Genome edited primary b cell and methods of making and using the same

Abstract

To provide the delivery of therapeutic genes to B cells using genome engineering approaches or the use of any targeted nuclease in primary human B cells.SOLUTION: The present invention provides genome-edited primary B cells, methods of making genome-edited primary B cells, a therapeutic cassette that can be introduced into primary B cells, and methods of using the genome-edited primary B cells and the therapeutic cassette.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] (Continuing application data) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 393,512, filed September 12, 2016, which is incorporated herein by reference.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office via EFS-Web as an ASCII text file entitled "110-05460201_ST25.txt," 90.3 kilobytes in size, created on September 12, 2017. By filing the Sequence Listing in electronic format, the Sequence Listing submitted electronically serves as both the paper copy required by 37 CFR 1.821(c) and the CRF required by 1.821(e). The information contained in the Sequence Listing is incorporated herein by reference. [Background technology]

[0003] B lymphocytes are a component of the adaptive immune system, a population of cells expressing clonally diverse cell surface immunoglobulin (Ig) receptors that recognize specific antigenic epitopes. The process of B cell maturation is largely conserved between humans and mice. Dysregulation of normal B cell development can lead to congenital immunodeficiencies, autoimmune diseases, and even leukemia or lymphoma. B cells can produce protective antibodies that persist for decades after initial antigen exposure. Following immunization, antigen-reactive Ig can be detected for years by the generation of long-lived plasma cells. These long-lived plasma cells can arise from long-lived antibody-producing cells that do not proliferate or from cells arising from germinal centers during antibody maturation. It is believed that such plasma cells can survive for years, even decades. Summary of the Invention

[0004] This disclosure describes genome-edited primary B cells, methods for making genome-edited primary B cells, therapeutic cassettes that can be introduced into primary B cells, and methods of using the genome-edited primary B cells and therapeutic cassettes.

[0005] In one aspect, the present disclosure describes genome-edited primary B cells. In some embodiments, the B cells include cells that express at least one of CD19, IgM, IgD, CD27, CD21, and CXCR5.

[0006] In some embodiments, the B cells comprise cells isolated from peripheral blood, umbilical cord cells, ascites, or solid tumors. In some embodiments, the B cells comprise non-clonal, proliferating, mammalian, and / or human cells.

[0007] In some embodiments of the genome-edited primary B cells, an endogenous gene is deleted, a gene contains a point mutation, and / or the cells contain a foreign gene. The gene may include a nucleic acid encoding at least a portion of a B cell receptor (BCR).

[0008] In some embodiments, the B cells exhibit reduced expression of the endogenous B cell receptor (BCR) compared to non-genome-edited primary B cells.

[0009] In some embodiments, the B cells comprise a modification that alters the expression or activity of CD 19. In some embodiments, the B cells comprise a modification in a non-coding region of the genome.

[0010] In some embodiments, the genome-edited primary B cells exhibit increased viability compared to non-genome-edited primary B cells.

[0011] In another aspect, the disclosure describes a method comprising administering to a subject a composition comprising genome-edited primary B cells. In some embodiments, the method comprises treating or preventing a disease in the subject, which can include, for example, an enzymopathy, cancer, a precancerous condition, or a pathogenic or viral infection.

[0012] In another aspect, the present disclosure describes a therapeutic cassette comprising a nucleic acid encoding a B cell receptor (BCR) and a nucleic acid encoding a gene to be overexpressed. The B cell receptor can include a transmembrane domain. The gene to be overexpressed can include a nucleic acid encoding an enzyme. In some embodiments, the enzyme includes an enzyme that is deficient in a subject having or diagnosed with an enzymopathy. In some embodiments, the nucleic acid encoding the BCR and the nucleic acid encoding the gene to be overexpressed are transcriptionally linked, translationally linked, or both.

[0013] In some embodiments, the therapeutic cassette comprises a promoter that drives transcription of a nucleic acid encoding a BCR and a nucleic acid encoding a gene to be overexpressed.

[0014] In another aspect, the present disclosure describes cells comprising a therapeutic cassette. In some embodiments, the cells comprise B cells and / or long-lived plasma cells. The cells may comprise modifications of nucleic acids encoding endogenous B cell receptors (BCRs).

[0015] In a further aspect, the disclosure describes a method comprising administering cells comprising a therapeutic cassette. In some embodiments, the method can also include administering to the subject an antigen, wherein the BCR of the therapeutic cassette is specific for the antigen.

[0016] In yet another aspect, the present disclosure describes a method comprising editing the genome of a primary B cell. The primary B cell can be a cell that expresses CD19; IgM or IgD or a combination thereof; CD27 + cells;CD21 + cells; and / or CXCR5 + In some embodiments, primary B cells may include cells isolated from peripheral blood, umbilical cord cells, ascites, or solid tumors; non-clonal cells; proliferating cells; mammalian cells; and / or human cells.

[0017] In some embodiments, the method includes introducing an exogenous protein or nucleic acid into primary B cells. The method may include electroporation of the cells. In some embodiments, the method includes introducing a targeted nuclease or a nucleic acid encoding the targeted nuclease (e.g., Cas9 or a nucleic acid encoding Cas9). In some embodiments, the method includes introducing a guide RNA (gRNA). The gRNA may include a chemically modified gRNA. The chemically modified gRNA may include 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), or 2'-O-methyl-3'-thiophosphonoacetate (MSP).

[0018] In some embodiments, the method includes introducing Natronobacterium gregoryi Argonaute (NgAgo) and guide DNA (gDNA).

[0019] In some embodiments, editing the genome includes, for example, editing a gene including a nucleic acid encoding CD19; editing a nucleic acid encoding a portion of a B cell receptor (BCR); and / or editing a non-coding region of the genome.

[0020] In some embodiments, the method further comprises selecting the B cells. In some embodiments, the selection is performed after editing the genome. In some embodiments, the B cells are selected for the edited genome.

[0021] In some embodiments, the method subjects primary B cells to at least one of the steps of activation, stimulation, and expansion.

[0022] The words "preferred" and "preferably" refer to embodiments of the invention that may offer certain benefits, under particular circumstances, although other embodiments may be preferred, under the same or different circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0023] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0024] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0025] Also herein, when numerical ranges are recited by endpoints, they include every number subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0026] For any method comprising individual steps disclosed herein, the steps may be performed in any order practicable, and, where appropriate, two or more steps may be performed simultaneously in any combination.

[0027] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

[0028] Unless otherwise expressly indicated, all numerical values ​​expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and claims are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0029] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, with the proviso that any numerical value inherently contains a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0030] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies exemplary embodiments. In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 shows delivery of mRNA encoding eGFP into stimulated primary human B cells. (A) Histogram showing the percentage of eGFP-positive B cells after gating on viable cells. (B) Flow plot quantifying the viability of B cells shown in panel A based on APC e-Fluor 780 Fixable Viability Dye. [Figure 2]This figure shows exemplary results of a surveyor nuclease assay from primary human B cells treated and stimulated with chemically modified gRNA and Cas9 mRNA or protein. DNA was extracted 3 days after transfection. Lanes without gene modification percentages are not shown for simplicity but represent 0% editing. Each lane is numbered 1-5 (left to right) with a key indicating the conditions used. The target gene is listed in parentheses below each image. [Figure 3] Figure 1 shows a schematic representation of several exemplary embodiments of targeted gene integration at the BCR heavy chain locus. (A) Schematic representation of the BCR heavy chain locus showing the enhancer, D, J, and constant exons. Proposed sites for transgene integration are indicated. (B) Schematic representation of two embodiments of cargo design for gene delivery at the BCR heavy chain locus. P2A: ribosomal skip sequence to transcriptionally link cDNA; pA: polyadenylation sequence; splice acc: strong splice acceptor element. [Figure 4]

[0023] Figure 1 shows exemplary results of lentiviral transduction of primary human B cells. Contour plots show the percentage of eGFP-expressing B cells after gating on viable cells based on APC e-Fluor 780 Fixable Viability Dye. Acutely stimulated (top row), chronically stimulated (middle row), and unstimulated (bottom row) B cell cultures were tested with BaEV-pseudotypes (Fusil et al., Molecular Therapy, 2015, 23(11):1734-47) (left column), VSVg-pseudotypes (middle column), and no lentivirus control (right column) as described in Example 2. In all conditions, BaEV-pseudotyped viruses transduced B cells more efficiently than cells transduced with standard VSVg pseudotypes. [Figure 5]Figure 1 shows exemplary cell surface expression of CD19 protein after CD19 gene knockout in primary human B cell cultures. Stimulated B cells were electroporated with Cas9 mRNA and chemically modified gRNA (TriLink) using the NEON transfection system (1400 volts, 10 milliseconds (ms), 3 pulses) to target the CD19 locus. Samples with Cas9 mRNA without gRNA and no electroporation were included to represent control levels of CD19. Five days after electroporation, CD19 expression was measured by flow cytometry. (A) Histogram showing CD19 expression in cells treated with Cas9 + rRNA (solid line) compared to cells treated with Cas9 alone (dashed line) and the no-electroporation control (plain gray background). (B) CD19 cell surface expression was reduced from 96–98% in cells treated with Cas9 alone and the no-electroporation control to 38% in B cells treated with Cas9 and CD19 gRNA. [Figure 6A-B]Figure 1 shows exemplary vector constructs for plasmids encoding therapeutic cassettes used to engineer primary human B cells to express antibodies. These plasmids express membrane-bound B cell receptors (BCRs) or secreted antibodies, depending on the maturation state of the B cells. (A) Schematic diagram of lentiviral vectors constructed to express anti-PE heavy and light chains and codon-optimized alpha-L-iduronidase (coIDUA) under the control of the MND promoter. The heavy and light chains and coIDUA were coexpressed by introducing the P2A peptide sequence. The sequences are shown in Table 3. (B) Schematic diagram of FAM1 and FAM2 lentiviral vectors (originally described in Fusil et al., Molecular Therapy, 2015, 23(11):1734-47) modified to express anti-PE B cell receptors (BCRs) / antibodies. The sequences are shown in Tables 6 and 8. (C) Schematic diagram of lentiviral vectors constructed to express B12 heavy and light chains and codon-optimized IDUA (coIDUA) under the control of the MND promoter. The P2A peptide sequence was introduced to allow coexpression of the heavy and light chains and coIDUA. The sequences are shown in Table 4. (D) Schematic diagram of the FAM1 and FAM2 lentiviral vectors (originally described in Fusil et al., Molecular Therapy, 2015, 23(11):1734-47) modified to express the B12 B cell receptor (BCR) / antibody. The sequences are shown in Tables 5 and 7. [Figure 6C-D]Figure 1 shows exemplary vector constructs for plasmids encoding therapeutic cassettes used to engineer primary human B cells to express antibodies. These plasmids express membrane-bound B cell receptors (BCRs) or secreted antibodies, depending on the maturation state of the B cells. (A) Schematic diagram of lentiviral vectors constructed to express anti-PE heavy and light chains and codon-optimized alpha-L-iduronidase (coIDUA) under the control of the MND promoter. The heavy and light chains and coIDUA were coexpressed by introducing the P2A peptide sequence. The sequences are shown in Table 3. (B) Schematic diagram of FAM1 and FAM2 lentiviral vectors (originally described in Fusil et al., Molecular Therapy, 2015, 23(11):1734-47) modified to express anti-PE B cell receptors (BCRs) / antibodies. The sequences are shown in Tables 6 and 8. (C) Schematic diagram of lentiviral vectors constructed to express B12 heavy and light chains and codon-optimized IDUA (coIDUA) under the control of the MND promoter. The P2A peptide sequence was introduced to allow coexpression of the heavy and light chains and coIDUA. The sequences are shown in Table 4. (D) Schematic diagram of the FAM1 and FAM2 lentiviral vectors (originally described in Fusil et al., Molecular Therapy, 2015, 23(11):1734-47) modified to express the B12 B cell receptor (BCR) / antibody. The sequences are shown in Tables 5 and 7. [Figure 7] Figure 1 shows the effect of exemplary electroporation settings on the efficacy of transfecting primary human B cells with DNA or RNA encoding eGFP using the NEON transfection system. (A) The conditions tested are listed: voltage (volts); pulse width (milliseconds (ms)); and pulse number. (B) GFP-encoding plasmid (left column) or GFP-encoding mRNA (right column) were used to test the effect of electroporation conditions on transfection efficacy (top row), cell viability (middle row), and total cell count (bottom row). [Figure 8]

[0023] Figure 1 shows exemplary results of indel formation at the BCL2 locus using the Alt-R CRISPR-Cas9 system as described in Example 2. Primary human B cells were electroporated with the Alt-R CRISPR-Cas9 system (Integrated DNA Technology, Coralville, Iowa) targeting the BCL-2 locus using the NEON transfection system (1400 volts, 10 ms, 3 pulses). Sequencing analysis using the TIDE program (available on the World Wide Web at tide.nki.nl) revealed a total editing efficiency of 11.3% (R2 = 0.89). The majority of edits observed included insertions of 1 nucleotide or deletions of 9 nucleotides. [Figure 9] This figure shows the effect of cell density on proliferation and expansion of mature naive-like B cells derived from CD19+ cells isolated from peripheral blood mononuclear cells after activation with CD40L cross-linking antibody (Miltenyi Biotech, San Diego, CA) and IL-4. Cells plated at a high density (1 × 106 cells / mL) in HSC Expansion Media (Miltenyi Biotech, San Diego, CA) showed minimal expansion by day 14 compared to the 30-fold increase observed with cells plated at a low density (2 × 105 cells / mL). [Figure 10-A] FIG. 1 shows exemplary (A) intracellular IDUA activity and (B) secreted IDUA activity in HEK 293T cells 3 days after electroporation as detailed in Example 2. [Figure 10-B] FIG. 1 shows exemplary (A) intracellular IDUA activity and (B) secreted IDUA activity in HEK 293T cells 3 days after electroporation as detailed in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0032] (Detailed explanation) B cells, with their long-term viability and inherent ability to produce large amounts of proteins (i.e., antibodies), appear to be an ideal platform for gene therapy, including the treatment of enzymopathies. B cells are also readily available in peripheral blood (accounting for 1–7% of all white blood cells), and cell expansion methods are readily available. Furthermore, evidence suggests that cells cross the blood-brain barrier more readily than proteins, making cell therapy preferable over enzyme replacement therapy for enzymopathies affecting the brain. However, neither the delivery of therapeutic genes into B cells using genome engineering methods nor the use of any targeted nucleases in primary human B cells has been reported.

[0033]

[0003] The present disclosure describes genome-edited primary B cells; methods for making genome-edited primary B cells; and methods of using genome-edited primary B cells, including, for example, administering the cells. The present disclosure further describes therapeutic cassettes that can be introduced into primary B cells, methods for making the therapeutic cassettes, methods for making B cells containing the therapeutic cassettes, and methods for using the therapeutic cassettes.

[0034] B cells In some embodiments, the B cells are CD19 + In some embodiments, the B cells may be primary B cells. As used herein, "primary B cells" are non-immortalized B cells. In some embodiments, "primary B cells" are freshly isolated B cells. In some embodiments, the B cells may be isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the B cells may be derived from iPSCs. In some embodiments, the B cells may be CD34 + It may be derived from a population of cells.

[0035] In some embodiments, a "primary B cell" is a B cell that has undergone up to 5 replications or divisions since isolation, up to 10 replications or divisions since isolation, up to 15 replications or divisions since isolation, up to 20 replications or divisions since isolation, up to 25 replications or divisions since isolation, up to 30 replications or divisions since isolation, up to 35 replications or divisions since isolation, or up to 40 replications or divisions since isolation.

[0036] In some embodiments, a "primary B cell" is a B cell that has undergone up to 5 replications or divisions since induction, up to 10 replications or divisions since induction, up to 15 replications or divisions since induction, up to 20 replications or divisions since induction, up to 25 replications or divisions since induction, up to 30 replications or divisions since induction, up to 35 replications or divisions since induction, or up to 40 replications or divisions since induction.

[0037] In some embodiments, the primary B cells are non-clonal cells. In some embodiments, the primary B cells are proliferative cells. In some embodiments, it is preferred to culture the B cells in the presence of CD40L.

[0038] In some embodiments, the B cells can be naive B cells. In some embodiments, "naive B cells" are those that express CD19 + , IgD + , IgM + , CD27 - , CD21 + and / or CXCR5 + In some embodiments, the B cells can be memory B cells. In some embodiments, "memory B cells" are CD19 + , IgD - , CD27 + , CD21 + and / or CXCR5 + In some embodiments, the B cells can be activated memory B cells. In some embodiments, "activated memory B cells" are CD19 + , IgD - , CD27+ , CD21 - and / or CXCR5 + In some embodiments, the B cells can be natural effector B cells. In some embodiments, "natural effector B cells" are CD19 + , IgD + , IgM + and / or CD27 + In some embodiments, the B cells can be plasmablasts. In some embodiments, "plasmablasts" are cells that express CD19 + , CXCR5 - , CD38 + , CD27 - and / or CD20 - is.

[0039] In some embodiments, the B cell can be a B cell that has undergone class switch recombination. In some embodiments, the B cell can be a B cell that has not undergone class switch recombination.

[0040] In some embodiments, the B cells are mammalian cells. In some embodiments, the B cells are preferably human cells. In some embodiments, the B cells are mouse cells.

[0041] Genome-edited primary B cells A primary B cell is "genomically edited" if it contains a modification in its genome compared to a non-genomically edited B cell. In some embodiments, the non-genomically edited B cell is a wild-type B cell. In some embodiments, the non-genomically edited B cell is a freshly isolated B cell.

[0042] In some embodiments, the genome-edited primary B cells comprise modifications in non-coding regions of the genome and / or coding regions of the genome (e.g., genes). In some embodiments, the non-coding regions of the genome may comprise sequences of small regulatory non-coding RNAs, including, for example, microRNAs (miRNAs). In some embodiments, the non-coding regions of the genome are preferably those involved in regulating B cell function, activation, and / or survival.

[0043] In some embodiments, a portion of genomic information and / or a gene may be deleted. In some embodiments, a portion of genomic information and / or a gene may be added. In some embodiments, the added genomic information and / or gene is foreign. In some embodiments, the "foreign" genomic information or "foreign" gene may be genomic information or a gene from a non-B cell. In some embodiments, the "foreign" genomic information or "foreign" gene may be an additional copy of genomic information or a gene already present in the B cell. In some embodiments, the "foreign" genomic information or "foreign" gene may be genomic information or a gene from a cell of a different species than the B cell being modified. In some embodiments, the "foreign" genomic information or "foreign" gene may be artificially engineered, including, for example, a nucleic acid encoding a chimeric antigen receptor. In some embodiments, the portion of genomic information and / or a gene may be altered, for example, by point mutation.

[0044] In some embodiments, the genome-edited primary B cells preferably comprise a modification that alters the expression or activity of the genome-edited primary B cells relative to non-genome-edited primary B cells. For example, in some embodiments, the genome-edited primary B cells may comprise a therapeutic cassette, as further described below.

[0045] In some embodiments, the genome-edited primary B cells preferably comprise a modification of a nucleic acid encoding an endogenous B cell receptor (BCR). In some embodiments, this modification results in a modification of the expression of the endogenous BCR. For example, the expression of the endogenous BCR may be suppressed compared to non-genome-edited primary B cells. In some embodiments, the expression of the endogenous BCR may be enhanced compared to non-genome-edited primary B cells. In some embodiments, the genome-edited primary B cells comprise a modification of a nucleic acid encoding CD19. In some embodiments, the genome-edited primary B cells comprise a modification of a nucleic acid encoding a light chain.

[0046] In some embodiments, the genome-edited primary B cells comprise a modification in a nucleic acid encoding a cytokine, which may include, for example, IL-10, IL-4, IL-7, IL-2, IL-15, IL-6, or IFN-γ, or a combination thereof.

[0047] In some embodiments, the genome-edited primary B cells comprise modifications of nucleic acids encoding members of the Bcl-2 family, including, for example, BAX and bcl-2, also known as bcl-2-like protein 4. In some embodiments, the modifications of nucleic acids encoding members of the Bcl-2 family enable increased viability of the genome-edited primary B cells, e.g., increased viability in culture.

[0048] In some embodiments, the genome-edited primary B cells comprise a modification of a nucleic acid encoding a B cell inhibitory receptor, including, for example, FcγRII, CD22, PD-1, CD5, CD66a, LAIR1, ILT2, or CD72, or a combination thereof. In some embodiments, the modification alters the expression or activity of the inhibitory receptor compared to non-genome-edited primary NK cells. For example, the expression of the inhibitory receptor may be decreased.

[0049] In some embodiments, the genome-edited primary B cells comprise a modification that affects the frequency or rate at which B cells undergo affinity maturation. In some embodiments, the genome-edited primary B cells comprise a modification of a nucleic acid encoding BCL6 and / or BLIMP1. BCL6 inhibits BLIMP1 expression, and BLIMP1 expression inhibits BCL6 expression. BCL6 expression can cause B cells to remain in germinal centers, where they continue affinity maturation. BLIMP1 differentiates B cells into plasma cells (both short- and long-lived). In some embodiments, modification of a nucleic acid encoding BCL6 and / or BLIMP1 can be used to affect the frequency or rate at which B cells undergo affinity maturation.

[0050] In some embodiments, the genome-edited primary B cells comprise modifications of nucleic acids encoding members of the endoplasmic reticulum (ER) stress response pathway, including, for example, IRE1, PERK, or ATF6, or combinations thereof, during their initial differentiation and under transient inhibition of caspase-dependent cell death. In some embodiments, the modifications of nucleic acids encoding members of the ER stress response pathway may affect the viability of the genome-edited primary B cells, e.g., may increase their viability in culture.

[0051] In some embodiments, the genome-edited primary B cells preferably comprise a modification that alters the viability of the genome-edited primary B cells relative to non-genome-edited primary B cells. In some embodiments, the gene-edited primary B cells exhibit an increased ability to expand relative to non-genome-edited primary B cells. Expansion can be, for example, in vivo or in vitro. In some embodiments, expansion can be in vitro following co-culture with cytokines, antibodies, antigens, cells expressing antigens, or combinations thereof.

[0052] B cell genome editing The present disclosure also describes methods for producing genome-edited B cells, including genome-edited primary B cells.

[0053] In some embodiments, the method involves a technique for introducing a protein or nucleic acid into primary B cells. Any suitable method for introducing a protein or nucleic acid may be used. In some embodiments, the method preferably involves electroporation, which introduces genetic material, including, for example, DNA, RNA, and / or mRNA, into primary B cells. As used herein, electroporation may include nucleofection. In some embodiments, the genetic material may be introduced via viral transduction, including, for example, adeno-associated virus (AAV), integrase-deficient lentivirus (IDLV), and the like. Adeno-associated virus may include any suitable serotype, including, for example, AAV2, AAV3, AAV4, AAV5, AAV6, and the like. Because plasmid DNA can be toxic to B cells, in some embodiments, mRNA- or protein-based genome editing methods are preferred. In some embodiments, techniques for introducing proteins or nucleic acids may include introducing proteins or nucleic acids by electroporation, microinjection, viral delivery, exosomes, liposomes, particle bombardment, jet injection, hydrodynamic injection, ultrasound, magnetic field-mediated gene transfer, electric pulse-mediated gene transfer, the use of nanoparticles, including lipid-based nanoparticles, incubation with endosomolytic agents, the use of cell-penetrating peptides, etc. In some embodiments, the method preferably involves electroporation of primary B cells using the NEON transfection system.

[0054] In some embodiments, the method includes editing a gene. Editing a gene can include introducing one or more copies of a gene, altering a gene, deleting a gene, upregulating the expression of a gene, downregulating the expression of a gene, mutating a gene, methylating a gene, demethylating a gene, acetylating a gene, and / or deacetylating a gene. Mutating a gene can include introducing an activating mutation, introducing an inactivating and / or inhibitory mutation, and / or introducing a point mutation.

[0055] In some embodiments, the method preferably comprises inducing a double-strand break in the genome of the primary B cell. The double-strand break may be introduced using a targeted nuclease, including, for example, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a CRISPR-associated nuclease, etc. In some embodiments, the double-strand break is preferably introduced using a CRISPR / Cas9 system. In some embodiments, the method preferably comprises introducing a CRISPR nuclease (e.g., including Cas9 and / or Cpf1) or DNA or RNA encoding a CRISPR nuclease (e.g., including DNA or RNA encoding Cas9 or Cpf1). In some embodiments, the method may comprise introducing a guide RNA (gRNA).

[0056] In some embodiments, the method includes introducing a DNA-guided DNase. In some embodiments, the method includes introducing Natronobacterium gregoryi Argonaute (NgAgo). In some embodiments, NgAgo can be used as a DNA-guided endonuclease. (Gao et al., Nature Biotechnology, 2016, doi:10.1038 / nbt.354.) The method can further include, for example, introducing guide DNA (gDNA).

[0057] The gRNA target or gDNA target can include any suitable target. In some embodiments, the target includes a portion of the B cell genome, including, for example, a gene or a portion of a gene. In some embodiments, the target gene or a portion of the gene enhances B cell function. For example, the gRNA target or gDNA target can include, for example, a heavy chain gene, a light chain gene, or a B cell receptor, including CD79; CD19; a B cell development regulator, including, for example, BLIMP1 or BCL6; adeno-associated virus integration site 1 (AAVS1); a B cell inhibitory receptor (e.g., FCyRII, CD22, PD1, CD5, CD66a, LAIR1, ILT2, CD72, etc.); and / or a member of the ER stress response pathway (e.g., IREI, PERK, ATF6, etc.).

[0058] In some embodiments, when the CRISPR / Cas9 system can be delivered using transfection, it may be preferable for the gRNA to comprise a chemically modified gRNA. In some embodiments, it is preferable for the chemical modification of the gRNA to reduce the cell's ability to degrade the RNA. In some embodiments, the chemically modified gRNA comprises one or more of the following modifications: 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), S-restrained ethyl (cEt), 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), and / or 2'-O-methyl-3'-thiophosphonoacetate (MSP). In some embodiments, the chemically modified gRNA may comprise the gRNA and / or chemical modifications described in Hendel et al., Nature Biotechnology, 2015, 33(9):985-989 or Rahdar et al., PNAS, 2015, 112(51):E7110-7.

[0059] In such embodiments, genome editing can occur via homologous recombination (HR) and / or non-homologous end joining (NHEJ) pathways, including, for example, by microhomology-mediated end joining (MMEJ).

[0060] In some embodiments, the method includes selecting B cells. In some embodiments, the selection is performed after editing the gene. In some embodiments, the B cells may be selected using one or more of the following methods: flow sorting (e.g., including for GFP expression); magnetic bead separation (e.g., including targeting a cell surface marker); transient drug resistance gene expression (e.g., including antibiotic resistance). In some embodiments, the selection may be for B cells with an edited genome.

[0061] In some embodiments, the method includes expanding the edited B cells. In some embodiments, the expansion may be performed after selecting the B cells. In some embodiments, the B cells may be expanded by co-incubation with an antigen recognized by the B cell receptor or cells expressing an antigen recognized by the B cell receptor. In some embodiments, the B cells may be expanded by co-incubation with a cytokine or ligand, including, for example, CD40L and / or IL-4.

[0062] Transfection method In some embodiments, the primary B cells upon which electroporation or transfection is performed are preferably stimulated cells, ie, cells that have been subjected to a step of activation, stimulation and / or proliferation.

[0063] In some embodiments, B cells may be stimulated for at least 12 hours, at least 18 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some embodiments, B cells may be stimulated for up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 12 days, up to 14 days, up to 3 weeks, up to 4 weeks, or up to 2 months. In some embodiments, B cells are preferably stimulated for 14 days.

[0064] In some embodiments, B cells may be stimulated with cytokines. Cytokines may include, for example, IL-4, IL-7, IL-21, and / or B cell activating factor (BAFF). In some embodiments, B cells may be stimulated by crosslinking cell surface receptors, including, for example, CD40 (e.g., ligated and / or crosslinked by CD40L or an anti-CD40 antibody).

[0065] In some embodiments, it is preferred to transfect B cells by electroporation, for example, the number of B cells per reaction (e.g., 100,000, 500,000, 1 million, 2 million, 3 million, 4 million, or 5 million cells), the number of pulses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), the voltage (e.g., 1000 volts, 1100 volts, 1200 volts, 1300 volts, 1500 volts, or 1600 volts), the amount of nucleic acid (e.g., , 0.5 μg, 1 μg, 2 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg or 50 μg) and the duration of the pulse(s) (e.g., 2 ms, 5 ms, 7 ms, 9 ms, 10 ms, 11 ms, 13 ms or 15 ms).

[0066] In some embodiments, B cells may be electroporated using the AMAXA nucleofector or NEON system. In some embodiments, 1 million B cells per reaction may be electroporated using the NEON platform, 25 μg of mRNA, and a protocol of 3 pulses at 1400 volts and 10 ms duration.

[0067] In some embodiments, it is preferable to test the transfection level 48 hours after transfection. In some embodiments, it is preferable to test the transfection level 72 hours after transfection. In some embodiments, it is preferable to use eGFP mRNA transfection and test the transfection level by performing flow cytometry analysis 48 hours and / or 72 hours after transfection to evaluate the eGFP expression level and cell viability.

[0068] In one embodiment, to test the efficiency and toxicity of mRNA delivery to primary human B cells, peripheral blood mononuclear cells were isolated from leukopaques using standard Ficoll-Paque separation. B cells were then isolated from PBMCs using the EasySep Human CD19 Positive Selection Kit (Stem Cell Technologies, Vancouver, Canada) and cultured in X-VIVO 20 medium (Lonza Group, Allendale, New Jersey) containing 10% human serum. B cells were electroporated with in vitro transcribed mRNA encoding eGFP (TriLink BioTechnologies, San Diego, California) using the AMAXA or NEON electroporation platform. Preliminary results using this method showed EGFP was detected 48 hours after electroporation as measured by flow cytometry analysis. + The result was disappointing, with B cells at less than 6%.

[0069] In contrast, when primary B cells were stimulated and expanded prior to electroporation, for example by culturing them with IL-4 and CD40L (Miltenyi Biotech, San Diego, CA) for 7 days, EGFP was detected 72 hours after electroporation. + An increase in the proportion of eGFP-positive B cells was observed, with a maximum of 97.6% and a maximum viability of 68.6%.

[0070] One of the major barriers to the application of CRISPR / Cas9 technology is the low rate of gene modification in some cell types, including cells that are difficult to transfect, various primary cells, and other cells that cannot be cloned (i.e., expanded from a single isolated cell). For example, initial attempts using unmodified gRNAs failed to produce CD34 genes in primary human T cells or CD34 cells. + It also failed to induce detectable double-strand breaks in cells.

[0071] To determine whether the CRISPR / Cas9 system can be used for targeted gene delivery into B cells, we investigated double-strand break (DSB) induction via homologous recombination (HR) or non-homologous end joining (NHEJ) pathways. Gene modification was induced using gRNAs synthesized as RNA oligonucleotides containing three tandem 2-O-methyl-3-phosphorothioate-modified bases at the 5' and 3' ends, and Cas9 mRNA or protein (Figure 2). These data demonstrate that targeted double-strand breaks can be induced in B cells.

[0072] Treatment cassette In some embodiments, the genome-edited primary B cells may comprise a therapeutic cassette. In some embodiments, the therapeutic cassette preferably comprises a nucleic acid encoding a BCR and a nucleic acid encoding a gene to be overexpressed. In some embodiments, the gene to be overexpressed preferably comprises a nucleic acid encoding an enzyme. The nucleic acid encoding the BCR and the nucleic acid encoding the gene to be overexpressed are preferably transcriptionally and / or translationally linked.

[0073] In some embodiments, it may be desirable to inactivate the endogenous BCR, as it may interfere with the function of the BCR transgene. In some embodiments, it may be preferable to insert a therapeutic cassette into the heavy chain locus of the endogenous BCR. In some embodiments, it may be preferable to edit the endogenous BCR using the transfection methods described herein. Because VDJ recombination removes some regions of the endogenous heavy chain locus, in some embodiments, genome editing, including the insertion of a therapeutic cassette, may be targeted near enhancers found in the constant region. In some embodiments, the therapeutic cassette may be targeted to the region shown in Figure 3A, which is a region that is retained in almost all heavy chain recombination events.

[0074] In some embodiments, the therapeutic cassette preferably comprises a nucleic acid encoding a BCR. In some embodiments, the BCR is specific for an antigen that can be administered to a subject by immunization. In some embodiments, the BCR preferably comprises a transmembrane domain and / or a membrane-bound antibody. As used herein, a BCR may comprise a membrane-anchored BCR or a soluble Ig, or both.

[0075] In some embodiments, the therapeutic cassette comprises a nucleic acid encoding a heavy chain. In some embodiments, transcription of the nucleic acid encoding the heavy chain may be driven by an endogenous promoter. In some embodiments, transcription of the nucleic acid encoding the heavy chain may be driven by an exogenous promoter. In some embodiments, the promoter may include, for example, an MND promoter, a CMV promoter, a CAG promoter, a PGK promoter, an EF1A promoter, a FEEK promoter, etc. In some embodiments, the nucleic acid encoding the heavy chain preferably encodes a single variable segment, a single diversity segment, a single binding segment, and a single C region. In some embodiments, the nucleic acid encoding the heavy chain preferably encodes a transmembrane region, including, for example, an M1 and / or M2 domain.

[0076] In some embodiments, the therapeutic cassette may include a nucleic acid encoding a light chain. In some embodiments, transcription of the nucleic acid encoding the light chain may be driven by an endogenous promoter. In some embodiments, transcription of the nucleic acid encoding the light chain may be driven by an exogenous promoter. In some embodiments, the promoter may include, for example, an MND promoter, a CMV promoter, a CAG promoter, a PGK promoter, an EF1A promoter, a FEEK promoter, etc. In some embodiments, the nucleic acid encoding the light chain preferably encodes a single variable segment, a single binding segment, and a single C region.

[0077] In some embodiments, expression of a nucleic acid encoding a light chain can be transcriptionally or translationally coupled to expression of a nucleic acid encoding a heavy chain, for example, by an internal ribosome entry site (IRES), a 2A peptide sequence, a "2A-like" sequence, a ribosome skipping and / or a CHYSEL (cis-acting hydrolase element) sequence. The 2A peptide sequence disrupts normal peptide bond formation by a ribosome skipping mechanism, allowing expression of two or more proteins without the introduction of an internal ribosome entry site (IRES) or an additional promoter. In some embodiments, the 2A peptide can be derived from porcine teschovirus-1 (P2A), foot-and-mouth disease virus (F2A), or Thosea asigna virus (T2A).

[0078] In some embodiments, the heavy and / or light chains of the therapeutic cassette are specific for an antigen that can be administered to a subject by immunization. For example, in some embodiments, the heavy and / or light chains can be specific for phycoerythrin (PE). As another example, in some embodiments, the heavy and / or light chains can be specific for B12, an anti-HIV envelope protein.

[0079] The therapeutic cassette comprises a nucleic acid encoding a gene to be overexpressed. In some embodiments, the transcription of the nucleic acid encoding the gene to be overexpressed is preferably driven by the same promoter as the promoter driving the transcription of at least one of the heavy chain or light chain of BCR. In some embodiments, the transcription of the nucleic acid encoding the gene to be overexpressed can be driven by a promoter different from the promoter driving the transcription of at least one of the heavy chain or light chain of BCR.

[0080] For example, if transcription of the nucleic acid encoding the gene to be overexpressed is driven by the same promoter as that driving transcription of at least one of the heavy and light chains of the BCR, overexpression of the gene can be controlled by immunizing the subject against an antigen recognized by the exogenous BCR. Furthermore, in some embodiments, whether the therapeutic cassette produces a membrane-anchored BCR or a soluble Ig-type BCR depends on the maturation state of the B cells targeted by the therapeutic cassette.

[0081] In some embodiments, a treatment cassette may include the components shown in Figure 3B. In some embodiments, a treatment cassette may include the components arranged as shown in Figure 3B. In some embodiments, a treatment cassette may include the components shown in at least one panel of Figure 6. In some embodiments, a treatment cassette may include the components arranged as shown in at least one panel of Figure 6.

[0082] In some embodiments, expression of the nucleic acid encoding the enzyme may be transcriptionally or translationally linked to BCR expression, for example, by an internal ribosome entry site (IRES), a 2A peptide sequence, a "2A-like" sequence, a ribosome skipping and / or a CHYSEL (cis-acting hydrolase element) sequence, etc. In some embodiments, a splice acceptor method or a constitutive promoter may be used to drive the nucleic acid encoding the BCR linked to the nucleic acid encoding the therapeutic enzyme.

[0083] In some embodiments, the overexpressed gene preferably comprises an enzyme and / or a therapeutic enzyme. The therapeutic enzyme may, for example, comprise an enzyme lacking in a subject with an enzymopathy. The enzymopathy may, for example, comprise Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, type II diabetes, adenosine deaminase deficiency, or Pompe disease. In some embodiments, the therapeutic enzyme comprises alpha-L-iduronidase (IDUA), an enzyme essential for the degradation of glycosaminoglycans (GAGs). Therapeutic enzymes may additionally or alternatively comprise enzymes whose expression improves the health of the subject, for example.

[0084] In some embodiments, the therapeutic cassette comprises nucleic acid encoding a marker gene, including, for example, a gene for GFP or a gene for drug resistance.

[0085] In some embodiments, the nucleic acid encoding the heavy chain, the nucleic acid encoding the light chain, and the nucleic acid encoding the gene to be overexpressed are contained in a therapeutic cassette and are transcriptionally linked, hi some embodiments, the therapeutic cassette further comprises one or more 2A peptides transcriptionally linked to the nucleic acid encoding the heavy chain, the nucleic acid encoding the light chain, and the therapeutic enzyme.

[0086] The therapeutic cassette can be encoded by a vector construct. In some embodiments, the vector construct comprises a plasmid. In some embodiments, the vector can comprise a lentiviral vector, including, for example, a BaEV-pseudotyped lentiviral vector, a VSVg-pseudotyped lentiviral vector, a FAM1 lentiviral vector, and / or a FAM2 lentiviral vector (see Fusil et al., Molecular Therapy, 2015, 23(11):1734-47). In some embodiments, the vector can comprise cis-acting DNA elements, including, for example, a gene encoding a posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE), a gene encoding mouse intracisternal type A particles, one or more copies of a gene encoding a constitutive transport element (CTE) from various simian retroviruses, etc.

[0087] Genome-edited plasma cells and methods for differentiating genome-edited primary B cells into genome-edited plasma cells The present disclosure further provides methods for differentiating genome-edited plasma cells and genome-edited primary B cells into long-term surviving plasma cells.

[0088] In some embodiments, the genome-edited primary B cells and genome-edited plasma cells preferably comprise a modification of a nucleic acid encoding an endogenous B cell receptor (BCR), in which expression of the endogenous BCR is suppressed relative to non-genome-edited primary B cells and expression of the exogenous BCR is enhanced relative to non-genome-edited primary B cells.

[0089] In some embodiments, the BCR of the genome-edited primary B cells is specific for an antigen that can be administered to a subject by immunization. For example, in some embodiments, the B cell receptor can be specific for phycoerythrin (PE). In another example, in some embodiments, the B cell receptor can be specific for B12, an anti-HIV envelope protein.

[0090] In some embodiments, a subject comprising genome-edited primary B cells is exposed to an antigen recognized by BCR. In some embodiments, if the BCR of genome-edited primary B cells is specific for a certain antigen, administering the antigen to the subject by immunization generates long-term living plasma cells. If the antigen-binding site of BCR binds to the antigen, the BCR is considered to be specific for that antigen.

[0091] By replacing the endogenous B cell receptor with a B cell receptor of known specificity, it becomes possible to regulate transcription of a nucleic acid under the same promoter as the B cell receptor component.

[0092] For example, genome-edited primary B cells containing a B cell receptor specific for phycoerythrin (PE) and a nucleic acid encoding alpha-L-iduronidase (IDUA) can be introduced into a subject. The subject may have IDUA deficiency. Immunizing the subject with PE can cause the genome-edited primary B cells to differentiate into long-lived plasma cells and transcribe the nucleic acid encoding IDUA, thereby increasing IDUA expression in the genome-edited B cells and throughout the patient's body, resulting in cross-correction of the disease. In other embodiments, the nucleic acid encoding a gene that alters and / or overexpresses the specificity of B cells can be modified.

[0093] Administration The present disclosure further provides methods for using the genome-edited primary B cells described herein. For example, the genome-edited primary B cells can be used to treat or prevent a disease in a subject. A method can include administering to a subject a composition comprising the genome-edited primary B cells described herein or the genome-edited primary B cells produced by the method described herein. The disease can include, for example, an enzymopathy, cancer, a precancerous condition, an infectious disease caused by a pathogen (including, for example, malaria), or an infectious disease caused by a virus.

[0094] The genome-edited primary B cells may be administered to a subject alone or in combination with one or more other therapies. For example, the genome-edited primary B cells may be administered to a subject in combination with a pharmaceutical composition comprising an active agent and a pharmaceutically acceptable carrier, and / or in combination with a cell therapy, including, for example, chimeric antigen receptor T cells (CAR-T). The B cells may be administered to a patient, preferably a mammal, more preferably a human, in an amount effective to achieve the desired effect. The B cells may be administered by various routes, including, for example, intravenously, intratumorally, intraarterially, percutaneously, by local delivery via a catheter or stent, by needle or other device for intratumoral injection, subcutaneously, etc. The B cells may be administered once or multiple times. A physician of ordinary skill can determine and prescribe the effective amount and dosage of the genome-edited primary B cells and, optionally, the necessary pharmaceutical composition.

[0095] Examples of cancer include bone cancer, brain tumor, breast cancer, cervical cancer, laryngeal cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, spinal cancer, stomach cancer, uterine cancer, hematopoietic system cancer and / or lymphatic system cancer. Examples of hematopoietic system cancer and / or lymphatic system cancer include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), chronic myelogenous leukemia (CML), Hodgkin's disease and / or multiple myeloma. The cancer may be metastatic cancer.

[0096] In a further aspect, the genome-edited primary B cells may be administered to inhibit the growth of a tumor in a subject. In some embodiments, the tumor may comprise a solid tumor.

[0097] Examples of viruses include herpesviruses, including, for example, CMV, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), or Kaposi's sarcoma-associated herpesvirus (KSHV); Flaviviridae viruses, including, for example, dengue virus or Zika virus; or lentiviruses, including, for example, human immunodeficiency virus (HIV).

[0098] The enzymopathies, in some embodiments, may include those currently treated by enzyme replacement therapy, including, for example, Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, type II diabetes, adenosine deaminase deficiency, or Pompe disease. The enzymopathies, in some embodiments, may include those currently treated by gene therapy.

[0099] The genome-edited primary B cells may be administered or prepared before, during, and / or after other treatments. Such combination therapy may include administering the genome-edited primary B cells before, during, and / or after other anticancer agents, other antiviral agents, or combinations of other anticancer and other antiviral agents. Such agents include, for example, cytokines; chemokines; therapeutic antibodies, including high-affinity anti-CMV IgG antibodies; NK cell receptor ligands, including BiKE or TRiKE; adjuvants; antioxidants; chemotherapeutic agents; and / or radiation. The administration or preparation of the genome-edited primary B cells may be separated from the administration of other anticancer and / or antiviral agents by hours, days, or even weeks. Additionally or alternatively, the administration or preparation may be combined with other biologically active agents or modalities, including, but not limited to, anti-neoplastic agents and non-drug therapies, such as, but not limited to, surgery.

[0100] In some embodiments, genome-edited primary B cells containing a B cell receptor of known specificity may be administered to a subject prior to immunizing the subject with an antigen recognized by the B cell receptor. In such embodiments, immunization with the antigen may allow transcriptional regulation of a nucleic acid under the same promoter as the B cell receptor component.

[0101] For example, genome-edited primary B cells containing a B cell receptor specific for a certain antigen and a nucleic acid encoding a certain enzyme can be introduced into a subject. The subject can have an enzyme deficiency. Immunization of the subject with the antigen is expected to cause the genome-edited primary B cells to differentiate into long-lived plasma cells and transcribe the nucleic acid encoding the enzyme, thereby increasing the expression of the enzyme in the genome-edited B cells.

[0102] Exemplary Embodiments of Genome-Edited Primary B Cells 1. Genome-edited primary B cells.

[0103] 2. The genome-edited primary B cells of embodiment 1, comprising cells that express CD19.

[0104] 3. The genome-edited primary B cells of any of embodiments 1 or 2, comprising cells that express IgM or IgD or a combination thereof.

[0105] 4.CD27 + The genome-edited primary B cell of any one of embodiments 1 to 3, comprising the cell.

[0106] 5. B cells express CD21 + 5. The genome-edited primary B cell of any one of embodiments 1 to 4, comprising the cell.

[0107] 6.CXCR5 + 6. The genome-edited primary B cell of any one of embodiments 1 to 5, comprising the cell.

[0108] 7. The genome-edited primary B cells of any one of embodiments 1 to 6, comprising cells isolated from peripheral blood, umbilical cord cells, ascites, or solid tumors.

[0109] 8. The genome-edited primary B cells of any one of embodiments 1 to 7, comprising non-clonal cells.

[0110] 9. The genome-edited primary B cells of any one of embodiments 1 to 8, comprising proliferative cells.

[0111] 10. The genome-edited primary B cell of any one of embodiments 1 to 9, which is a mammalian cell.

[0112] 11. The genome-edited primary B cell of any one of embodiments 1 to 10, which is a human cell.

[0113] 12. The genome-edited primary B cell of any one of embodiments 1 to 11, wherein an endogenous gene of the genome-edited primary B cell is deleted.

[0114] 13. The genome-edited primary B cell of any one of embodiments 1 to 12, wherein an endogenous gene of the genome-edited primary B cell comprises a point mutation.

[0115] 14. The genome-edited primary B cell of any one of embodiments 1 to 13, comprising an exogenous gene.

[0116] 15. The genome-edited primary B cell of any one of embodiments 12-14, wherein the gene comprises a nucleic acid encoding at least a portion of a B cell receptor (BCR).

[0117] 16. The genome-edited primary B cells of any one of embodiments 1 to 15, which exhibit reduced expression of endogenous B cell receptor (BCR) compared to non-genome-edited primary B cells.

[0118] 17. The genome-edited primary B cell of any one of embodiments 1-16, comprising a modification that alters the expression or activity of CD19.

[0119] 18. The genome-edited primary B cell of any one of embodiments 1-17, comprising a modification of a non-coding region of the genome.

[0120] 19. The genome-edited primary B cells of any one of embodiments 1 to 18, which exhibit increased viability compared to non-genome-edited primary B cells.

[0121] 20. The genome-edited primary B cell of any one of embodiments 1 to 19, comprising a therapeutic cassette comprising a nucleic acid encoding a B cell receptor (BCR) and a nucleic acid encoding a gene to be overexpressed.

[0122] 21. A method for treating or preventing a disease in a subject, comprising administering to the subject a composition comprising the genome-edited primary B cells of any one of embodiments 1 to 20.

[0123] 22. The method of embodiment 21, wherein the disease comprises an enzymopathy, cancer, a precancerous condition, an infection by a pathogen, or a viral infection.

[0124] Exemplary Treatment Cassette Embodiments 1. A therapeutic cassette comprising a nucleic acid encoding a B cell receptor (BCR) and a nucleic acid encoding a gene to be overexpressed.

[0125] 2. The therapeutic cassette of embodiment 1, wherein the BCR comprises a transmembrane domain.

[0126] 3. The therapeutic cassette of embodiment 1 or embodiment 2, wherein the gene to be overexpressed comprises a nucleic acid encoding an enzyme.

[0127] 4. The therapeutic cassette of embodiment 3, wherein the enzyme comprises an enzyme that is deficient in a subject having an enzymopathy.

[0128] 5. The therapeutic cassette of embodiment 4, wherein the enzyme comprises alpha-L-iduronidase (IDUA).

[0129] 6. The therapeutic cassette of any one of embodiments 1 to 5, wherein the nucleic acid encoding the BCR and the nucleic acid encoding the gene to be overexpressed are transcriptionally linked, translationally linked, or both.

[0130] 7. The therapeutic cassette of any one of embodiments 1 to 6, comprising a promoter driving transcription of a nucleic acid encoding a BCR and a nucleic acid encoding a gene to be overexpressed.

[0131] 8. The therapeutic cassette of any one of embodiments 1-7, wherein the BCR comprises a BCR specific for phycoerythrin (PE).

[0132] 9. The therapeutic cassette of any one of embodiments 1-7, wherein the BCR comprises a BCR specific for B12.

[0133] 10. A vector comprising the therapeutic cassette of any one of embodiments 1 to 9.

[0134] 11. The vector of embodiment 10, comprising a lentiviral vector.

[0135] 12. The vector of either embodiment 10 or embodiment 11, comprising at least one of a BaEV-pseudotyped lentiviral vector, a VSVg-pseudotyped lentiviral vector, a FAM1 lentiviral vector, and a FAM2 lentiviral vector.

[0136] 13. The vector of any one of embodiments 10-12, comprising a cis-acting DNA element.

[0137] 14. A cell comprising the therapeutic cassette of any one of embodiments 1 to 9.

[0138] 15. A cell comprising the vector of any one of embodiments 10 to 13.

[0139] 16. The cell of any of embodiments 14 or 15, comprising a modification of a nucleic acid encoding an endogenous B cell receptor (BCR).

[0140] 17. The cell of embodiment 16, wherein the modification comprises a modification that reduces expression of endogenous BCR.

[0141] 18. The cell of any one of embodiments 15-17, comprising a B cell.

[0142] 19. The cells of any one of embodiments 15-18, comprising long-term viable plasma cells.

[0143] 20. A method comprising administering to a subject the cells of any one of embodiments 14-19.

[0144] 21. The method of embodiment 20, further comprising administering to the subject an antigen, wherein the BCR of the therapeutic cassette is specific for the antigen.

[0145] Exemplary embodiments of methods for editing the genome of primary B cells 1. A method comprising editing the genome of a primary B cell.

[0146] 2. The method of embodiment 1, wherein the primary B cells comprise cells that express CD19.

[0147] 3. The method of any of embodiments 1 or 2, wherein the primary B cells comprise cells that express IgM or IgD or a combination thereof.

[0148] 4. Primary B cells express CD27 + 4. The method of any of embodiments 1 or 3, comprising cells.

[0149] 5. Primary B cells express CD21 + 5. The method of any one of embodiments 1 to 4, comprising cells.

[0150] 6. Primary B cells express CXCR5 + 6. The method of any one of embodiments 1 to 5, comprising cells.

[0151] 7. The method of any one of embodiments 1-6, wherein the primary B cells comprise cells isolated from peripheral blood, umbilical cord cells, ascites, or solid tumors.

[0152] 8. The method of any one of embodiments 1-7, wherein the primary B cells comprise non-clonal cells.

[0153] 9. The method of any one of embodiments 1-8, wherein the primary B cells comprise proliferative cells.

[0154] 10. The method of any one of embodiments 1-9, wherein the primary B cells are mammalian cells.

[0155] 11. The method of any one of embodiments 1-10, wherein the primary B cells are human cells.

[0156] 12. The method of any one of embodiments 1-11, comprising introducing an exogenous protein or nucleic acid into the primary B cells.

[0157] 13. The method of any one of embodiments 1-12, comprising electroporation of the cells.

[0158] 14. The method of any one of embodiments 1 to 13, comprising introducing a targeted nuclease or a nucleic acid encoding a targeted nuclease.

[0159] 15. The method of any one of embodiments 1 to 14, comprising introducing a guide RNA (gRNA).

[0160] 16. The method of embodiment 15, wherein the gRNA comprises a chemically modified gRNA.

[0161] 17. The method of embodiment 16, wherein the chemically modified gRNA comprises 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), or 2'-O-methyl-3'-thiophosphonoacetate (MSP).

[0162] 18. The method of any one of embodiments 1-17, comprising introducing Natronobacterium gregoryi Argonaute (NgAgo) and guide DNA (gDNA).

[0163] 19. The method of any one of embodiments 1-18, wherein editing the genome comprises editing the gene for CD19.

[0164] 20. The method of any one of embodiments 1-19, wherein editing the genome comprises editing a nucleic acid encoding a portion of a B cell receptor (BCR).

[0165] 21. The method of any one of embodiments 1-20, wherein editing the genome comprises editing a non-coding region of the genome.

[0166] 22. The method of any one of embodiments 1-21, further comprising selecting B cells.

[0167] 23. The method of embodiment 22, wherein selection is performed after editing the genome.

[0168] 24. The method of any of embodiments 22 or 23, wherein B cells are selected for an edited genome.

[0169] 25. The method of any one of embodiments 1-24, further comprising subjecting the primary B cells to at least one of the steps of activation, stimulation, and proliferation.

[0170] 26. The method of embodiment 25, wherein subjecting the primary B cells to at least one of the steps of activation, stimulation, and proliferation comprises exposing the B cells to a cytokine.

[0171] 27. The method of any of embodiments 25 or 26, wherein subjecting the primary B cells to at least one of the steps of activation, stimulation, and proliferation comprises exposing the B cells to CD40L.

[0172] 28. The method of any one of embodiments 25-27, wherein the primary B cells are subjected to at least one of the steps of activation, stimulation and proliferation prior to introducing the exogenous protein or nucleic acid into the primary B cells.

[0173] 29. The method of any one of embodiments 1-28, further comprising introducing into the primary B cells a therapeutic cassette comprising a nucleic acid encoding a B cell receptor (BCR) and a nucleic acid encoding a gene to be overexpressed.

[0174] The present invention is illustrated by the following examples, with the understanding that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0175] (Example) Example 1 Culture medium Culture medium: -X-VIVO 20 (Lonza Group, Allendale, New Jersey) -10% human serum or -HSC Expansion Media XF (Miltenyi Biotec, San Diego, CA) -5% human serum Freezing medium: - 45 mL of heat-inactivated fetal bovine serum (FBS) -DMSO 5mL

[0176] method Guide RNA design and construction Using the CRISPR Design Program (Zhang Lab, MIT 2015), guide RNAs (gRNAs) were designed for the desired region of the gene, as shown in Table 1. Multiple gRNAs were selected based on the highest ranking determined by off-target locations. The gRNAs were arranged in the form of an oligonucleotide pair: 5'-CACCG-gRNA sequence-3' and 5'-AAAC-reverse complementary gRNA sequence-C-3'. The gRNAs were cloned together using a modified target sequence cloning protocol (Zhang Lab, MIT). The oligonucleotide pairs were phosphorylated and annealed together in a thermocycler using T4 PNK (NEB) and 10x T4 Ligation Buffer (NEB) at 37°C for 30 minutes, 95°C for 5 minutes, and then gradually decreased to 25°C at 5°C / minute. The ligation reaction used pENTR1 vector digested with FastDigest BbsI (Fermentas), FastAP (Fermentas), and 10x FastDigest Buffer. The digested pENTR1 vector was ligated with the phosphorylated and annealed oligo duplex (dilution 1:200) from the previous step using T4 DNA Ligase and Buffer (NEB). The ligation was incubated at room temperature for at least 1 hour, then transformed and miniprepped (GeneJET Plasmid Miniprep Kit, Life Technologies). Plasmids were sequenced to confirm proper insertion.

[0177] [Table 1]

[0178] gRNA validation 293T cells in a 24-well plate at 1 x 10 5Cells were seeded at a density of 100 cells / well. 150 μL of Opti-MEM medium was mixed with 1.5 μg of gRNA plasmid, 1.5 μg of Cas9 plasmid, and 100 ng of GFP. 5 μL of Lipofectamine 2000 Transfection Reagent (Invitrogen, Life Technologies) was added to another 150 μL of Opti-MEM medium. These solutions were mixed and incubated at room temperature for 10–15 minutes. The DNA-lipid complex was added dropwise to one well of a 24-well plate. The cells were incubated at 37°C for 3 days, and then genomic DNA was collected using the GeneJET Genomic DNA Purification Kit (Thermo Scientific). gRNA activity was quantified by Surveyor Digest, gel electrophoresis, and densitometry (Guschin et al., Methods Mol Biol. 2010, 649:247–56).

[0179] Isolation of peripheral blood mononuclear cells (PBMCs) from leukopacks Human PBMCs (Stem Cell Technologies, Vancouver, Canada) were diluted 3:1 with chilled 1x PBS. The diluted blood was added dropwise (very slowly) onto 15 mL of Lymphoprep (Stem Cell Technologies, Vancouver, Canada) in a 50 mL conical tube. The cells were centrifuged at 400 x g for 25 minutes without the brake. The buffy coat was removed and placed in a new conical tube. The cells were washed with chilled 1x PBS and centrifuged at 400 x g for 10 minutes with the brake. The supernatant was removed, and the cells were resuspended in freezing medium, counted, and frozen.

[0180] CD19 + B cell isolation Thaw and count PBMCs to ensure a cell density of 1 x 10 8The concentration was adjusted to 100 cells / mL, and the cells were transferred to a 14 mL polystyrene round-bottom tube. B cells were selected using the EasySep Human CD19 Positive Selection Kit (Stem Cell Technologies, Vancouver, Canada) according to the manufacturer's protocol. Collected cells were centrifuged at 400 × g for 5 minutes and resuspended in growth medium.

[0181] CD19 + B cell activation and stimulation Isolated CD19 for stimulating B cells + Count B cells and place 1 x 10 in a 24-well plate. 6 Cells were plated at a density of 1000 cells / mL. Cells were plated with CD40L and IL-4 according to the manufacturer's protocol (Miltenyi Biotec, San Diego, CA). Cells were incubated at 37°C for 1 week and then counted using a hemocytometer.

[0182] CD19 + NEON transfection of B cells Unstimulated or stimulated B cells were electroporated using the NEON Transfection System (100 μL Kit, ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions, with any modifications noted below. Cells were counted and diluted to 1 × 10 in 100 μL of Resuspension Buffer T. 6 The cells were resuspended at a density of 1000 kJ / well. 5 μg of GFP plasmid or mRNA or 15 μg of Cas9 and 10 μg of plasmid or mRNA gRNA (dissolved in molecular water) were added to the cell mixture. The cells were electroporated at 1400 V, 10 ms, for three pulses. After transfection, the cells were seeded in 2 mL of culture medium in a 6-well plate.

[0183] Unstimulated or stimulated B cells were nucleoporated using AMAXA NUCLEOFECTOR and Human B Cell NUCLEOFECTOR Kits (Lonza Cologne, Cologne, Germany) according to the manufacturer's instructions.

[0184] Flow cytometry 24–72 hours after transfection, electroporated B cells were analyzed for GFP expression by flow cytometry. Cells were prepared by washing with chilled 1x PBS containing 0.5% FBS and stained with Viability Dye eFlour 780 (eBiosciences, San Diego, CA). Cells were analyzed using an LSR II (BD Biosciences, San Jose, CA) and FlowJo v.9.

[0185] CD19 + B cell homologous recombination Stimulated CD19 + B cells were electroporated using the NEON transfection system (100 μL Kit, ThermoFisher Scientific, Waltham, MA). Cells were counted and 1.0–3.0 × 10 cells were transfected in 100 μL of Resuspension Buffer T. 6Cells were resuspended at a density of 1000 x g. HR was examined using 15 μg of mRNA Cas9 (TriLink BioTechnologies, San Diego, CA), 10 μg of mRNA gRNA (TriLink BioTechnologies), and 10 μg of a homologous recombination (HR) targeting vector. 10 μg of the HR targeting vector alone or 15 μg of Cas9 and 10 μg of mRNA gRNA were used as controls. After electroporation, cells were seeded in 2 mL of culture medium in 6-well plates. Cells were counted every 3 days using a Countess II Automated Cell Counter (ThermoFisher Scientific, Waltham, MA) to monitor proliferation under the various conditions. To monitor HR, cells were analyzed by flow cytometry and tested by PCR. Flow cytometry analysis was performed weekly for 3 weeks. B cells were stained with Fixable Viability Dye eFluor 780 (eBiosciences, San Diego). Cells were analyzed using LSR II (BD Biosciences, San Jose, CA) and FlowJo v.9. To test for HR by PCR, gDNA was isolated from B cells and amplified by PCR using accuprime taq DNA polymerase high fidelity (ThermoFisher Scientific, Waltham, MA). Primers shown in Table 2 were designed against both ends of the CCR5 gene and the HR targeting vector to search for appropriate homologous recombination.

[0186] [Table 2]

[0187] The results are shown in Figures 1 and 2.

[0188] Example 2 method gRNA validation 293T cells in a 24-well plate at 1 x 10 5Cells were seeded at a density of 100 cells / well. 150 microliters (μL) of Opti-MEM medium was mixed with 1.5 micrograms (μg) of gRNA plasmid, 1.5 μg of Cas9 plasmid, and 100 nanograms (ng) of GFP. 5 μL of Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA; Life Technologies, Carlsbad, CA) was added to another 150 μL of Opti-MEM medium. These solutions were mixed and incubated at room temperature for 10–15 minutes. The DNA-lipid complex was added dropwise to one well of a 24-well plate. The cells were incubated at 37°C for 3 days, and then genomic DNA was collected using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA).

[0189] In Figures 5 and 8, gRNA activity was quantified using the Tracking of Indels by Decomposition (TIDE) algorithm (available on the World Wide Web at tide.nki.nl). Briefly, edited regions were amplified by PCR using region-specific primers and sent to ACGT (Wheeling, IL) for Sanger sequencing. Chromatogram files returned by ACGT were uploaded to the TIDE website to analyze editing efficiency.

[0190] Lentiviral vector production and titration BaEV-pseudotyped lentiviral vectors and VSVg-pseudotyped lentiviral vectors were generated by transient transfection of 293T cells using Lipofectamine 2000 Transfection Reagent (Invitrogen, Waltham, MA) according to the manufacturer's instructions. BaEV-pseudotyped virus and VSVg-pseudotyped virus were constructed by mixing 15 μg of BaEV glycoprotein (Fusil et al., Molecular Therapy, 2015, 23(11):1734-47) or 15 μg of VSV glycoprotein with 20 μg of gagpol packaging and cargo plasmids, respectively. 18 hours after transfection, the medium was replaced with Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1x penicillin-streptomycin. After 24 hours, virus titers were obtained and filtered to remove cellular debris. Lentivirus titers were determined using a qPCR Lentivirus Titration Kit (Applied Biological Materials, Vancouver, Canada) according to the manufacturer's instructions, and the results are shown in Figure 4.

[0191] Two additional lentiviral vectors were constructed to express the heavy and light chains of B12 or anti-PE and codon-optimized IDUA (coIDUA) under the control of the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer). Coexpression of the heavy and light chains and coIDUA was achieved by introducing the P2A peptide sequence. These vectors use alternative splicing of mRNA transcripts to induce the expression of functional BCRs in mature naive B cells or functional soluble antibodies in plasma cells, thereby determining the maturation status of the B cells. A schematic diagram of the vectors is shown in Figure 6. The vector sequences are listed in Tables 3–8.

[0192] Acute and chronic activation of B cells Both acutely and chronically activated B cells were activated with the B Cell Expansion Kit (Miltenyi Biotec, San Diego, CA), which activates B cells by crosslinking CD40. Acutely activated cells were stimulated for 12 hours before addition of the lentiviral construct, and chronically activated cells were activated for 14 days before addition of the lentiviral construct.

[0193] Targeted knockout of CD19 expression The CRISPR / Cas9 system was used to knock out CD19 expression in primary human B cells. Chronically activated B cells were transfected with 1.5 μg of chemically modified mRNA encoding Cas9 protein (TriLink BioTechnologies, San Diego, CA) and 1 μg of chemically modified CD19 gRNA4 oligo1 (TriLink BioTechnologies) using the NEON Transfection System (1400 volts, 10 ms, 3 pulses). The combination of Cas9 protein and CD19 gRNA creates a double-strand break, resulting in indel formation and frameshift mutations that abrogate gene expression and protein levels.

[0194] Assessment of CD19 expression Five days after electroporation, engineered primary human B cells were stained with APC e-Fluor 780 Fixable Viability Dye and BV421-conjugated anti-CD19 antibody (BioLegend, San Diego, CA). Cells were run on an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo Version 9 (Tree Star, Ashland, OR). Results are shown in Figure 5.

[0195] Examination of electroporation conditions for B cells B cells activated for 14 days were electroporated with plasmid DNA or mRNA encoding eGFP by NEON transfection under various voltage, width, and pulse settings (Figure 7A). Two days after electroporation, the percentage of transfection was determined by eGFP expression and measured using an LSRII flow cytometer. Cell counts and cell viability were determined by trypan blue exclusion. The results are shown in Figure 7B.

[0196] CD19 + B cell gene knockout Stimulated CD19 + B cells were electroporated using the NEON Transfection Kit and System (Invitrogen, Carlsbad, CA). Gene editing (gRNA6 Oligo1) at the BCL2 locus was performed using the Alt-R CRISPR-Cas9 reagent from Integrated DNA Technologies (Coralville, IA). Briefly, 1.1 uL of 200 uM Alt-R CRISPR-Cas9 crRNA, 1.1 uL of Alt-R tracrRNA, and 2.8 uL of nuclease-free duplex buffer were incubated at 95°C for 5 minutes and then cooled to room temperature (RT) to form the crRNA:tracrRNA duplex. 0.5 uL of 22 picomoles (pmol) of crRNA:tracrRNA duplex and 0.5 uL of 18 pmol of Alt-R Cas9 enzyme were incubated at RT for 20 minutes to form the Alt-R CRISPR-Cas9 system. The Alt-R CRISPR-Cas9 system was mixed with electroporation enhancer (Invitrogen, Carlsbad, CA) and then added to 360,000 chronically activated B cells, with a final volume of 12 μL in T buffer. Using a 10 μL pipette tip, cells were electroporated with three pulses of 1400 volts for 10 ms. After culturing the electroporated cells for 5 days, gene editing was measured using the TIDE assay as described above. The results are shown in Figure 8.

[0197] B cell expansion Sorted CD19 cells were cultured using a B cell expansion kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer's protocol. + B cells were expanded for 14 days. A low density starting B cell concentration (1 × 10 5 cells / mL) and high density starting B concentration (1 x 10 6 Both 100% ethanol (100% ethanol / mL) and 100% ethanol (100% ethanol / mL) were tested. Cell counts were determined at the indicated time points using trypan blue exclusion. The results are shown in Figure 9.

[0198] IDUA activity Using a NEON System (1400 volts, 10 ms, 3 pulses), 293T cells were electroporated with GFP mRNA alone (control) or GFP mRNA and the pLL MND B12-IDUA expression plasmid, which contains the expression cassette shown in Figure 6C.

[0199] To measure intracellular IDUA activity, cells were harvested 3 days after electroporation. IDUA activity (nmol / (h·mg protein)) was measured using the IDUA assay described in Ou et al., 2014, Mol Genet Metab., 2014;111(2):113-5. HEK 293T cells transfected with the pLL MND B12-IDUA expression plasmid exhibited significantly higher intracellular IDUA activity (40-fold) than controls. The results are shown in Figure 10A.

[0200] To measure IDUA activity in the cell culture medium, the medium was collected 3 days after electroporation. IDUA activity (nmol / (h·mL)) was measured using the IDUA assay described in Ou et al., 2014, Mol Genet Metab., 2014;111(2):113-5. The medium containing HEK 293T cells transfected with the pLL MND B12-IDUA expression plasmid exhibited significantly higher IDUA activity (14-fold) than the control. The results are shown in Figure 10B.

[0201] The entire disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions from, e.g., GenBank and RefSeq, and amino acid sequence submissions from, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions from GenBank and RefSeq), are incorporated by reference. In the event of any inconsistency between the disclosure of this application and the disclosure(s) of any document incorporated herein by reference, the disclosure of this application shall govern. The foregoing detailed description and examples have been given solely for clarity of understanding. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, since variations obvious to those skilled in the art will be within the scope of the invention, as defined by the claims.

[0202] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0203] [Table 4-1] [Table 4-2] [Table 4-3] Table 4-4 Table 4-5 Table 4-6

[0204] Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7

[0205] Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7

[0206] Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7

[0207] Table 8-1 Table 8-2 Table 8-3 Table 8-4 Table 8-5 Table 8-6 Table 8-7

Claims

1. Genome-edited primary B cells.

2. CD19, IgM, IgD, CD27 ＋ , CD21 ＋ and CXCR5 ＋ The genome-edited primary B cells of claim 1, comprising cells that express at least one of:

3. The genome-edited primary B cells of claim 1 or 2, comprising cells isolated from peripheral blood, umbilical cord cells, ascites, or solid tumors.

4. The genome-edited primary B cells of any one of claims 1 to 3, comprising non-clonal cells.

5. The genome-edited primary B cells of any one of claims 1 to 4, comprising proliferative cells.

6. The genome-edited primary B cell of any one of claims 1 to 5, which is a mammalian cell.

7. The genome-edited primary B cell of any one of claims 1 to 6, wherein an endogenous gene is deleted.

8. The genome-edited primary B cell of any one of claims 1 to 7, wherein the endogenous gene comprises a point mutation.

9. The genome-edited primary B cell of any one of claims 1 to 8, comprising an exogenous gene.

10. The genome-edited primary B cell of any one of claims 7 to 9, wherein at least one of the endogenous gene and the exogenous gene comprises a nucleic acid encoding at least a portion of a B cell receptor (BCR).

11. The genome-edited primary B cells of any one of claims 1 to 10, which exhibit reduced expression of endogenous B cell receptor (BCR) compared to non-genome-edited primary B cells.

12. The genome-edited primary B cell of any one of claims 1 to 11, comprising a modification that alters the expression or activity of CD19.

13. The genome-edited primary B cells of any one of claims 1 to 12, comprising a therapeutic cassette comprising a nucleic acid encoding a B cell receptor (BCR) and a nucleic acid encoding a gene to be overexpressed.

14. A method comprising administering to a subject a composition comprising the genome-edited primary B cells of any one of claims 1 to 13.

15. 15. The method of claim 14, comprising treating or preventing a disease in a subject, wherein the disease comprises an enzymopathy, cancer, a precancerous condition, an infection by a pathogen, or a viral infection.

16. A therapeutic cassette comprising a nucleic acid encoding a B cell receptor (BCR) and a nucleic acid encoding a gene to be overexpressed.

17. 17. The therapeutic cassette of claim 16, wherein the gene to be overexpressed comprises a nucleic acid encoding an enzyme.

18. 18. The therapeutic cassette of claim 17, wherein the enzyme comprises an enzyme that is deficient in a subject with an enzymopathy.

19. The therapeutic cassette of any one of claims 16 to 18, wherein the nucleic acid encoding the BCR and the nucleic acid encoding the gene to be overexpressed are transcriptionally linked, translationally linked, or both.

20. The therapeutic cassette of any one of claims 16 to 19, comprising a promoter that drives transcription of the nucleic acid encoding the BCR and the nucleic acid encoding the gene to be overexpressed.

21. A vector comprising the therapeutic cassette of any one of claims 16 to 20.

22. 21. The vector of claim 20, comprising a lentiviral vector.

23. 23. The vector of claim 21 or 22, comprising at least one of a BaEV-pseudotyped lentiviral vector, a VSVg-pseudotyped lentiviral vector, a FAM1 lentiviral vector, and a FAM2 lentiviral vector.

24. A cell comprising the therapeutic cassette of any one of claims 16 to 20.

25. 25. The cell of claim 24, comprising a primary B cell.

26. A method comprising administering to a subject the cells of claim 24 or 25.

27. 27. The method of claim 26, further comprising administering to the subject an antigen, wherein the BCR of the therapeutic cassette is specific for the antigen.

28. 1. A method comprising editing the genome of a primary B cell, wherein the primary B cell expresses CD19, IgM, IgD, CD27 ＋ , CD21 ＋ and CXCR5 ＋ The method comprises a cell expressing at least one of:

29. 29. The method of claim 28, comprising introducing an exogenous protein or nucleic acid into the primary B cells.

30. 30. The method of claim 28 or 29, comprising electroporation of the cells.

31. The method of any one of claims 28 to 30, comprising introducing a targeted nuclease or a nucleic acid encoding a targeted nuclease.

32. The method of any one of claims 28 to 31, further comprising subjecting the primary B cells to at least one of the steps of activation, stimulation and proliferation.

33. subjecting the primary B cells to at least one of the steps of activation, stimulation, and proliferation; electroporating the primary B cells to introduce an exogenous protein or exogenous nucleic acid into the cells; 33. The method of claim 32, comprising:

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