Prism v10 software to calculate a standard curve using a 4-parameter or 5-parameter sigmoidal algorithm. The curve with a higher r-squared was selected and results were interpolated for each sample. PGDM1400 and CAP256V2LS were detected with a modified protocol. Briefly, recombinant HIV-1 Env trimer, BG505 SOSIP, was biotinylated with the EZ-Link™-Biotinylation Kit (Thermo Fisher Scientific, cat.: 21435) as previously described.85 Nunc MaxiSorp plates were coated with 4 1 hour. Plates were then blocked with ChonBlock (Thermo Fisher Scientific, cat.: 50-152- 6971) overnight at 4°C. The next da /mL biotinylated BG505 SOSIP trimer was added for 1 hour at room temperature. Samples were diluted in PBS with 0.1% BSA and 0.05% Tween-20 and incubated on the coated plates for 1 hour. Plates were then detected with HRP- conjugated anti-human IgG-Fc antibody as described above. Results were analyzed as described above. [0202] Western Blotting of Purified Antibodies. Purified antibodies (0.5 ug each) were denatured by adding 4X LaemmLi sample buffer (Bio-Rad, cat.: 1610747) and 2- mercaptoethanol and heating at 100°C for 5 minutes. Samples were loaded onto a 4-15% Mini- PROTEAN TGX precast protein gel (Bio-Rad, cat.: 4561084). Following electrophoresis, protein from gel was transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, cat.: 1704150). Subsequently, the membrane was blocked using Blotting- Grade Blocker (Bio-Rad, cat.: 1706404) in phosphate-buffered saline with 0.02% Tween 20 (PBS-T) for 30 minutes at room temperature. Membrane was then incubated in 1:5000 primary antibody (HRP-conjugated Goat pAb to human IgG, Abcam, cat.: 7153) overnight at 4°C. The next day, the membrane was washed three times with PBS-T. SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat.: 34579) was used for detection and blots were imaged using a Bio-Rad ChemiDoc XRS+ gel imager using ImageLab software. [0203] TZM-bl Infection Assay for Determining IC50. The TZM-bl HIV-1 pseudotype infection assay was adapted from previously described protocols.86,87 TZM-bl cells were obtained through the NIH AIDS Reagent Program (cat.: 8129) and cultured in DMEM with 10% BGS, and 1% penicillin-streptomycin (complete DMEM) at 37°C, 5% CO2, and ambient oxygen levels. HIV-1 env pseudotyped lentivirus was produced as previously described using a pNL4- env plasmids provided by the NIH AIDS Reagents Program (cat.: 11100 and cat.: 12670, respectively).88 Briefly, 5x103 cells were plated in black- walled, clear-bottom 96-well plates (Corning, Corning, NY, USA, cat.: 07-200-565) and incubated overnight. The next day, each antibody was incubated with HIV-1 pseudovirus for 1 -5 -fold serial dilution in -dextran (Sigma-Aldrich, D9885). Culture media was aspirated from the TZM-bl cells and replaced with antibody-virus mixture. Virus only (no antibodies added) and cells only (no virus or antibodies) wells were included for determining 100% and 0% infection readouts. Cells were incubated for 48 hours then measured for luciferase signal corresponding to infection. Briefly, cells were lysed with Reporter Lysis Buffer (Promega, Madison, WI, USA, cat.: E3971) and freeze-thawed at -80°C. Lysed samples were read for relative luminescence units (RLU) using a Synergy H1 plate reader (BioTek, Winooski, VT, USA) that automatically injected luciferin solution consisting of the following: Firefly Luciferase Signal Enhancer -luciferin (Biosynth Chemistry & Biology, Staad, Switzerland, cat.: L8220). Percent infection was determined by normalizing RLU values to the average RLU of virus-only and cells-only wells using GraphPad Prism v10. IC50 was calculated using a linear regression dose-response curve fit for inhibitor versus response (three parameters) based on the average of technical duplicate wells using GraphPad Prism v10. [0204] TZM-bl Infection Assay with B cell supernatant. Six days post-editing, B cells were plated at 1×106 cells/mL and antibody was allowed to accumulate in supernatant for five days. Culture supernatant was collected following centrifugation to remove cells. A modified TZM-bl assay was performed as described above with the following changes. Culture supernatant was diluted in complete DMEM with two-fold serial dilutions. Then, 50uL of each dilution was mixed with 50uL of HIV-1 pseudovirus + complete DMEM for final dilutions of -dextran (Sigma-Aldrich, cat.: D9885). Samples were incubated for 1 hour at 37°C before being applied to TZM-bl cells. [0205] Colony forming unit (CFU) assay and colony genotyping. Two days post- electroporation 500 HSPCs were plated in each well of a SmartDish 6-well plate (STEMCELL Technologies, Vancouver, Canada, cat.: 27370) containing MethoCult H4434 Classic (STEMCELL Technologies, cat.: 04444). After 14 days, the wells were imaged using the STEMvision Hematopoietic Colony Counter (STEMCELL Technologies). Colonies were counted and scored with manual correction to determine the number of BFU-E, CFU-GM, and CFU-GEMM colonies. Individual colonies were picked and gDNA was extracted using QuickExtract DNA extraction solution (Biosearch Technologies, cat.: QE09050). Knock-in was determined by ddPCR and INDELs were determined by ICE analysis as described above. [0206] Mice. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and NOD.Cg-KitW-41JTyr+Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in the Stanford University barrier facility. All experiments were completed under the Administrative Panel on Laboratory Animal Care (APLAC Protocol #25065). [0207] CD34+ HSPC transplantation in to immunodeficient mice. For the experiment in NSG mice, 6-8 week old mice were irradiated with 2Gy approximately 4 hours prior to transplantation.8.5×105 mock or gene-edited cells were transplanted into each mouse via retro- orbital injection. For the experiment in NSGBW mice, 6-8 week old mice received no conditioning and 5×105 mock or gene-edited cells were transplanted into each mouse via retro- orbital injection. [0208] Assessment of human HSPC engraftment. Human engraftment was assessed at 12- (NSGBW) or 16-weeks (NSG) post-transplantation. Mice were euthanized and bone marrow and spleen were harvested from recipient mice. Mononuclear cells from bone marrow samples were isolated via Ficoll gradient centrifugation. Spleen samples were treated with RBC Lysis Buffer (IBI Scientific, Dubuque, IA, USA, cat.: IB47620) to eliminate mature red blood cells. All samples were blocked for non-specific binding with FcR Blocking Reagent for human (Miltenyi Biotec, cat.: 130-059-901) and mouse (Miltenyi Biotec, cat.: 130-092-575) for 10 min at room temperature and then stained for 30 min at 4°C with the following antibody cocktail: anti-mouse CD45.1 (Biolegend, cat.: 110735), anti-mouse TER-119 (eBiosciences, San Diego, CA, USA, cat.: 15-5921-82), anti-human CD45 (Biolegend, cat.: 368540), anti- human HLA-ABC (Biolegend, cat.311426), anti-human CD33 (BD, cat.: 555450), anti-human CD19 (Biolegend, cat.: 302212), anti-human CD3 (Biolegend, cat.: 300328), and viability dye Ghost Dye™ Violet 540 (Tonbo Bioscience, San Diego, CA, USA, cat.130879T100). Samples were analyzed on a CytoFLEX flow cytometer (Beckman-Coulter, Indianapolis, IN, USA). Data was subsequently analyzed using FlowJo (v.10.9.0). Humam CD19+ cells were isolated from the bone marrow using positive selection with the CD19 MicroBeads, human kit (Miltenyi Biotec, cat.: 130-050-301). Knock-in and INDEL frequencies were determined by ddPCR and ICE analysis as described above. [0209] Statistical analysis. All statistical analysis was performed using GraphPad Prism v10 software. Example 2. Engineered Hematopoietic Stem Cells Give Rise to Therapeutic Antibody Secreting B Cells [0210] The therapeutic antibody market dominates modern medicine, with monoclonal antibodies representing half of the top ten selling drugs with global sales of >$200B. Despite their proven efficacy across a wide variety of diseases (i.e. cancer, autoimmunity, metabolism, etc.) they still require repeated administration to remain effective. Conversely, cell-based medicines have entirely different pharmacokinetics and pharmacodynamics than traditional medicines, including the potential to have lifetime durability. [0211] To this end, we have developed a genetically engineered stem cell-based therapy that enables continuous antibody production from a single dose. Using CRISPR/Cas9 homology directed repair mediated editing, we engineered hematopoietic stem and progenitor cells (HSPCs) (Figure 14A) such that when they differentiate into B cells, they will secrete therapeutic levels of antibodies. [0212] We insert the antibody expression cassette in the CCR5 safe-harbor locus under the control of a synthetic B cell promoter, enabling antibody expression across B cell subtypes while preserving normal hematopoiesis and endogenous B cell function. We validated this platform using two clinically approved antibodies: anti- - PCSK9 for hypercholesterolemia. We achieved 35-40% targeted integration of the gene- targeted antibodies (GT-Ab) in human HSPCs (Figure 14B) and demonstrated robust engraftment (up to 85% chimerism) of these cells in sub-lethally irradiated NSG mice with maintenance of edited alleles (average 35%) in the bone marrow 16 weeks post-transplant. [0213] Because immunodeficient mice lack an immune system and do not reconstitute with mature IgG-secreting B cells, we used two other models to demonstrate antibody secretion using our system. First, we engineered GT-Ab human B cells in vitro and demonstrated efficient expression of anti- -PCSK9 antibodies into the culture supernatant (Figure 14C). We achieved 18-30% targeted integration of the gene-targeted antibodies (GT- Ab) in human B cells 3 days post editing (Figure 14D), and up to 3000 ng/mL of the anti- PCSK9 antibody expression in B cell culture supernatant 5 days post re-plating (Figure 14E). [0214] We then modeled autologous hematopoietic stem cell transplant (HSCT) by transplanting engineered GT-Ab murine HSPCs into lethally irradiated C57BL/6 immunocompetent mice. We detected therapeutic antibodies in the serum of mice by week 4 post-transplant, with week 16 concentrations reaching 125- for anti-PCSK9 (Figure 15A) and 4- - – meeting or exceeding therapeutic requirements in humans. Notably, these levels were achieved with less than 10% gene- modified cells in the peripheral blood (Figure 15C), suggesting compatibility of the system with reduced-intensity, non-toxic conditioning regimens. In terms of safety, we observed normal B cell development in the mice, including formation of IgM+IgD+ mature B cells (Figure 15D), with stable maintenance of the engineered GT-Ab alleles in B cells sorted from the spleen (Figure 15E). [0215] The high levels of antibodies in the mouse studies prompted us to develop a system in which the levels could be modulated.92, 93 By fusing the antibody to a destabilization domain, we demonstrated that levels of antibody secretion could be controlled through small molecule regulation. Figure 16 shows the levels of anti-PCSK9 antibodies in B cell culture supernatant of cells editing with anti-PCSK9 antibody expression cassette with fused destabilization domain with and without addition of small molecule stabilizer (1uM Shield-1). This ability to regulate antibody level may be essential to achieve both safety and efficacy of sustained cell- based delivery of monoclonal antibodies. [0216] Overall, this platform represents a modular approach for treating chronic diseases currently reliant on repeated administration of therapeutic antibodies, offering the potential for a durable production of antibodies from a single treatment. 10. References: 1. 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(2022). Human B-cell subset identification and changes in inflammatory diseases. Clinical and Experimental Immunology 210, 201-216. 92. Chu BW, Banaszynski LA, Chen L chun, Wandless TJ. Recent progress with FKBP- derived destabilizing domains. Bioorganic & Medicinal Chemistry Letters. 2008;18(22):5941-5944. doi:10.1016/j.bmcl.2008.09.043 93. Banaszynski LA, Chen L chun, Maynard-Smith LA, Ooi AGL, Wandless TJ. A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules. Cell. 2006;126(5):995-1004. doi:10.1016/j.cell.2006.07.025 Exemplary embodiments [0217] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments: [0218] Embodiment 1. A method of genetically modifying a cell from a subject, the method comprising: introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting a selected locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein: the sgRNA binds to the nuclease and directs it to a target sequence at the selected locus in a genome, whereupon the nuclease cleaves the locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. [0219] Embodiment 2. The method of Embodiment 1, wherein the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, the RNA- guided nuclease, and the homologous donor template. [0220] Embodiment 3. The method of Embodiment 1 or 2, wherein the sgRNA comprises chemical modifications at one or more nucleotides. [0221] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the -O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides. [0222] Embodiment 5. The method of Embodiment -O-methyl- - [0223] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the selected locus is a safe harbor locus. [0224] Embodiment 7. The method of Embodiment 6, wherein the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. [0225] Embodiment 8. The method of Embodiment 7, wherein the safe harbor locus is the CCR5 locus and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1. [0226] Embodiment 9. The method of Embodiment 8, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4. [0227] Embodiment 10. The method of Embodiment 8 or 9, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof. [0228] Embodiment 11. The method of any one of Embodiments 8 to 10, wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. [0229] Embodiment 12. The method of Embodiment 7, wherein the safe harbor locus is the AAVS1 locus, and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39. [0230] Embodiment 13. The method of Embodiment 12, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40. [0231] Embodiment 14. The method of Embodiment 12 or 13, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof. [0232] Embodiment 15. The method of any one of Embodiments 12 to 14, wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. [0233] Embodiment 16. The method of any one of Embodiments 1 to 5, wherein the selected locus is an immunoglobulin-associated locus. [0234] Embodiment 17. The method of Embodiment 16, wherein the immunoglobulin- associated locus is an IgH locus, an [0235] Embodiment 18. The method of Embodiment 17, wherein the immunoglobulin- associated locus is the IgH locus, and wherein the target sequence of the sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 27, 31 and 35. [0236] Embodiment 19. The method of Embodiment 18, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 28, 32 or 36. [0237] Embodiment 20. The method of Embodiment 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 28, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29, or a fragment thereof. [0238] Embodiment 21. The method of Embodiment 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof. [0239] Embodiment 22. The method of Embodiment 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof. [0240] Embodiment 23. The method of any one of Embodiments 1 to 22, wherein the RNA-guided nuclease is a Cas9. [0241] Embodiment 24. The method of any one of Embodiments 1 to 23, wherein the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP). [0242] Embodiment 25. The method of Embodiment 24, wherein the RNP is introduced into the cell by electroporation. [0243] Embodiment 26. The method of any one of Embodiments 1 to 25, wherein the transgene is present within an expression cassette. [0244] Embodiment 27. The method of Embodiment 26, wherein the expression cassette comprises a coding sequence for the therapeutic antibody, operably linked to a promoter, and an exogenous polyadenylation (polyA) fragment. [0245] Embodiment 28. The method of Embodiment 27, wherein the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. [0246] Embodiment 29. The method of Embodiment 28, wherein the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain. [0247] Embodiment 30. The method of any one of Embodiments 27 to 29, wherein the promoter is a B-cell specific promoter. [0248] Embodiment 31. The method of Embodiment 30, wherein the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof. [0249] Embodiment 32. The method of Embodiment 31, wherein the EEK promoter comprises a sequence having at least 80% identity to SEQ ID NO: 7. [0250] Embodiment 33. The method of Embodiment 31, wherein the B29 promoter comprises a sequence having at least 80% identity to SEQ ID NO: 8. [0251] Embodiment 34. The method of Embodiment 31, wherein the IgH promoter comprises a sequence having at least 80% identity to SEQ ID NO: 9 or 10. [0252] Embodiment 35. The method of Embodiment 27, wherein the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment. [0253] Embodiment 36. The method of any one of Embodiments 27 to 35, wherein the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic antibody. [0254] Embodiment 37. The method of any one of Embodiments 1 to 36, wherein the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. [0255] Embodiment 38. The method of any one of Embodiments 1 to 37, wherein the transgene encodes a therapeutic antibody against HIV infection. [0256] Embodiment 39. The method of any one of Embodiments 1 to 38, wherein the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain. [0257] Embodiment 40. The method of Embodiment 39, wherein the at least one light chain and the at least one heavy chain are linked by a linker. [0258] Embodiment 41. The method of Embodiment 40, wherein the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6. [0259] Embodiment 42. The method of any one of Embodiments 1 to 41, wherein the therapeutic antibody comprises an amino acid sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. [0260] Embodiment 43. The method of any one of Embodiments 1 to 42, wherein the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26. [0261] Embodiment 44. The method of any one of Embodiments 1 to 43, wherein the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector. [0262] Embodiment 45. The method of Embodiment 44, wherein the recombinant adeno- associated virus is serotype 6 (rAAV6). [0263] Embodiment 46. The method of any one of Embodiments 1 to 45, wherein the cell is a hematopoietic stem and progenitor cell (HSPC). [0264] Embodiment 47. The method of any one of Embodiments 1 to 45, wherein the cell is a primary B cell. [0265] Embodiment 48. The method of any one of Embodiments 1 to 47, wherein the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9 or a destabilization domain fused to the therapeutic antibody. [0266] Embodiment 49. The method of Embodiment 48, wherein the inducible Caspase 9 is a Caspase 9-FKBPF36V. [0267] Embodiment 50. A method of treating a subject in need thereof, comprising (i) genetically modifying a cell from the subject using the method of any one of Embodiments 1 to 49, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject. [0268] Embodiment 51. The method of Embodiment 50, wherein the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection. [0269] Embodiment 52. The method of Embodiment 50, wherein the cell is reintroduced into the subject by systemic transplantation. [0270] Embodiment 53. The method of Embodiment 50, wherein the cell is reintroduced into the subject by local transplantation. [0271] Embodiment 54. The method of Embodiment 53, wherein the local transplantation is intrafemoral or intrahepatic. [0272] Embodiment 55. The method of any one of Embodiments 50 to 54, wherein the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject. [0273] Embodiment 56. A sgRNA that specifically targets a CCR5 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 1. [0274] Embodiment 57. The sgRNA of Embodiment 56, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 4. [0275] Embodiment 58. A sgRNA that specifically targets an AAVS1 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 39. [0276] Embodiment 59. The sgRNA of Embodiment 58, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 40. [0277] Embodiment 60. A sgRNA that specifically targets an IgH locus, wherein the target sequence of the sgRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27, 31, and 35. [0278] Embodiment 61. The sgRNA of Embodiment 60, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 28, 32, and 36. [0279] Embodiment 62. The sgRNA of any one of Embodiments 56 to 61, wherein the sgRNA comprises chemical modifications at one or more nucleotides. [0280] Embodiment 63. The sgRNA of Embodiment 62, wherein the sgRNA comprises -O-methyl- -phosphorothioate (MS) modifications at one or more nucleotides. [0281] Embodiment 64. The sgRNA of Embodiment -O-methyl- - [0282] Embodiment 65. The sgRNA of Embodiment 64, wherein the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47. [0283] Embodiment 66. A homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic antibody, operably linked to (b) a promoter and (c) a polyadenylation signal at the 3’ end of the coding sequence; (ii) a first homology region located to one side of the expression cassette within the donor template; and (iii) a second homology region located to the other side of the expression cassette within the donor template. [0284] Embodiment 67. The donor template of Embodiment 66, wherein the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic. [0285] Embodiment 68. The donor template of Embodiment 66 or 67, wherein the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. [0286] Embodiment 69. The donor template of Embodiment 68, wherein the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain. [0287] Embodiment 70. The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. [0288] Embodiment 71. The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. [0289] Embodiment 72. The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 30 or a fragment thereof. [0290] Embodiment 73. The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34 or a fragment thereof. [0291] Embodiment 74. The donor template of any one of Embodiments 66 to 69, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 38 or a fragment thereof. [0292] Embodiment 75. The donor template of any one of Embodiments 66 to 71, wherein the expression cassette encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. [0293] Embodiment 76. The donor template of any one of Embodiments 66 to 75, wherein the expression cassette encodes a therapeutic antibody against HIV infection. [0294] Embodiment 77. The donor template of any one of Embodiments 66 to 76, wherein the therapeutic antibody encoded by the expression cassette comprises at least one light chain and at least one heavy chain. [0295] Embodiment 78. The donor template of Embodiment 77, wherein the at least one light chain and the at least one heavy chain are linked by a linker. [0296] Embodiment 79. The donor template of Embodiment 78, wherein the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6. [0297] Embodiment 80. The donor template of any one of Embodiments 66 to 79, wherein the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. [0298] Embodiment 81. The donor template of any one of Embodiments 66 to 80, wherein the coding sequence comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26. [0299] Embodiment 82. An HSPC comprising the sgRNA of any one of Embodiments 56 to 64 and/or the homologous donor template of any one of Embodiments 66 to 81. [0300] Embodiment 83. A B cell comprising the sgRNA of any one of Embodiments 56 to 64 and/or the homologous donor template of any one of Embodiments 66 to 81. [0301] Embodiment 84. A genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody. [0302] Embodiment 85. The genetically modified cell of Embodiment 84, wherein the selected locus is a safe harbor locus. [0303] Embodiment 86. The genetically modified cell of Embodiment 85, wherein the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. [0304] Embodiment 87. The genetically modified cell of Embodiment 84, wherein the selected locus is an immunoglobulin-associated locus. [0305] Embodiment 88. The genetically modified cell of Embodiment 87, wherein the immunoglobulin- [0306] Embodiment 89. The genetically modified cell of any one of Embodiments 84 to 88, wherein the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. [0307] Embodiment 90. The genetically modified cell of any one of Embodiments 84 to 89, wherein the cell was modified using the method of any one of Embodiments 1 to 49. [0308] Embodiment 91. pharmaceutical composition comprising a plurality of HSPCs of Embodiment 82, a plurality of B cells of Embodiment 83, or a plurality of genetically modified cells of any one of Embodiments 84 to 90. [0309] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Informal Sequence Listing SEQ ID NO: 1 CCR5 sgRNA target sequence (without PAM sequence) 5’-TGACATCAATTATTATACAT-3’ SEQ ID NO: 2 Left homology arm of CCR5 5’- tgggcgacagagtgagaccctgtctcacaacaacaacaacaacaacaaaaaggctgagctgcaccatgcttgacccag tttcttaaaattgttgtcaaagcttcattcactccatggtgctatagagcacaagattttatttggtgagatggtgctttcatgaattcccccaa cagagccaagctctccatctagtggacagggaagctagcagcaaaccttcccttcactacaaaacttcattgcttggccaaaaagaga gttaattcaatgtagacatctatgtaggcaattaaaaacctattgatgtataaaacagtttgcattcatggagggcaactaaatacattctag gactttataaaagatcactttttatttatgcacagggtggaacaag-3’ SEQ ID NO: 3 Right homology arm of CCR5 5’- catcggagccctgccaaaaaatcaatgtgaagcaaatcgcagcccgcctcctgcctccgctctactcactggtgttcatc tttggttttgtgggcaacatgctggtcatcctcatcctgataaactgcaaaaggctgaagagcatgactgacatctacctgctcaacctgg ccatctctgacctgtttttccttcttactgtccccttctgggctcactatgctgccgcccagtgggactttggaaatacaatgtgtcaactctt gacagggctctattttataggcttcttctctggaatcttcttcatcatcctcctgacaatcgataggtacctggctgtcgtccatgctgtgtttg ctttaaaagccaggacggtcacctttggggtggtgacaag-3’ SEQ ID NO: 4 CCR5 sgRNA sequence 5'- UGACAUCAAUUAUUAUACAUGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU UUU-3’ SEQ ID NO: 5 Linker 1 (AA) GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGT ATAGASSGS SEQ ID NO: 6 Linker 2 (AA) GGSSGSGSGSNWSHPQFEKGGGGSNWSHPQFEKGGGGSNWSHPQFEKGSG GGGSAGG SEQ ID NO: 7 EEK promoter 5’- taaaccggtgagtttcatggttacttgcctgagaagattaaaaaaagtaatgctaccttatgagggagagtcccagggacc aagatagcaactgtcatagcaaccgtcacactgctttggtcaaggagaagaccctttggggaactgaaaacagaaccttgagcacatc tgttgctttcgctcccatcctcctccaacagggctgggtggagcactccacaccctttcaccggtcgtacggctcagccagagtaaaaat cacacccatgacctggccactgagggcttgatcaattcactttgaatttggcattaaataccattaaggtatattaactgattttaaaataag atatattcgtgaccatgtttttaactttcaaaaatgtagctgccagtgtgtgattttatttcagttgtacaaaatatctaaacctatagcaatgtg attaataaaaacttaaacatattttccagtaccttaattctgtgataggaaaattttaatctgagtattttaatttcataatctctaaaatagtttaat gatttgtcattgtgttgctgtcgtttaccccagctgatctcaaaagtgatatttaaggagattattttggtctgcaacaacttgatagggctca gcctctcccacccaacgggtggaatcccccagagggggatttccaagaggccacctggcagttgctgagggtcagaagtgaagcta gccacttcctcttaggcaggtggccaagattacagttgacccgtacgtgcagctgtgcccagcctgccccatcccctgctcatttgcatg ttcccagagcacaacctcctgccctgaagccttattaataggctggtcacactttgtgcaggagtcagactcagtcaggacacagct-3’ SEQ ID NO: 8 B29 promoter 5’- aaacggagggttgtgaggagagtgagaggtggacagagggcaccgacgatttagcatctcttcctctcctgggggtc gaggatgagagacaaaaaagaagctgccaggaaacataaaattcagagggctcagctgcagggctgaggtctgcaagcatgctgtg tacacttgtgcatgttgtgccctgcacaagggcatctctgaaggggctgcactggacccaggggcaggggcgcaaaggtgagtttata tcagttcctgagcactgtggctccatccagcactctgaggacaggcaggatacagctggaggacctgagggctcccccacaccagct cctgttccctgcccaagaccccctggacctgcagacaacaattcaacgcactcagagtcccacagttaagaactccctgaagaagccc ccagtggctgcgtggtggattttcgcaaagctgtctccacctacatccaccctgtttggcagcccctacatactctttcacagcatgagga agggaggcctctcaccaagacctggactgaatcttctcccagtggctgccacacctgacctgctcttgctccagaacctctgtggctcc catcctccacagggtcaacttccaacatggctgcctgcactccagccaagaggctctgctctgggcccctccagatgcctgacctggg tctgtggctgccctgtccttcttcagtgctcctcttcccgctgggtgaggaatagttcaggacagaggagctaagttcaggttcattcatag gacaggtgcctatttcgctcacggcccaggaatagagacttgccgggctcggcccttcggggagttggcagacggcagaggggag gctggctggcccaggggatgaccaccggtggggtaagcacagacagaggggagcacaggcttcccccagaagactgagaggcc ccccagaggcatccacagaggaccccagctgtgctgcccaagctgggcgaccgccaaaccttagcggcccagctgacaaaagcct gccctcccccagggtccccggagagctggtgcctcccctgggtcccaatttgcatggcaggaaggggcctggtgaggaagaggcg gggaggggacaggctgcagccggtgcagttacacgttttcctccaaggagcctcggacgttgtcacgggtttggggtcggggacag agcggtgacc-3’ SEQ ID NO: 9 IgH promoter (option 1) 5’- cgtaatctttaggccaataaaatgtgggttcacagtgaggagtgcatcctggggttggggtttgttctgcagcgggaaga gcgctgtgcacagaaagcttagaaatggggcaagagatgcttttcctcaggcaggatttagggcttggtctctcagcatcccacacttgt acagctgatgtggcatctgtgttttctttctcatcctagatcaggctttgagctgtgaaataccctgcctcatgcatatgcaaataacctgag gtcttctgagataaatatagatatattggtgccctgagagcatcacgccgccacc-3’ SEQ ID NO: 10 IgH promoter (option 2) 5’- gtaacgagtggccaccttttcagtgttaccagtgagctctgagtgttcctaatgggaccaggatgggtctaggtgcctgc tcaatgtcagagacagcaatggtcccacaaaaaacccaggtaatctttaggccaataaaatgtgggttcacagtgaggagtgcatcctg gggttggggtttgttctgcagcgggaagagtgctgtgcacagaaagcttagaaatggggcaagagatgcttttcctcaggcaggattta gggcttggtctctcagcatcccacacttgtacagctgatgtggcatctgtgttttctttctcatcctagatcaggctttgagctgtgaaatacc ctgcctcatgcatatgcaaataacctgaggtcttctgagataaatatagatatattggtgccctgagagcatcacataacaaccacattcct cctctgaagaagcccctgggagcacagctcatcacc-3’ SEQ ID NO: 11 anti-HIV Ab 10-1074 (LC-L-HC) AA MGWSCIILFLVATATGVHSSYVRPLSVALGETARISCGRQALGSRAVQWYQ HRPGQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWD SRSGFSWSFGGATRLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTV EKTVAPTECSGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSA GGTATAGASSGSQVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKG LEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQ RIYGVVSFGEFFYYYSMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID NO: 12 anti-CD4 Ab Ibalizumab (LC-L-HC) AA MGWSCIILFLVATATGVHSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYST NQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVA VYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGS GGGGSAGGTATAGASSGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWV RQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVY YCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK SEQ ID NO: 13 anti-HIV Ab 3BNC117 (LC-L-HC) AA MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDTVTITCQANGYLNWY QQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEF VVPGTRLDLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATA GASSGSQVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVG WINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRS DYWDFDVWGSGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID NO: 14 anti-HIV Ab PGDM1400 (LC-L-HC) AA MGWSCIILFLVATATGVHSDFVLTQSPHSLSVTPGESASISCKSSHSLIHGDR NNYLAWYVQKPGRSPQLLIYLASSRASGVPDRFSGSGSDKDFTLKISRVETEDVGTY YCMQGRESPWTFGQGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGS GGGGSAGGTATAGASSGSQVHLTQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWV RSVPGQGLQWMGWISHEGDKKVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAV YYCAKGSKHRLRDYALYDDDGALNWAVDVDYLSNLEFWGQGTAVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 15 anti-HIV Ab 1-18 (LC-L-HC) AA MGWSCIILFLVATATGVHSEVVLTQSPAILSVSPGDRVILSCRASQGLDSSHL AWYRFKRGQIPTLVIFGTSNRARGTPDRFSGSGSGADFTLTISRVEPEDFATYYCQRY GGTPITFGGGTTLDKKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSA GGTATAGASSGSQGRLFQSGAEVKRPGASVRISCRADDDPYTDDDTFTKYWTHWIR QAPGQRPEWLGVISPHFARPIYSYKFRDRLTLTRDSSLTAVYLELKGLQPDDSGIYFC ARDPFGDRAPHYNYHMDVWGGGTAVIVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID NO: 16 anti-HIV Ab CAP256V2LS (LC-L-HC) AA MGWSCIILFLVATATGVHSQSVLTQPPSVSAAPGQKVTISCSGNTSNIGNNF VSWYQQRPGRAPQLLIYETDKRPSGIPDRFSASKSGTSGTLAITGLQTGDEADYYCAT WAASLSSARVFGTGTKVIVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGG GSAGGTATAGASSGSQVQLVESGGGVVQPGTSLRLSCAASQFRFDGYGMHWVRQA PGKGLEWVASISHDGIKKYHAEKVWGRFTISRDNSKNTLYLQMNSLRPEDTALYYC AKDLREDECEEWWSDYYDFGAQLPCAKSRGGLVGIADNWGQGTMVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 17 anti-hyperlipidemia Ab Evolocumab (LC-L-HC) AA MGWSCIILFLVATATGVHSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNS VSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCN SYTSTSMVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECSGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAG GTATAGASSGSQVQLVQSGAEVKKPGASVKVSCKASGYTLTSYGISWVRQAPGQGL EWMGWVSFYNGNTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARG YGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 18 anti-TNF-a Ab Infliximab (LC-L-HC) AA MGWSCIILFLVATATGVHSDILLTQSPAILSVSPGERVSFSCRASQFVGSSIH WYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHS WPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG TATAGASSGSEVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLE WVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYY GSTYDYWGQGTTLTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK SEQ ID NO: 19 10-1074 (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCTCCTATGTGAGACCTCTGTCCGTCGCTCTGGGCGAGACCGCCAGGAT CTCTTGTGGCAGACAGGCCCTGGGCAGCAGAGCCGTGCAGTGGTATCAGCACAG ACCTGGCCAGGCCCCTATCCTGCTGATCTACAACAATCAGGACAGACCAAGCGG CATCCCAGAGAGATTCAGCGGCACACCTGACATCAACTTCGGCACCAGGGCCAC CCTGACAATCTCCGGCGTGGAGGCCGGCGATGAGGCCGACTACTATTGCCACAT GTGGGATAGCAGGTCTGGCTTTAGCTGGTCCTTTGGCGGCGCCACCAGACTGACA GTGCTGGGCCAGCCCAAGGCCGCCCCAAGCGTGACCCTGTTTCCTCCATCCAGCG AGGAGCTGCAGGCCAACAAGGCCACACTGGTGTGCCTGATCTCCGACTTCTACCC AGGCGCCGTGACAGTGGCCTGGAAGGCCGATTCCTCTCCAGTGAAGGCCGGCGT GGAGACAACCACACCTTCCAAGCAGAGCAATAACAAGTACGCCGCCTCCTCTTA CCTGTCCCTGACCCCTGAGCAGTGGAAGTCTCACAGATCCTACAGCTGCCAGGTC ACCCACGAAGGAAGCACTGTCGAAAAAACCGTCGCTCCCACCGAATGTAGTGGG GGAAGTAGTGGGTCAGGGTCTGGAAGTACCGGGACGTCTTCCTCTGGAACAGGC ACTAGCGCTGGAACTACCGGTACGAGTGCCTCTACGAGCGGTAGCGGTAGTGGT GGCGGAGGTGGTAGCGGCGGAGGTGGATCTGCGGGTGGAACTGCCACAGCGGGC GCCAGCTCTGGTAGTCAGGTCCAGCTGCAGGAGTCAGGCCCCGGCCTGGTGAAG CCATCTGAGACCCTGTCCGTGACCTGCAGCGTGTCCGGCGATTCTATGAACAACT ATTACTGGACATGGATCAGGCAGTCTCCCGGCAAGGGCCTGGAGTGGATCGGCT ATATCTCCGATAGAGAGTCTGCCACATACAACCCTTCCCTGAACTCTAGGGTGGT AATCTCTCGGGATACCTCCAAGAACCAGCTGTCCCTGAAGCTGAACAGCGTGACC CCAGCCGATACCGCCGTGTACTATTGCGCCACCGCCAGAAGAGGCCAGAGGATC TATGGCGTGGTGAGCTTTGGCGAGTTCTTTTACTATTACTCTATGGACGTGTGGG GCAAGGGCACCACCGTGACAGTGTCTAGCGCCTCCACAAAGGGCCCAAGCGTGT TTCCCCTGGCCCCAAGCTCCAAGTCTACATCCGGCGGCACAGCCGCCCTGGGCTG CCTGGTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCC CTGACAAGCGGCGTGCACACCTTTCCTGCCGTGCTGCAGTCCTCTGGCCTGTATA GCCTGTCTTCCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCCAGACCTACAT CTGCAATGTGAATCACAAGCCCTCCAACACCAAGGTGGATAAGAAGGTGGAGCC AAAGTCTTGTGACAAGACCCACACCTGCCCACCTTGTCCCGCCCCAGAGCTGCTG GGCGGCCCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAGGATACCCTGATGATCT CCAGAACCCCAGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCAG AGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACAA AGCCTAGAGAGGAGCAGTACAACAGCACATATCGGGTGGTGTCTGTGCTGACAG TGCTGCACCAGGACTGGCTGAATGGCAAGGAGTATAAGTGCAAGGTGTCCAACA AGGCCCTGCCAGCCCCTATCGAGAAGACAATCAGCAAGGCCAAGGGCCAGCCAC GCGAGCCTCAGGTGTACACCCTGCCACCTAGCAGAGACGAGCTGACCAAGAACC AGGTGTCTCTGACATGTCTGGTGAAGGGCTTTTATCCCTCTGACATCGCCGTGGA GTGGGAGAGCAACGGCCAGCCCGAGAATAACTACAAGACCACACCTCCAGTGCT GGACAGCGATGGCTCCTTTTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGA TGGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAAGCCCTGCACAAC CATTACACCCAGAAGAGCCTGTCCCTGAGCCCCGGAAAATAG SEQ ID NO: 20 Ibalizumab (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCGACATCGTGATGACCCAGTCTCCAGATAGCCTGGCCGTGTCCCTGGG AGAGAGGGTGACCATGAACTGTAAGAGCAGCCAGTCCCTGCTGTACTCTACAAA CCAGAAGAATTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGTCCCCCAAGCT GCTGATCTATTGGGCCAGCACCCGGGAGTCCGGCGTGCCTGACAGATTCTCTGGC AGCGGCTCCGGCACAGACTTCACCCTGACAATCTCTAGCGTGCAGGCCGAGGAC GTGGCCGTGTACTATTGCCAGCAGTACTATAGCTACAGGACCTTCGGCGGCGGCA CAAAGCTGGAGATCAAGAGGACCGTGGCAGCACCTTCCGTGTTCATCTTTCCCCC TTCTGACGAGCAGCTGAAGTCTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAA CTTCTACCCACGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTC CGGCAATTCTCAGGAGAGCGTGACCGAGCAGGACTCCAAGGATTCTACATATAG CCTGTCCTCTACCCTGACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTAT GCATGCGAGGTGACCCACCAGGGACTGAGCAGCCCAGTGACAAAGAGCTTTAAT AGGGGCGAGTGTGGGGGAAGTAGTGGGTCAGGGTCTGGAAGTACCGGGACGTCT TCCTCTGGAACAGGCACTAGCGCTGGAACTACCGGTACGAGTGCCTCTACGAGC GGTAGCGGTAGTGGTGGCGGAGGTGGTAGCGGCGGAGGTGGATCTGCGGGTGGA ACTGCCACAGCGGGCGCCAGCTCTGGTAGTCAGGTGCAGCTGCAGCAGTCCGGA CCAGAGGTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGC TACACCTTCACAAGCTATGTGATCCACTGGGTGCGGCAGAAGCCAGGACAGGGC CTGGACTGGATCGGCTACATCAACCCTTATAATGATGGCACCGACTACGATGAGA AGTTTAAGGGCAAGGCCACCCTGACATCTGACACCTCCACATCTACCGCCTATAT GGAGCTGAGCAGCCTGAGGAGCGAGGACACAGCCGTGTACTATTGCGCCCGCGA GAAGGATAACTACGCAACCGGAGCATGGTTCGCATATTGGGGACAGGGCACACT GGTGACCGTGTCTAGCGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCC TGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG TGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAG ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC CCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCT GTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCC TGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTC TCCCTGTCTCTGGGTAAATAG SEQ ID NO: 21 3BNC117 (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCGACATTCAGATGACTCAGAGCCCTTCTTCACTGTCCGCCTCTGTGGG CGATACAGTGACCATCACATGCCAGGCCAATGGCTACCTGAACTGGTATCAGCA GAGAAGGGGCAAGGCCCCAAAGCTGCTGATCTACGATGGCTCTAAGCTGGAGAG AGGCGTGCCAAGCAGATTCTCTGGCAGGAGATGGGGCCAGGAGTACAACCTGAC CATCAACAATCTGCAGCCAGAGGACATCGCCACCTACTTCTGTCAGGTGTACGAG TTCGTGGTGCCCGGCACAAGACTGGATCTGAAGAGGACCGTGGCAGCACCTTCC GTGTTCATCTTTCCCCCTTCTGACGAGCAGCTGAAGTCTGGCACAGCCAGCGTGG TGTGCCTGCTGAACAACTTCTACCCACGGGAGGCCAAGGTGCAGTGGAAGGTGG ATAACGCCCTGCAGTCCGGCAATTCTCAGGAGAGCGTGACCGAGCAGGACTCCA AGGATTCTACATATAGCCTGTCCTCTACCCTGACACTGTCTAAGGCCGATTACGA GAAGCACAAGGTGTATGCATGCGAGGTGACCCACCAGGGACTGAGCAGCCCAGT GACAAAGAGCTTTAATAGGGGCGAGTGTGGGGGAAGTAGTGGGTCAGGGTCTGG AAGTACCGGGACGTCTTCCTCTGGAACAGGCACTAGCGCTGGAACTACCGGTAC GAGTGCCTCTACGAGCGGTAGCGGTAGTGGTGGCGGAGGTGGTAGCGGCGGAGG TGGATCTGCGGGTGGAACTGCCACAGCGGGCGCCAGCTCTGGTAGTCAGGTGCA GCTGCTGCAGTCTGGAGCAGCAGTGACCAAGCCAGGAGCCTCCGTGCGCGTGTC TTGTGAGGCCAGCGGCTACAACATCAGAGACTACTTCATCCACTGGTGGAGGCA GGCACCAGGACAGGGACTGCAGTGGGTGGGCTGGATCAATCCTAAGACCGGCCA GCCAAACAATCCAAGGCAGTTCCAGGGACGCGTGAGCCTGACCAGGCACGCCTC CTGGGACTTTGATACATTCTCCTTTTACATGGACCTGAAGGCCCTGCGGTCTGAC GATACCGCCGTGTACTTCTGCGCCCGGCAGAGAAGCGATTATTGGGACTTTGACG TGTGGGGCTCCGGCACCCAGGTGACCGTGAGCAGCGCCTCCACAAAGGGCCCAA GCGTGTTTCCCCTGGCCCCAAGCTCCAAGTCTACATCCGGCGGCACAGCCGCCCT GGGCTGCCTGGTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCCTGGAACTCT GGCGCCCTGACAAGCGGCGTGCACACCTTTCCTGCCGTGCTGCAGTCCTCTGGCC TGTATAGCCTGTCTTCCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCCAGAC CTACATCTGCAATGTGAATCACAAGCCCTCCAACACCAAGGTGGATAAGAAGGT GGAGCCAAAGTCTTGTGACAAGACCCACACCTGCCCACCTTGTCCCGCCCCAGAG CTGCTGGGCGGCCCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAGGATACCCTGA TGATCTCCAGAACCCCAGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGG ACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCA AGACAAAGCCTAGAGAGGAGCAGTACAACAGCACATATCGGGTGGTGTCTGTGC TGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTATAAGTGCAAGGTGT CCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACAATCAGCAAGGCCAAGGGCC AGCCACGCGAGCCTCAGGTGTACACCCTGCCACCTAGCAGAGACGAGCTGACCA AGAACCAGGTGTCTCTGACATGTCTGGTGAAGGGCTTTTATCCCTCTGACATCGC CGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAATAACTACAAGACCACACCTCC AGTGCTGGACAGCGATGGCTCCTTTTTCCTGTACTCCAAGCTGACCGTGGACAAG TCCAGATGGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAAGCCCTGC ACAACCATTACACCCAGAAGAGCCTGTCCCTGAGCCCCGGAAAATAG SEQ ID NO: 22 PGDM1400 (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCGATTTTGTCCTGACTCAGTCTCCACACTCTCTGTCCGTCACCCCTGGA GAGTCGGCCTCCATCTCCTGCAAGTCTAGTCACAGCCTCATTCATGGTGATAGGA ACAATTATTTGGCTTGGTACGTACAGAAGCCAGGGCGGTCTCCACAACTCCTGAT CTATTTGGCTTCCAGTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGA TCGGACAAAGATTTTACACTGAAGATCAGCAGAGTGGAGACTGAGGATGTTGGG ACGTATTACTGCATGCAAGGTCGAGAAAGTCCCTGGACGTTCGGCCAAGGGACC AAGGTGGACATCAAAAGGACCGTGGCAGCACCTTCCGTGTTCATCTTTCCCCCTT CTGACGAGCAGCTGAAGTCTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACT TCTACCCACGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCCG GCAATTCTCAGGAGAGCGTGACCGAGCAGGACTCCAAGGATTCTACATATAGCC TGTCCTCTACCCTGACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGC ATGCGAGGTGACCCACCAGGGACTGAGCAGCCCAGTGACAAAGAGCTTTAATAG GGGCGAGTGTGGGGGAAGTAGTGGGTCAGGGTCTGGAAGTACCGGGACGTCTTC CTCTGGAACAGGCACTAGCGCTGGAACTACCGGTACGAGTGCCTCTACGAGCGG TAGCGGTAGTGGTGGCGGAGGTGGTAGCGGCGGAGGTGGATCTGCGGGTGGAAC TGCCACAGCGGGCGCCAGCTCTGGTAGTCAGGTGCATCTGACGCAGTCTGGGCCT GAGGTGAGGAAGCCTGGGACCTCCGTAAAGGTCTCCTGCAAGGCCCCTGGAAAC ACATTGAAGACTTATGATCTACACTGGGTGCGCAGCGTCCCTGGACAAGGCCTTC AGTGGATGGGATGGATAAGCCATGAGGGCGACAAGAAGGTCATTGTGGAAAGAT TCAAGGCCAAAGTCACCATTGATTGGGACAGGTCCACCAATACGGCCTATCTCCA ACTGAGCGGCCTCACATCTGGCGACACGGCCGTCTATTATTGTGCGAAAGGCTCA AAACACAGGCTGCGAGATTACGCTCTCTACGACGACGACGGCGCATTGAATTGG GCTGTCGATGTTGACTACCTTTCGAACTTGGAATTCTGGGGCCAAGGGACCGCCG TCACCGTCTCTTCAGCCTCCACAAAGGGCCCAAGCGTGTTTCCCCTGGCCCCAAG CTCCAAGTCTACATCCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGATTAC TTCCCAGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGC ACACCTTTCCTGCCGTGCTGCAGTCCTCTGGCCTGTATAGCCTGTCTTCCGTGGTG ACAGTGCCTTCCTCTAGCCTGGGCACCCAGACCTACATCTGCAATGTGAATCACA AGCCCTCCAACACCAAGGTGGATAAGAAGGTGGAGCCAAAGTCTTGTGACAAGA CCCACACCTGCCCACCTTGTCCCGCCCCAGAGCTGCTGGGCGGCCCCTCCGTGTT CCTGTTTCCACCCAAGCCTAAGGATACCCTGATGATCTCCAGAACCCCAGAGGTG ACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCAGAGGTGAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACAAAGCCTAGAGAGGAGCA GTACAACAGCACATATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTG GCTGAATGGCAAGGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCC TATCGAGAAGACAATCAGCAAGGCCAAGGGCCAGCCACGCGAGCCTCAGGTGTA CACCCTGCCACCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCTCTGACATG TCTGGTGAAGGGCTTTTATCCCTCTGACATCGCCGTGGAGTGGGAGAGCAACGGC CAGCCCGAGAATAACTACAAGACCACACCTCCAGTGCTGGACAGCGATGGCTCC TTTTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGCAACG TGTTTAGCTGCTCCGTGATGCACGAAGCCCTGCACAACCATTACACCCAGAAGAG CCTGTCCCTGAGCCCCGGAAAATAG SEQ ID NO: 23 1-18 (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCGAGGTAGTGTTGACACAGAGCCCGGCCATTCTTTCCGTTTCTCCGGG GGACAGGGTTATCTTGAGTTGCCGCGCCTCCCAAGGACTTGATTCATCACACCTT GCGTGGTACAGGTTCAAGAGGGGCCAGATACCTACGCTCGTTATATTCGGCACTT CAAACAGGGCACGCGGAACACCCGACCGCTTTTCCGGGTCCGGTTCTGGGGCAG ATTTTACCTTGACCATTTCAAGGGTGGAGCCGGAGGATTTTGCTACCTATTACTGT CAGAGGTATGGCGGTACTCCAATCACGTTCGGCGGTGGTACGACCCTCGACAAA AAAAGGACCGTGGCAGCACCTTCCGTGTTCATCTTTCCCCCTTCTGACGAGCAGC TGAAGTCTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCACGGGA GGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCCGGCAATTCTCAGGA GAGCGTGACCGAGCAGGACTCCAAGGATTCTACATATAGCCTGTCCTCTACCCTG ACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGCATGCGAGGTGACC CACCAGGGACTGAGCAGCCCAGTGACAAAGAGCTTTAATAGGGGCGAGTGTGGG GGAAGTAGTGGGTCAGGGTCTGGAAGTACCGGGACGTCTTCCTCTGGAACAGGC ACTAGCGCTGGAACTACCGGTACGAGTGCCTCTACGAGCGGTAGCGGTAGTGGT GGCGGAGGTGGTAGCGGCGGAGGTGGATCTGCGGGTGGAACTGCCACAGCGGGC GCCAGCTCTGGTAGTCAAGGCCGCCTCTTCCAGTCAGGAGCAGAAGTAAAAAGA CCAGGAGCATCCGTGAGGATTTCCTGCCGGGCAGATGATGACCCGTACACGGAT GACGACACGTTTACTAAATACTGGACCCACTGGATTAGACAGGCTCCGGGGCAG CGGCCTGAGTGGCTCGGCGTTATTAGTCCGCATTTTGCGAGGCCGATATACTCCT ACAAATTTAGAGATAGGCTCACACTTACGCGGGATAGCTCACTTACTGCGGTTTA CCTGGAGTTGAAGGGCTTGCAACCGGACGACAGCGGGATCTATTTCTGTGCACG GGACCCATTTGGTGATAGGGCACCCCACTATAACTATCACATGGACGTCTGGGGC GGAGGTACTGCTGTGATTGTAAGCTCAGCCTCCACAAAGGGCCCAAGCGTGTTTC CCCTGGCCCCAAGCTCCAAGTCTACATCCGGCGGCACAGCCGCCCTGGGCTGCCT GGTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTG ACAAGCGGCGTGCACACCTTTCCTGCCGTGCTGCAGTCCTCTGGCCTGTATAGCC TGTCTTCCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCCAGACCTACATCTG CAATGTGAATCACAAGCCCTCCAACACCAAGGTGGATAAGAAGGTGGAGCCAAA GTCTTGTGACAAGACCCACACCTGCCCACCTTGTCCCGCCCCAGAGCTGCTGGGC GGCCCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAGGATACCCTGATGATCTCCA GAACCCCAGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCAGAGG TGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACAAAGC CTAGAGAGGAGCAGTACAACAGCACATATCGGGTGGTGTCTGTGCTGACAGTGC TGCACCAGGACTGGCTGAATGGCAAGGAGTATAAGTGCAAGGTGTCCAACAAGG CCCTGCCAGCCCCTATCGAGAAGACAATCAGCAAGGCCAAGGGCCAGCCACGCG AGCCTCAGGTGTACACCCTGCCACCTAGCAGAGACGAGCTGACCAAGAACCAGG TGTCTCTGACATGTCTGGTGAAGGGCTTTTATCCCTCTGACATCGCCGTGGAGTG GGAGAGCAACGGCCAGCCCGAGAATAACTACAAGACCACACCTCCAGTGCTGGA CAGCGATGGCTCCTTTTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGATGG CAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAAGCCCTGCACAACCATT ACACCCAGAAGAGCCTGTCCCTGAGCCCCGGAAAATAG GCTGAAGTCTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCACGG GAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCCGGCAATTCTCAG GAGAGCGTGACCGAGCAGGACTCCAAGGATTCTACATATAGCCTGTCCTCTACCC TGACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGCATGCGAGGTGA CCCACCAGGGACTGAGCAGCCCAGTGACAAAGAGCTTTAATAGGGGCGAGTGTG GGGGAAGTAGTGGGTCAGGGTCTGGAAGTACCGGGACGTCTTCCTCTGGAACAG GCACTAGCGCTGGAACTACCGGTACGAGTGCCTCTACGAGCGGTAGCGGTAGTG GTGGCGGAGGTGGTAGCGGCGGAGGTGGATCTGCGGGTGGAACTGCCACAGCGG GCGCCAGCTCTGGTAGTcaggtgcagttggtggagtctgggggaggcgtggtccagcctgggacgtccctgagact ctcctgtgcagcctctcaattcaggtttgatggttatggcatgcactgggtccgccaggccccaggcaaggggctggagtgggtggca tctatatcacatgatggaattaaaaagtatcacgcagaaaaagtgtggggccgcttcaccatctccagagacaattccaagaacacact gtatctacaaatgaacagcctgcgacctgaggacacggctctctactactgtgcgaaagatttgcgagaagacgaatgtgaagagtgg tggtcggattattacgattttggggcacaactcccttgcgcaaagtcacgcggcggcttggttggaattgctgataactggggccaagg gacaatggtcaccgtctcttcaGCCTCCACAAAGGGCCCAAGCGTGTTTCCCCTGGCCCCAAG CTCCAAGTCTACATCCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGATTAC TTCCCAGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGC ACACCTTTCCTGCCGTGCTGCAGTCCTCTGGCCTGTATAGCCTGTCTTCCGTGGTG ACAGTGCCTTCCTCTAGCCTGGGCACCCAGACCTACATCTGCAATGTGAATCACA AGCCCTCCAACACCAAGGTGGATAAGAAGGTGGAGCCAAAGTCTTGTGACAAGA CCCACACCTGCCCACCTTGTCCCGCCCCAGAGCTGCTGGGCGGCCCCTCCGTGTT CCTGTTTCCACCCAAGCCTAAGGATACCCTGATGATCTCCAGAACCCCAGAGGTG ACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCAGAGGTGAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACAAAGCCTAGAGAGGAGCA GTACAACAGCACATATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTG GCTGAATGGCAAGGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCC TATCGAGAAGACAATCAGCAAGGCCAAGGGCCAGCCACGCGAGCCTCAGGTGTA CACCCTGCCACCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCTCTGACATG TCTGGTGAAGGGCTTTTATCCCTCTGACATCGCCGTGGAGTGGGAGAGCAACGGC CAGCCCGAGAATAACTACAAGACCACACCTCCAGTGCTGGACAGCGATGGCTCC TTTTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGCAACG TGTTTAGCTGCTCCGTGCTGCACGAAGCCCTGCACAGCCATTACACCCAGAAGAG CCTGTCCCTGAGCCCCGGAAAATAG SEQ ID NO: 25 Evolocumab (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACA GTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTCT GTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGG TCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTAC TGCAATTCATATACAAGCACCAGCATGGTATTCGGCGGAGGGACCAAGCTGACC GTCCTAcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgt gtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccac cacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagct acagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcaGGGGGAAGTAGT GGGTCAGGGTCTGGAAGTACCGGGACGTCTTCCTCTGGAACAGGCACTAGCGCT GGAACTACCGGTACGAGTGCCTCTACGAGCGGTAGCGGTAGTGGTGGCGGAGGT GGTAGCGGCGGAGGTGGATCTGCGGGTGGAACTGCCACAGCGGGCGCCAGCTCT GGTAGTCAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCC TCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTAACCAGCTATGGTATCA GCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGGTCAGTT TTTATAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGGCACCATGA CCACAGACCCATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTG ACGACACGGCCGTGTATTACTGTGCGAGAGGCTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCT SEQ ID NO: 26 Infliximab (S-LC-L-HC) NA ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAG TGCACAGCGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGG AGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGTTCGTTGGCTCAAGCATCCAC TGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTG AGTCTATGTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTT TACTCTTAGCATCAACACTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAA CAAAGTCATAGCTGGCCATTCACGTTCGGCTCGGGGACAAATTTGGAAGTAAAA AGGACCGTGGCAGCACCTTCCGTGTTCATCTTTCCCCCTTCTGACGAGCAGCTGA AGTCTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCACGGGAGG CCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCCGGCAATTCTCAGGAGA GCGTGACCGAGCAGGACTCCAAGGATTCTACATATAGCCTGTCCTCTACCCTGAC ACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGCATGCGAGGTGACCCA CCAGGGACTGAGCAGCCCAGTGACAAAGAGCTTTAATAGGGGCGAGTGTGGGGG AAGTAGTGGGTCAGGGTCTGGAAGTACCGGGACGTCTTCCTCTGGAACAGGCAC TAGCGCTGGAACTACCGGTACGAGTGCCTCTACGAGCGGTAGCGGTAGTGGTGG CGGAGGTGGTAGCGGCGGAGGTGGATCTGCGGGTGGAACTGCCACAGCGGGCGC CAGCTCTGGTAGTGAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACC TGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCATTTTCAGTAACCACT GGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAA TTAGATCAAAATCTATTAATTCTGCAACACATTATGCGGAGTCTGTGAAAGGGAG GTTCACCATCTCAAGAGATGATTCCAAAAGTGCTGTCTACCTGCAAATGACCGAC TTAAGAACTGAAGACACTGGCGTTTATTACTGTTCCAGGAATTACTACGGTAGTA CCTACGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCGCCTCCACAAAGGG CCCAAGCGTGTTTCCCCTGGCCCCAAGCTCCAAGTCTACATCCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCCTGGA ACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCTGCCGTGCTGCAGTCCTC TGGCCTGTATAGCCTGTCTTCCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCC AGACCTACATCTGCAATGTGAATCACAAGCCCTCCAACACCAAGGTGGATAAGA AGGTGGAGCCAAAGTCTTGTGACAAGACCCACACCTGCCCACCTTGTCCCGCCCC AGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAGGATACC CTGATGATCTCCAGAACCCCAGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACG AGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACG CCAAGACAAAGCCTAGAGAGGAGCAGTACAACAGCACATATCGGGTGGTGTCTG TGCTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTATAAGTGCAAGG TGTCCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACAATCAGCAAGGCCAAGG GCCAGCCACGCGAGCCTCAGGTGTACACCCTGCCACCTAGCAGAGACGAGCTGA CCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGGCTTTTATCCCTCTGACAT CGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAATAACTACAAGACCACACC TCCAGTGCTGGACAGCGATGGCTCCTTTTTCCTGTACTCCAAGCTGACCGTGGAC AAGTCCAGATGGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAAGCC CTGCACAACCATTACACCCAGAAGAGCCTGTCCCTGAGCCCCGGAAAATAG SEQ ID NO: 27 B cell locus 1 sgRNA target sequence 5' GTCTCAGGAGCGGTGTCTGT 3' SEQ ID NO: 28 B cell locus 1 sgRNA sequence 5’ GUCUCAGGAGCGGUGUCUGUGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU UUU 3’ SEQ ID NO: 29 B cell locus 1 Left homology arm 5’ tgtgacgcccggagacagaaggtctctgggtggctgggtttttgtggggtgaggatggacattctgccattgtgattac tactactactactacatggacgtctggggcaaagggaccacggtcaccgtctcctcaggtaagaatggccactctagggcctttgttttc tgctactgcctgtggggtttcctgagcattgcaggttggtcctcggggcatgttccgaggggacctgggcggactggccaggagggg atgggcactggggtgccttgaggatctgggagcctctgtggattttccgatgcctttggaaaatgggactcaggttgggtgcgtctgatg gagtaactgagcctgggggcttggggagccacatttggacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtg tctcaggagcggtgtct 3’ SEQ ID NO: 30 B cell locus 1 Right homology arm 5’ gtaggactgcaagatcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggtgcgctgtgtgtcaacct gcatcttaaattctttattggctggaaagagaactgtcggagtgggtgaatccagccaggagggacgcgtagccccggtcttgatgag agcagggttgggggcaggggtagcccagaaacggtggctgccgtcctgacaggggcttagggaggctccaggacctcagtgcctt gaagctggtttccatgagaaaaggattgtttatcttaggaggcatgcttactgttaaaagacaggatatgtttgaagtggcttctgagaaaa atggttaagaaaattatgacttaaaaatgtgagagattttcaagtatattaatttttttaactgtccaagtatttgaaattcttatcatttgattaac acccatg 3’ SEQ ID NO: 31 B cell locus 2 sgRNA target sequence 5' AGGCATCGGAAAATCCACAG 3' SEQ ID NO: 32 B cell locus 2 sgRNA sequence 5’ AGGCAUCGGAAAAUCCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAA SEQ ID NO: 33 B cell locus 2 Left homology arm 5’ accacggtcaccgtctcctcaggtaagaatggccactctagggcctttgttttctgctactgcctgtggggtttcctgagcat tgcaggttggtcctcggggcatgttccgaggggacctgggcggactggccaggaggggatgggcactggggtgccttgaggatctg ggagcctctg 3’ SEQ ID NO: 34 B cell locus 2 Right homology arm 5’ tggattttccgatgcctttggaaaatgggactcaggttgggtgcgtctgatggagtaactgagcctgggggcttggggagc cacatttggacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtgtctcaggagcggtgtctgtaggactgcaag atcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggtgcgctgtgtgtcaacctgcatcttaaattctttattggctggaa agagaactgtcggagtgggtgaatccagccaggagggacgcgtagccccggtcttgatgagagcagggttgggggcaggggtag cccagaaacggtggctgccgtcctgacaggggcttagggaggctccaggacctcagtgccttgaagctggtttccatgagaaaagga ttgtttatcttaggaggcatgcttactgttaaaagacaggatatgtttgaagtggcttctgagaaaaatggttaagaaaattatgac 3’ SEQ ID NO: 35 B cell locus 3 sgRNA target sequence 5' TCTTGATGAGAGCAGGGTTG 3' SEQ ID NO: 36 B cell locus 3 sgRNA sequence 5’ UCUUGAUGAGAGCAGGGUUGGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU UUU 3’ SEQ ID NO: 37 B cell locus 3 Left homology arm 5’ ctcggggcatgttccgaggggacctgggcggactggccaggaggggatgggcactggggtgccttgaggatctggg agcctctgtggattttccgatgcctttggaaaatgggactcaggttgggtgcgtctgatggagtaactgagcctgggggcttggggagc cacatttggacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtgtctcaggagcggtgtctgtaggactgcaag atcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggtgcgctgtgtgtcaacctgcatcttaaattctttattggctggaa agagaactgtcggagtgggtgaatccagccaggagggacgcgtagccccggtc 3’ SEQ ID NO: 38 B cell locus 3 Right homology arm 5’Agcagggttgggggcaggggtagcccagaaacggtggctgccgtcctgacaggggcttagggaggctccagga cct cagtgccttgaagctggtttccatgagaaaaggattgtttatcttaggaggcatgcttactgttaaaagacaggatatgtttgaagtggctt ctgagaaaaatggttaagaaaattatgacttaaaaatgtgagagattttcaagtatattaatttttttaactgtccaagtatttgaaattcttatc atttgattaacacccatgagtgatatgtgtctggaattgaggccaaagcaagctcagctaagaaatactagcacagtgctgtcggcccc gatgcgggactgcgttttgaccatcataaatcaagtttattttttta 3’ SEQ ID NO: 39 AAVS1 sgRNA target sequence 5’ GGGGCCACTAGGGACAGGAT 3’ SEQ ID NO: 40 AAVS1 sgRNA sequence 5’ GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU UUU 3’ SEQ ID NO: 41 AAVS1 Left homology arm 5’Agaactcaggaccaacttattctgattttgtttttccaaactgcttctcctcttgggaagtgtaaggaagctgcagcacca ggatcagtgaaacgcaccagacagccgcgtcagagcagctcaggttctgggagagggtagcgcagggtggccactgagaaccgg gcaggtcacgcatcccccccttccctcccaccccctgccaagctctccctcccaggatcctctctggctccatcgtaagcaaaccttag aggttctggcaaggagagagatggctccaggaaatgggggtgtgtcaccagataaggaatctgcctaacaggaggtgggggttaga cccaatatcaggagactaggaaggaggaggcctaaggatggggcttttctgtcaccaatc 3’ SEQ ID NO: 42 AAVS1 Right homology arm 5’Ctgtccctagtggccccactgtggggtggaggggacagataaaagtacccagaaccagagccacattaaccggcc ctgggaatataaggtggtcccagctcggggacacaggatccctggaggcagcaaacatgctgtcctgaagtggacataggggcccg ggttggaggaagaagactagctgagctctcggacccctggaagatgccatgacagggggctggaagagctagcacagactagaga ggtaaggggggtaggggagctgcccaaatgaaaggagtgagaggtgacccgaatccacaggagaacggggtgtccaggcaaag aaagcaagaggatggagaggtggctaaagccagggagacggggtactttggggttgtccagaaaaacggtg 3’ SEQ ID NO: 43 CCR5 sgRNA sequence (MS modified) 5'[mU](ps)[mG](ps)[mA](ps)CAUCAAUUAUUAUACAUGUUUUAGAGCUAG AAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCUU[mU](ps) [mU](ps)[mU](ps)U-3’ SEQ ID NO: 44 B cell locus 1 sgRNA sequence (MS modified) 5’ [mG](ps)[mU](ps)[mC](ps)UCAGGAGCGGUGUCUGUGUUUUAGAGCUAGAA AUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUU[mU](ps)[mU](ps)[mU](ps)U 3’ SEQ ID NO: 45 B cell locus 2 sgRNA sequence (MS modified) 5’ [mA](ps)[mG](ps)[mG](ps)CAUCGGAAAAUCCACAGGUUUUAGAGCUAGA AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUU[mU](ps)[mU](ps)[mU](ps)U 3’ SEQ ID NO: 46 B cell locus 3 sgRNA sequence (MS modified) 5’ [mU](ps)[mC](ps)[mU](ps)UGAUGAGAGCAGGGUUGGUUUUAGAGCUAGA AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUU[mU](ps)[mU](ps)[mU](ps)U 3’ SEQ ID NO: 47 AAVS1 sgRNA sequence (MS modified) 5’ [mG](ps)[mG](ps)[mG](ps)GCCACUAGGGACAGGAUGUUUUAGAGCUAGA AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUU[mU](ps)[mU](ps)[mU](ps)U 3’ SEQ ID NO: 48 CCR5-BGH Forward Primer (FP) 5’-GGGAGGATTGGGAAGACA-3’ SEQ ID NO: 49 CCR5-BGH Reverse Primer (RP) 5’-AGGTGTTCAGGAGAAGGACA-3’ SEQ ID NO: 50 CCR5-BGH Probe 5’-6-FAM/AGCAGGCATGCTGGGGATGCGGTGG/3IABkFQ-3’ SEQ ID NO: 51 CCR5-IgG1 FP 5’-CCTGAGCCCCGGAAAATAG-3’ SEQ ID NO: 52 CCR5-IgG1 RP 5’-AGGTGTTCAGGAGAAGGACA-3’ SEQ ID NO: 53 CCR5-IgG1 Probe 5’-6-FAM/AGCAGGCATGCTGGGGATGCGGTGG/3IABkFQ-3’ SEQ ID NO: 54 CCR5-IgG4 FP 5’-CCTCTCCCTGTCTCTGGGTA-3’ SEQ ID NO: 55 CCR5-IgG4 RP 5’-AGGTGTTCAGGAGAAGGACA-3’ SEQ ID NO: 56 CCR5-IgG4 Probe 5’-6-FAM/AGCAGGCATGCTGGGGATGCGGTGG/3IABkFQ-3’ SEQ ID NO: 57 CCRL2 FP 5’- GCTGTATGAATCCAGGTCC-3’, SEQ ID NO: 58 CCRL2 RP 5’- CCTCCTGGCTGAGAAAAAG -3’ SEQ ID NO: 59 CCRL2 Probe 5’- HEX/TGTTTCCTC/ZEN/CAGGATAAGGCAGCTGT/3IABkFQ -3’ SEQ ID NO: 60 CCR5 FP 5’-CAGGGAAGCTAGCAGCAAACC-3’ SEQ ID NO: 61 CCR5 RP 5’-AGACGCAAACACAGCCACC-3’ SEQ ID NO: 62 Ibalizumab FP 5’-ACAGTCCTCAGGACTCTACTCC-3’ SEQ ID NO: 63 10-1074 FP 5’-TATGGCGTGGTGAGCTTTGG-3’ SEQ ID NO: 64 PGDM1400 FP 5’-CTGGGACCTCCGTAAAGGTCT-3’ SEQ ID NO: 65 CCR5 RP 5’-AGACGCAAACACAGCCACC-3’

Claims

WHAT IS CLAIMED IS: 1. A method of genetically modifying a cell from a subject, the method comprising: introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting a selected locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic antibody, wherein: the sgRNA binds to the nuclease and directs it to a target sequence at the selected locus in a genome, whereupon the nuclease cleaves the locus at the target sequence; the homologous donor template comprises a first homology region to one side of the transgene, and a second homology region to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved locus; and the integrated transgene directs the expression of the therapeutic antibody in the cell. 2. The method of claim 1, wherein the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, the RNA-guided nuclease, and the homologous donor template. 3. The method of claim 1 or 2, wherein the sgRNA comprises chemical modifications at one or more nucleotides. 4. The method of claim 1 -O-methyl- - phosphorothioate (MS) modifications at one or more nucleotides. 5. The method of claim 4 -O-methyl- -phosphorothioate 6. The method of claim 1, wherein the selected locus is a safe harbor locus. 7. The method of claim 6, wherein the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus.
8. The method of claim 7, wherein the safe harbor locus is the CCR5 locus and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 1. 9. The method of claim 8, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 4. 10. The method of claim 8 or 9, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof. 11. The method of claim 8, wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. 12. The method of claim 7, wherein the safe harbor locus is the AAVS1 locus, and wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 39. 13. The method of claim 12, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 40. 14. The method of claim 12 or 13, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof. 15. The method of claim 12, wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. 16. The method of claim 1, wherein the selected locus is an immunoglobulin-associated locus. 17. The method of claim 16, wherein the immunoglobulin-associated locus is an IgH locus, an an 18. The method of claim 17, wherein the immunoglobulin-associated locus is the IgH locus, and wherein the target sequence of the sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 27, 31 and 35. 19. The method of claim 18, wherein the sgRNA comprises a sequence having 80% or greater identity to SEQ ID NO: 28, 32 or 36.
20. The method of claim 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 28, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29, or a fragment thereof. 21. The method of claim 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34, or a fragment thereof. 22. The method of claim 18 or 19, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 36, or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37, or a fragment thereof. 23. The method of claim 1, wherein the RNA-guided nuclease is a Cas9. 24. The method of claim 1, wherein the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP). 25. The method of claim 24, wherein the RNP is introduced into the cell by electroporation. 26. The method of claim 1, wherein the transgene is present within an expression cassette. 27. The method of claim 26, wherein the expression cassette comprises a coding sequence for the therapeutic antibody, operably linked to a promoter, and an exogenous polyadenylation (polyA) fragment. 28. The method of claim 27, wherein the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. 29. The method of claim 28, wherein the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain.
30. The method of claim 27, wherein the promoter is a B-cell specific promoter. 31. The method of claim 30, wherein the B-cell specific promoter is an EEK promoter, a B29 promoter, a IgH promoter, or a variant thereof. 32. The method of claim 31, wherein the EEK promoter comprises a sequence having at least 80% identity to SEQ ID NO: 7. 33. The method of claim 31, wherein the B29 promoter comprises a sequence having at least 80% identity to SEQ ID NO: 8. 34. The method of claim 31, wherein the IgH promoter comprises a sequence having at least 80% identity to SEQ ID NO: 9 or 10. 35. The method of claim 27, wherein the exogenous polyA fragment is a bovine growth hormone (BGH) polyA fragment. 36. The method of claim 27, wherein the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic antibody. 37. The method of claim 1, wherein the transgene encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. 38. The method of claim 1, wherein the transgene encodes a therapeutic antibody against HIV infection. 39. The method of claim 1, wherein the therapeutic antibody encoded by the transgene comprises at least one light chain and at least one heavy chain. 40. The method of claim 39, wherein the at least one light chain and the at least one heavy chain are linked by a linker. 41. The method of claim 40, wherein the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6.
42. The method of claim 1, wherein the therapeutic antibody comprises an amino acid sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. 43. The method of claim 1, wherein the transgene comprises a nucleotide sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26. 44. The method of claim 1, wherein the homologous donor template is introduced into the cell using a recombinant adeno-associated virus (rAAV) vector. 45. The method of claim 44, wherein the recombinant adeno-associated virus is serotype 6 (rAAV6). 46. The method of claim 1, wherein the cell is a hematopoietic stem and progenitor cell (HSPC). 47. The method of claim 1, wherein the cell is a primary B cell. 48. The method of claim 1, wherein the method further comprises introducing into the cell a sequence encoding an inducible Caspase 9 or a destabilization domain fused to the therapeutic antibody. 49. The method of claim 48, wherein the inducible Caspase 9 is a Caspase 9-FKBPF36V. 50. A method of treating a subject in need thereof, comprising (i) genetically modifying a cell from the subject using the method of any one of claims 1 to 49, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject. 51. The method of claim 50, wherein the subject has a viral infection, a cancer, an immunodeficiency disorder, a cytokine release syndrome, a bacterial infection, or a pathogen infection. 52. The method of claim 50, wherein the cell is reintroduced into the subject by systemic transplantation. 53. The method of claim 50, wherein the cell is reintroduced into the subject by local transplantation.
54. The method of claim 53, wherein the local transplantation is intrafemoral or intrahepatic. 55. The method of claim 50 , wherein the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject. 56. A sgRNA that specifically targets a CCR5 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 1. 57. The sgRNA of claim 56, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 4. 58. A sgRNA that specifically targets an AAVS1 locus, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 39. 59. The sgRNA of claim 58, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to SEQ ID NO: 40. 60. A sgRNA that specifically targets an IgH locus, wherein the target sequence of the sgRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27, 31, and 35. 61. The sgRNA of claim 60, wherein the sgRNA comprises a nucleotide sequence having 80% or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 28, 32, and 36. 62. The sgRNA of claim 56, wherein the sgRNA comprises chemical modifications at one or more nucleotides. 63. The sgRNA of claim 62 -O-methyl- - phosphorothioate (MS) modifications at one or more nucleotides. 64. The sgRNA of claim 63 -O-methyl- -phosphorothioate 65. The sgRNA of claim 64, wherein the MS modified sgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 43-47.
66. A homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic antibody, operably linked to (b) a promoter and (c) a polyadenylation signal at the 3’ end of the coding sequence; (ii) a first homology region located to one side of the expression cassette within the donor template; and (iii) a second homology region located to the other side of the expression cassette within the donor template. 67. The donor template of claim 66, wherein the expression cassette further comprises a signal sequence encoding a signal peptide at the 5’ end of the coding sequence for the therapeutic. 68. The donor template of claim 66 or 67, wherein the coding sequence for the therapeutic antibody comprises a sequence encoding a light chain and a sequence encoding a heavy chain. 69. The donor template of claim 68, wherein the coding sequence further comprises a linker sequence between the sequence encoding the light chain and the sequence encoding the heavy chain. 70. The donor template of claim 66, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 2 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 3 or a fragment thereof. 71. The donor template of claim 66, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 41 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 42 or a fragment thereof. 72. The donor template of claim 66, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 29 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 30 or a fragment thereof.
73. The donor template of claim 66, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 33 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 34 or a fragment thereof. 74. The donor template of claim 66, wherein the first homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 37 or a fragment thereof, and wherein the second homology region comprises a sequence having 80% or greater identity to SEQ ID NO: 38 or a fragment thereof. 75. The donor template of claim 66, wherein the expression cassette encodes a therapeutic antibody that binds to a virus, a cancer cell, an immune checkpoint inhibitor, a cytokine, a bacterium, or a pathogen. 76. The donor template of claim 66, wherein the expression cassette encodes a therapeutic antibody against HIV infection. 77. The donor template of claim 66, wherein the therapeutic antibody encoded by the expression cassette comprises at least one light chain and at least one heavy chain. 78. The donor template of claim 77, wherein the at least one light chain and the at least one heavy chain are linked by a linker. 79. The donor template of claim 78, wherein the linker comprises a sequence having 80% or greater identity to SEQ ID NO: 5 or 6. 80. The donor template of claim 66, wherein the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11-18. 81. The donor template of claim 66, wherein the coding sequence comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 19-26. 82. An HSPC comprising the sgRNA of claim 56 and/or the homologous donor template of claim 66. 83. A B cell comprising the sgRNA of claim 56 and/or the homologous donor template of claim 66.
84. A genetically modified cell comprising an integrated transgene at a selected locus, wherein the integrated transgene comprises a coding sequence for a therapeutic antibody. 85. The genetically modified cell of claim 84, wherein the selected locus is a safe harbor locus. 86. The genetically modified cell of claim 85, wherein the safe harbor locus is a CCR5 locus, an AAVS1 locus, or a ROSA26 locus. 87. The genetically modified cell of claim 84, wherein the selected locus is an immunoglobulin-associated locus. 88. The genetically modified cell of claim 87, wherein the immunoglobulin- associated locus is an IgH locus, an an 89. The genetically modified cell of claim 84, wherein the therapeutic antibody comprises a sequence having 80% or greater identity to any one of SEQ ID NOs: 11- 18. 90. The genetically modified cell of claim 84 , wherein the cell was modified using the method of any one of claims 1 to 49. 91. A pharmaceutical composition comprising a plurality of HSPCs of claim 82, a plurality of B cells of claim 83, or a plurality of genetically modified cells of claim 84.
PCT/US2025/018226 2024-03-04 2025-03-03 Genetic engineering of cells for secretion of therapeutic antibodies Pending WO2025188676A1 (en)

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