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WO2024238710A1 - Compositions et procédés de production de cellules immunitaires modifiées - Google Patents

Compositions et procédés de production de cellules immunitaires modifiées Download PDF

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Publication number
WO2024238710A1
WO2024238710A1 PCT/US2024/029546 US2024029546W WO2024238710A1 WO 2024238710 A1 WO2024238710 A1 WO 2024238710A1 US 2024029546 W US2024029546 W US 2024029546W WO 2024238710 A1 WO2024238710 A1 WO 2024238710A1
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Prior art keywords
cell
lineage
engineered
cells
plasma
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Inventor
Timothy James MULLEN
Sarah Grace LEACH
Jasmine Nicole EDELSTEIN
Sean Philip Leary ARLAUCKAS
Anja Fides Hohmann
Richard A. Morgan
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Be Biopharma Inc
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Be Biopharma Inc
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • Cell therapy is an emerging from of treatment that comprises administration of viable human cells to a subject to act as “living drugs.”
  • certain cell therapies make use of engineered immune cells, to combat disease. Culturing and methods of administration of these engineered adaptive immune cells are critical both in clinical and pre-clinical settings for treatment in a patient population.
  • compositions, methods, and administration of said compositions comprising modified immune cells comprising modified immune cells (e.g., B lineage cells).
  • modified immune cells e.g., B lineage cells
  • present disclosure also encompasses, among other things, culturing methods and compositions for enhanced genetic integration of engineered immune cells (e.g., B lineage cells).
  • the present disclosure provides methods of preparing an engineered B lineage cell population with a relatively high incidence of genetic integration, a method comprising steps of: (a) isolating primary B cells so that a primary B cell population is obtained;
  • methods described herein comprise a step of expanding an engineered B lineage cell population.
  • methods described herein comprise a step of differentiating an engineered B lineage cell population into a population of plasmablasts.
  • a differentiation step comprises contacting an engineered B lineage cell population with culture media comprising: (a) IL-2; (b) IL-6; (c) IL- 10; and/or (d) IL-15.
  • methods described herein comprise a step of differentiating a population of plasmablasts into a population of plasma cells.
  • a differentiation step comprises contacting a population of plasmablasts with culture media comprising: (a) IL-6; (b) IL-15; and/or (c) IFN-alpha-2-beta (IFNa-2P).
  • a step of engineering further comprises introducing a donor construct comprising a transgene into a primary B cell population.
  • a donor construct comprises: (a) a 5’ homology arm that is at least 95% identical to a sequence 5’ to a double-stranded break site; and (b) a 3’ homology arm that is at least 95% identical to a sequence 3’ to a double- stranded break site.
  • a 5’ homology arm comprises or is: (i) a length about 450-850 base pairs in size, (ii) a PAM site or absence of a PAM site, and/or (iii) symmetrical or asymmetrical in length with a 3’ homology arm.
  • a 3’ homology arm comprises or is: (i) a length about 450-850 base pairs in size, (ii) a PAM site or absence of PAM site, and/or (iii) symmetrical or asymmetrical in length with a 5’ homology arm.
  • a donor construct is or comprises an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • a step of engineering comprises contacting a primary B cell population with a targeted nuclease capable of introducing double- stranded breaks.
  • a targeted nuclease is or comprises a CRISPR-associated (Cas) protein, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALEN), or meganuclease.
  • a Cas protein (a) is or comprises Cas9, Casl2a, or Casl3a, or a variant thereof; and/or (b) is complexed with a guide RNA (gRNA).
  • gRNA guide RNA
  • gRNA guide RNA
  • a gRNA is a single guide RNA (sgRNA).
  • a cell culture media that varies in serum in step (d) lacks serum or is substantially free of serum.
  • serum is or comprises human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof.
  • a cell culture media that varies in plasma in step (d) lacks plasma or is substantially free of plasma.
  • plasma is or comprises human plasma, bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma, or a combination thereof.
  • a step of engineering comprises: (a) electroporation of a primary B cell population, and/or (b) transduction of a primary B cell population with a donor construct.
  • electroporation is performed on a composition comprising: (a) a Cas protein complexed with a gRNA; and (b) a primary B cell population.
  • a primary B cell population is maintained in cell culture media without serum for a duration of about 24 hours.
  • an engineered B lineage cell population exhibits an incidence of genetic integration at a desired locus.
  • a desired locus is or comprises a CCR5 site.
  • methods described herein comprise a step of assessing using an assay one or more of: (a) percent of homology -directed repair; (b) percent of indels; and (c) amount of a reporter protein.
  • a reporter protein is or comprises of: green fluorescent protein (GFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), and/or luciferase.
  • an assay comprises or is one or more of: fluorescence-activated cell sorting (FAC-sort), Western Blot, flow cytometry, enzyme-linked immunosorbent spot assay (ELISpot), quantitative polymerase chain reaction (qPCR), reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), droplet digital PCR (ddPCR), and inference of CRISPR editing (ICE).
  • FAC-sort fluorescence-activated cell sorting
  • Western Blot flow cytometry
  • enzyme-linked immunosorbent spot assay enzyme-linked immunosorbent spot assay (ELISpot)
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RT-qPCR reverse transcriptase quantitative polymerase chain reaction
  • ELISA enzyme-linked immunosorbent assay
  • ddPCR droplet digital PCR
  • ICE inference of CRISPR editing
  • the present disclosure provides populations of engineered B lineage cells comprising a transgene sequence, wherein a transgene is integrated in at least 10% of an engineered B lineage cells. Tn some embodiments, a transgene is expressed from an endogenous CCR5 locus.
  • the present disclosure provides methods of preparing an engineered B lineage cell population by a process that comprises maintaining a B lineage cell population in a culture medium that comprises serum, an improvement comprising: a maintenance step in a method comprising a period of time during which a B lineage cell population is maintained in an otherwise comparable cell culture medium that lacks serum.
  • compositions comprising one or more B lineage cells selected from a population of engineered B lineage cells of any aspect or embodiment described herein, and one or more pharmaceutically acceptable excipients.
  • the present disclosure provides methods of administration of a pharmaceutical composition, a method comprising a step of: administering a pharmaceutical composition comprising: (a) a population of B lineage cells selected from a population of engineered B lineage cells of any aspect or embodiment described herein; and (b) one or more pharmaceutically acceptable excipients; wherein a pharmaceutical composition is administered to a subject.
  • a population of B lineage cells of step (a) are selected based on a higher percentage of integration relative to a total population of engineered B lineage cells.
  • a population of B lineage cells are characterized by at least 10%, at least 20%, at least 30%, or higher integration of a transgene.
  • the present disclosure provides methods of treating a disease, disorder, or condition in a subject, a method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition described herein, thereby treating the disease, disorder, or condition in a subject.
  • a pharmaceutical composition is administered intravenously.
  • a pharmaceutical composition is administered to an adult subject.
  • a pharmaceutical composition is administered to a pediatric subject.
  • the present disclosure provides methods of characterizing a population of engineered B lineage cells of any aspect or embodiment described herein, comprising assessing one or more of (a)-(g) using an assay: (a) presence of a CD38 marker; (b) presence of a CD138 marker; (c) presence of at least 50% of a CD27 marker; (d) secretion of at least 0.5 pg/ccll/day of IgG; (c) secretion of at least 2.5 pg/ccll/day of IgM; and (f) secretion of at least 0.5 pg/cell/day of IgD.
  • an assay comprises or is one or more of: fluorescence-activated cell sorting (FAC-sort), Western Blot, flow cytometry, enzyme-linked immunosorbent spot assay (ELISpot), quantitative polymerase chain reaction (qPCR), reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA).
  • FAC-sort fluorescence-activated cell sorting
  • Western Blot flow cytometry
  • enzyme-linked immunosorbent spot assay enzyme-linked immunosorbent spot assay
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RT-qPCR reverse transcriptase quantitative polymerase chain reaction
  • ELISA enzyme-linked immunosorbent assay
  • one or more B lineage cells selected from a population of engineered B lineage cells engraft within bone marrow of a subject.
  • the present disclosure provides methods of monitoring engraftment of engineered B lineage cells within a subject, comprising one or more of: bioluminescence, ELISpot, quantitative polymerase chain reaction (qPCR), reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), flow cytometry, and enzyme-linked immunosorbent assay.
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RT-qPCR reverse transcriptase quantitative polymerase chain reaction
  • flow cytometry comprising one or more of: bioluminescence, ELISpot, quantitative polymerase chain reaction (qPCR), reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), flow cytometry, and enzyme-linked immunosorbent assay.
  • Figure 1 shows a schematic of an exemplary process of B cell activation, genome engineering, expansion, and differentiation into engineered antibody-secreting cells (ASCs).
  • ASCs engineered antibody-secreting cells
  • Figure 2A shows measurements of antibody-secreting cell expansion of a B lineage cell population over time (fold change) using methods disclosed herein.
  • Figure 2B shows percentage of a B lineage cell population that is antibody-secreting cells over time using methods disclosed herein.
  • Figure 2C shows percentage of antibody-secreting cells of a B lineage cell population that were either engineered or non-engineered to express luciferase using methods disclosed herein.
  • Figure 3A shows percentage of indels at the CCR5 locus using methods of culturing and gene editing disclosed herein to demonstrate gene editing efficiency of a B lineage cell population.
  • Figure 3B demonstrates percentage of homology directed repair (HDR) as measured by droplet digital PCR (ddPCR) after editing with luciferase at the CCR5 locus.
  • Figure 4A demonstrates engraftment of a B lineage cell population engineered to express luciferase when compared to a negative control (PBS) over time at 15 days post-transfer and 104 days post-transfer in a murine model (NOG human IL-6 transgenic mice).
  • Figure 4B shows flux measurements of bioluminescence over time for engrafted engineered B lineage cells when compared to engrafted non-engineered B lineage cells and a PBS only control over 125 days.
  • Figure 5A demonstrates percentage HDR as measured by ddPCR for B lineage cell populations engineered with anti-CD19/CD3 bispecific T-cell engager (composed of single chain variable fragments, scFvs) containing expression cassettes targeting the CCR5 locus using the methods described herein.
  • Figure 5B shows secretion of anti-CD19/CD3 bispecific T-cell engager as measured by both CD19 ELISA and Jurkat T cell reporter assay.
  • Figure 5C demonstrates percent of tumor killing as a result of a tumor killing assay involving co-culture of CD8 + T cells and Raji tumor cells with the supernatant of B lineage cells that were either nonengineered or engineered with the aforementioned anti-CD19/CD3 expression cassette.
  • Figure 6A demonstrates percentage HDR as measured by ddPCR for B lineage cell populations engineered with expression cassettes as described herein targeting the CCR5 locus.
  • Figure 6B shows acid sphingomyelinase (ASM) secretion measured via ELISA in B lineage cell populations engineered with an exemplary ASM-encoding expression cassette with CCR5 -targeting guide RNA (gCCR5_232).
  • ASM acid sphingomyelinase
  • Figure 7 shows sphingomyelin phosphodiesterase 1 (SMPD1) activity in wild type HAP1 cells (control) or SMPD1 knockout (KO) HAP1 cells cultured with either of two different concentrations of recombinant human SMPD1 (rhSMPDl), supernatant from a B lineage cell population engineered with gCCR5 RNP only, or supernatant from a B lineage cell population engineered with an exemplified SMPD1 -containing expression cassette.
  • rhSMPDl recombinant human SMPD1
  • Figure 8 shows sphingomyelin content in wild type HAP1 cells (control) or SMPD1 knockout (KO) HAP1 cells cultured with either recombinant human acid sphingomyelinase (rhASM) supernatant from a B lineage cell population engineered with gCCR5 RNP only or supernatant from a B lineage cell population engineered with an exemplified SMPD1 -containing expression cassette.
  • rhASM human acid sphingomyelinase
  • Figure 9 shows fluorescence micrographs of sphingomyelin in HAP1 cells that were cither wild-type untreated (left), SMPD1 knockout (middle), or SMPD1-KO treated with supernatant from a B lineage cell population engineered with an exemplified SMPD1 -containing expressing cassette.
  • Figure 10A demonstrates percentage HDR as measured by ddPCR for B lineage cell populations engineered with expression cassettes as described herein targeting the CCR5 locus.
  • Figure 10B shows FIX secretion measured via ELlspot in B lineage cell populations engineered with the exemplary FIX expression cassettes with CCR5 -targeting guide RNA (gCCR5_232).
  • Figure 10C demonstrates FIX specific activity of B lineage cell populations engineered with expression cassettes disclosed herein that were cultured overnight with either vitamin K or media (control).
  • Figure 10D demonstrates activated partial thromoboplastin clotting time (aPPT) to quantify enzymatic activity of secreted FIX from engineered B lineage cells cultured in the presence or absence of Vitamin K.
  • aPPT activated partial thromoboplastin clotting time
  • Figure HA shows measurements of human FIX (huFIX) over time for mice after administration of two donors of B lineage cell populations that were engineered with methods and expression cassettes as disclosed herein.
  • Figure 1 IB shows measurements of human immunoglobulin G (hlgG) and human immunoglobulin M (hlgM) over time for mice after administration of two donors of B lineage cell populations that were engineered with methods and expression cassettes as disclosed herein.
  • Figures 12A-B shows two different donor measurements of percentage HDR and production of FIX for B lineage cell populations engineered to express FIX and cultured with or without serum variation methods as disclosed herein.
  • Figure 13A shows percentage of HDR in B lineage cell populations engineered to express alpha-galactosidase (GLA) with either a CCR5 or JCHAIN guide RNA.
  • B lineage cell populations either were cultured with serum variation (-FBS) or without serum variation (+FBS).
  • Figure 13B shows production of GLA as measured by ELISA in B lineage cell populations engineered to express GLA with either a CCR5 or JCHAIN guide RNA.
  • B lineage cell populations either were cultured with serum variation (-FBS) or without serum variation (+FBS).
  • Figure 14A shows percentage of HDR in B lineage cell populations engineered to express SMPD1 with two different JCHAIN guide RNAs.
  • B lineage cell populations either were cultured with serum variation (-FBS) or without serum variation (+FBS).
  • Figure 14B shows production of SMPD1 as measured by ELISA in B lineage cell populations engineered to express SMPD1 with either a CCR5 or JCHAIN guide RNA.
  • B lineage cell populations either were cultured with serum variation (-FBS) or without serum variation (+FBS).
  • Figure 15 shows percentage of HDR in B lineage cell populations engineered to express human leukocyte antigen-E (HLA-E) single chain fusion with a B2M guide RNA.
  • B lineage cell populations either were cultured with serum variation (-FBS) or without serum variation (+FBS).
  • Figure 16 shows percentage of HDR in engineered B lineage cell populations.
  • B lineage cell populations were cultured in indicated media compositions for a 24-hour transduction period with AAV vectors comprising Factor IX (FIX) for integration at CCR5.
  • Media compositions are described further in Table 1.
  • Figure 17 shows percentage of HDR in B lineage cell populations prepared from two different donor cell precursors (LKP220555 and LKP22099).
  • B lineage cell populations were cultured in indicated media compositions for a 24-hour transduction period with AAV vectors comprising FIX for integration at CCR5.
  • Media compositions are described further in Table 2.
  • Figure 18 shows percentage of HDR in B lineage cell populations.
  • B lineage cell populations were cultured in indicated media compositions for a 24-hour transduction period with AAV vectors comprising FIX for integration at CCR5.
  • Media compositions are described further in Table 3.
  • Figure 19 shows percentage of HDR in B lineage cell populations.
  • B lineage cell populations were cultured in indicated media compositions for a 24-hour transduction period with AAV vectors comprising FIX for integration at CCR5.
  • Media compositions are described further in Table 4.
  • Activation refers to the state of a cell, for example a B cell that has been sufficiently stimulated to induce detectable cellular proliferation or has been stimulated to exert its effector function. Activation can also be associated with induced cytokine production, cell signaling, differentiation, and/or antigen processing and presentation.
  • Administration typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment.
  • a subject or system e.g., that is or comprises one or more cells, tissues, organisms, etc
  • administration may be ocular, oral, parenteral, topical, etc.
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
  • bronchial e.g., by bronchial instillation
  • buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc
  • enteral intra-arterial, intradermal, intragastric,
  • administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • agent refers to a molecule that may be expressed, released, secreted or delivered to a target by a modified cell described herein.
  • An agent includes, but is not limited to, a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or fragments thereof, an antibody agent or fragments thereof, a growth factor, a cytokine, an enzyme, a protein (e.g., an RNAse inhibitor), a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combinations thereof.
  • An agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a cell, where it may act
  • Alloantigen refers to an antigen associated with allorecognition and/or graft rejection (e.g., an antigen against which a rejection immune response is directed).
  • alloantigens are agents that are present in or on tissue from one individual (e.g., a donor individual) of a particular species, but not in or on tissue from another individual (e.g., a recipient individual, for example who is genetically different from the donor individual) of the species, so that transfer of tissue from the donor individual to the recipient individual risks and/or results in a rejection immune response.
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.
  • an alloantigen is or comprises a polypeptide.
  • a variety of polypeptides are known in the art whose amino acid sequences can vary between and among individuals of the same species such that they might act as alloantigens.
  • Allogeneic refers to any material (e.g., a population of cells) derived from a different animal of the same species.
  • Allorecognition typically refers to an immune response mounted by the immune system of an individual (i.c., a recipient) who receives a tissue graft from another individual (i.e., a donor, who for example is genetically distinct from the recipient individual) of the same species, which immune response involves recognition of an alloantigen on the grafted tissue.
  • allorecognition involves T cell recognition of the alloantigen.
  • T cells recognize an alloantigen peptide, for example, encoded by a polymorphic gene whose sequence differs between the donor and recipient individuals.
  • Amelioration refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does not require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury).
  • a disease, disorder or condition e.g., radiation injury
  • Antigen refers to a molecule that is capable of provoking an immune response. This immune response may involve either antibody production, activation of specific immunologically-competent cells, or both. A skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • Antibody agent refers to a polypeptide that may be expressed, released, secreted, or delivered to a target by a modified cell described herein.
  • the polypeptide includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen.
  • an antibody agent comprises of an antibody.
  • antibodies as produced in nature are approximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure.
  • Each heavy chain comprises at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem).
  • VH amino-terminal variable
  • CH2 amino-terminal variable
  • CH3 carboxy-terminal CH3
  • Each light chain comprises two domains - an aminoterminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”.
  • Intact antibody agent tetramers comprises two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and a tetramer is formed.
  • Antibody agents are also glycosylated, typically on the CH2 domain.
  • Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel.
  • Each variable domain contains three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • the Fc region of naturally -occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. Affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification.
  • antibodies produced and/or utilized in accordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.
  • any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody agent”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology.
  • an antibody agent is polyclonal.
  • an antibody agent is monoclonal.
  • an antibody agent has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • antibody agent sequence elements are humanized, primatized, chimeric, etc, as is known in the art.
  • an antibody agent can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation.
  • an antibody agent may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody agent may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].
  • Autologous refers to any material derived from an individual to which it is later to be re-introduced into the same individual.
  • Biologically active refers to an observable biological effect or result achieved by an agent or entity of interest.
  • a specific binding interaction is a biological activity.
  • modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity.
  • presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.
  • Biomarker The term “biomarker” is used herein, consistent with its use in the art, to refer to a to an entity, event, or characteristic whose presence, level, degree, type, and/or form, correlates with a particular biological event or state of interest, so that it is considered to be a “marker” of that event or state.
  • a biomarker may be or comprise a marker for a particular' disease state, or for likelihood that a particular disease, disorder or condition may develop, occur, or reoccur.
  • a biomarkcr may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof.
  • a biomarker is predictive, in some embodiments, a biomarker is prognostic, in some embodiments, a biomarker is diagnostic, of the relevant biological event or state of interest.
  • a biomarker may be or comprise an entity of any chemical class, and may be or comprise a combination of entities.
  • a biomarker may be or comprise a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g., a metal or ion), or a combination thereof.
  • a biomarker is a cell surface marker.
  • a biomarker is intracellular.
  • a biomarker is detected outside of cells (e.g., is secreted or is otherwise generated or present outside of cells, e.g., in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc.
  • a biomarker may be or comprise a genetic or epigenetic signature.
  • a biomarker may be or comprise a gene expression signature.
  • Bispecific antibody refers to a bispecific binding agent in which at least one, and typically both, of the binding moieties is or comprises an antibody component.
  • a variety of different bi-specific antibody structures are known in the ail.
  • each binding moiety in a bispecific antibody that is or comprises an antibody component includes VH and/or VL regions; in some such embodiments, such VH and/or VL regions are those found in a particular monoclonal antibody.
  • each contains two antibody component-binding moieties, each includes VH and/or VL regions from different monoclonal antibodies.
  • a bispecific antibody contains two antibody component binding moieties, wherein one of the two antibody component binding moieties includes an immunoglobulin molecule having VH and/or VL regions that contain CDRs from a first monoclonal antibody, and one of the two antibody component binding moieties includes an antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.) having VH and/or VL regions that contain CDRs from a second monoclonal antibody.
  • an antibody fragment e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.
  • Conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into an antibody compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • Engineered' refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in an engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with a second coding sequence.
  • a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and/or if produced other than natural expression in a cell.
  • a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • such manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a particular agent of interest e.g., a protein, a nucleic acid, and/or a particular form thereof
  • progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • Endogenous refers to any material from or produced inside a particular organism, cell, tissue or system.
  • Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
  • suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Exogenous refers to any material introduced from or produced outside a particular organism, cell, tissue or system.
  • Expand refers to increasing in number, as in an increase in the number of cells, for example, monocytes, macrophages, and/or dendritic cells.
  • monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to the number originally present in a culture.
  • monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to other cell types in a culture.
  • expansion may occur in vivo.
  • the term "ex vivo,” as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).
  • a gene product can be a transcript.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cisacting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • fragment refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
  • a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide.
  • monomeric units e.g., nucleic acids
  • a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide.
  • the whole material or entity may in some embodiments be referred to as the “parent” of the whole.
  • a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • expression product generally refers to an RNA transcribed from a gene (pre-and/or postprocessing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from a gene.
  • homology refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences.
  • Calculation of the percent homology between two nucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • the percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Identity refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • Immune cell refers to a cell that is involved in an immune response, e.g., promotion of an immune response.
  • immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, T-lymphocytes, plasma cells, plasmablasts, or B -lymphocytes.
  • a source of immune cells e.g., macrophages, monocytes, or dendritic cells
  • a source of immune cells can be obtained from a subject.
  • Immune response refers to a cellular and/or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
  • Immunoglobulin refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as a BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.
  • IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts.
  • IgG is the most common circulating antibody.
  • IgM is the main immunoglobulin produced in the primary immune response in most subjects.
  • IgD is an immunoglobulin that has no known antibody function, but may serve as an antigen receptor.
  • IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (2) designed, produced, prepared, and/or manufactured by the hand of man.
  • a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated).
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of a substance is calculated without including such carriers or excipients.
  • carriers or excipients e.g., buffer, solvent, water, etc.
  • a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature.
  • a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide.
  • a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
  • a marker refers to an entity or moiety whose presence or level is a characteristic of a particular state or event.
  • presence or level of a particular marker may be characteristic of presence or stage of a disease, disorder, or condition.
  • the term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, stage of tumor, etc.
  • a presence or level of a particular marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular class of tumors.
  • the statistical significance of presence or absence of a marker may vary depending upon a particular marker.
  • detection of a marker is highly specific in that it reflects a high probability that such tumor is of a particular subclass. Such specificity may come at the cost of sensitivity (i.e., a negative result may occur even if the tumor is a tumor that would be expected to express the marker). Conversely, markers with a high degree of sensitivity may be less specific that those with lower sensitivity. Those skilled in the art will appreciate that, in many embodiments, a useful marker need not distinguish with 100% accuracy.
  • Modified refers to a changed state or structure of a molecule or cell of the invention.
  • Molecules may be modified in many ways, including chemically, structurally, and functionally.
  • Cells may be modified through the introduction of nucleic acids.
  • Modulating refers to mediating a detectable increase or decrease in the level of a response and/or a change in the nature of a response in a subject compared with the level and/or nature of a response in the subject in the absence of a treatment or compound, and/or compared with the level and/or nature of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • nucleic acid refers to a polymer of at least three nucleotides.
  • a nucleic acid comprises DNA.
  • a nucleic acid comprises RNA.
  • a nucleic acid is single stranded.
  • a nucleic acid is double stranded.
  • a nucleic acid comprises both single and double stranded portions.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages.
  • a nucleic acid comprises a backbone that comprises both phosphodiester and non- phosphodiester linkages.
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine, guanine, thymine, uracil).
  • a nucleic acid comprises one or more, or all, non-natural residues.
  • a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).
  • a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • operably linked refers to functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Payload In general, the term “payload”, as used herein, refers to an agent that may be delivered or transported by association with another entity.
  • association may be or include a covalent linkage; in some embodiments such association may be or include non-covalent interaction(s).
  • association may be direct; in some embodiments, association may be indirect.
  • a payload is not limited to a particular chemical identity or type; for example, in some embodiments, a payload may be or comprise, for example, an entity of any chemical class including, for example, a lipid, a metal, a nucleic acid (e.g., a transgene), a polypeptide, a saccharide (e.g., a polysaccharide), small molecule, or a combination or complex thereof.
  • a payload may be or comprise a biological modifier, a detectable agent (e.g., a dye, a fluorophore, a radiolabel, etc.), a detecting agent, a nutrient, a therapeutic agent, etc., or a combination thereof.
  • a payload may be or comprise a cell or organism, or a fraction, extract, or component thereof.
  • a payload may be or comprise a natural product in that it is found in and/or is obtained from nature; alternatively or additionally, in some embodiments, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
  • an payload may be or comprise an agent in isolated or pure form; in some embodiments, such agent may be in crude form.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
  • Polynucleotide As used herein, the term “polynucleotide” refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • polypeptide refers to any polymeric chain of residues (c.g., amino acids) that arc typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids.
  • a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof.
  • such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
  • a polypeptide may be cyclic, and/or may comprise a cyclic portion.
  • a polypeptide is not cyclic and/or does not comprise any cyclic portion.
  • a polypeptide is linear.
  • a polypeptide may be or comprise a stapled polypeptide.
  • the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a common sequence motif e.g., a characteristic sequence element
  • shares a common activity in some embodiments at a comparable level or within a designated range
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
  • a useful polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
  • Protein refers to a polypeptide (i.e. , a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the ail will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • Reference' As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities arc present to justify reliance on and/or comparison to a particular possible reference or control.
  • a response to treatment may refer to a beneficial alteration in a subject’s condition that occurs as a result of or correlates with treatment.
  • alteration may be or comprise stabilization of a condition (e.g., prevention of deterioration that would have taken place in the absence of a treatment), amelioration of symptoms of a condition, and/or improvement in prospects for cure of a condition, etc.
  • the term “response” may refer to a response of a particular system or components thereof (e.g., of a particular cell, tissue, organism, or subject).
  • sample typically refers to an aliquot of material obtained or derived from a source of interest, as described herein.
  • a source of interest is a biological or environmental source.
  • a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human).
  • a source of interest is or comprises biological tissue or fluid.
  • a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and/or combinations or componcnt(s) thereof.
  • a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
  • a biological fluid may be or comprise a plant exudate.
  • a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
  • a biological sample is or comprises cells obtained from an individual.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
  • a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
  • a sample may be a “crude” sample in that it has been subjected to relatively little processing and/or is complex in that it includes components of relatively varied chemical classes.
  • Signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.
  • the term “significant” typically refers to the context wherein the difference or relationship between two variables (e.g., sequence identity, protein production, spatiotemporal conditions, etc.) are certain and exist. Significance can be statistically measured e.g., statistically significant) by various mathematical formulas and models as understood by one skilled in the art. These methods include, but are not limited to, student t-test, two-tailed test, analysis of variance (ANOVA), etc. Furthermore, significance can impart differences within structure and chemistry between two different entities.
  • a sample molecule may be compared to a reference molecule, and exhibits a structural difference from said reference molecule that is significant, e.g., in the presence or absence or in the level of one or more biological or chemical moieties as compared to the reference entity.
  • Source typically refers to a context in which an agent of interest (e.g., that may be or comprise a carbohydrate, a lipid, a nucleic acid, a metal, polypeptide, a small molecule, or a combination thereof) may be found in nature, or from which such agent can be or has been obtained (e.g., isolated).
  • a source may be or comprise a biological source (e.g., an organism, tissue, or cell, or sample thereof); in some embodiments, a source may be an environmental source.
  • a source may be or comprise a primary sample from an organism (e.g., which may be or comprise a tissue or fluid of such organism, and/or may be or comprise cell(s) of such organism).
  • an organism may be or comprise a prokaryotic organism (e.g., a bacterium) or a eukaryotic organism (e.g., a fungus or yeast, an insect, a mammal, a plant, a reptile, etc.).
  • an infectious agent such as a virus or phage may be considered an organism for purposes of this disclosure, and in particular with respect to being a source.
  • a source may be or comprise an engineered source, such as a cell line or culture, an in vitro system, etc.
  • Subject refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog).
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein.
  • a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition.
  • a subject displays one or more symptoms of a disease, disorder, or condition.
  • a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject docs not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Substantial identity refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.
  • two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues.
  • the relevant stretch is a complete sequence.
  • the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
  • reference to “substantial identity” typically refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to that of a reference CDR.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture a potential lack of completeness inherent in many biological and chemical phenomena.
  • substantially purified refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they arc naturally associated in their natural state.
  • the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
  • Target refers to a cell, tissue, organ, or site within the body that is the subject of provided methods, systems, and/or compositions, for example, a cell, tissue, organ or site within a body that is in need of treatment or is preferentially bound by.
  • Target locus may refer to a specific site or location on a chromosome of interest.
  • a target locus may be a site to be “engineered” or “altered” by the hand of man.
  • an engineered polynucleotide comprises homology to a target locus in order to allow further alterations at a specific site (e.g., CCR5 as a target locus, whose homologous sequence may be part of a guide RNA to result in incorporation of an edit via CRISPR/Cas -mediated gene editing).
  • target locus may interchangeably refer to a target gene of interest for manipulation by man.
  • such target locus manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols.
  • Target site refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • Therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • Transfected As used herein, the term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
  • treat refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and/or reduction in incidence and/or severity of one or more symptoms or features of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and/or condition (e.g., may be prophylactic).
  • treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits established, severe, and/or late-stage signs of the disease, disorder, or condition.
  • Variant refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more biological or chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In some embodiments, a variant differs structurally but performs the same or similar function as its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule.
  • any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function;
  • a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • moieties e.g., carbohydrates, lipids, phosphate groups
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid is a truncated form of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide that is a truncated form of the reference polypeptide may demonstrate comparable, identical, or greater levels of one or more biological activities as compared to the reference polypeptide or nucleic acid.
  • a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular- positions.
  • Vector refers to a composition of matter that comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the ail including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but arc not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • the present disclosure encompasses culturing and engineering in order to produce highly viable and productive (e.g., relatively higher genetic integration of a desired payload) B lineage cell populations for administration to a subject.
  • Cell-based therapeutics are an emerging class of medicine that make use of innate cellular machinery to combat disease. Unlike many traditional treatment methods, cell therapies make use of cellular localization, migration, and proliferation within the body, which can translate to improved biodistribution and targeted delivery of therapeutics. Cell therapies also benefit from cellular ability to sense and respond to various extrinsic signals within a subject, including, e.g., small molecules, other cells, physical forces, and/or marker proteins. Cellular persistence in vivo also enables cell therapies to survive, differentiate, function, etc. within a subject over extended time periods. These innate qualities may also lead to improved safety and efficacy for cell therapies as compared to other biologies or pharmaceutical compounds, providing long-lived, specifically targeted, adjustable, and/or responsive treatment for disease.
  • Cell-based therapies may have potential applications for a broad range of diseases, including those that have proved intractable or difficult to manage with traditional treatment options.
  • Diseases that have been targeted for cell-based treatment include, e.g., various cancers, autoimmune diseases, central nervous system (CNS) diseases, neurodegenerative disorders, and cardiovascular diseases, among others.
  • Cellular therapeutics offer an alternative over other treatment options for diseases where highly specific targeting (e.g., to a particular tissue type, area of the body, etc.) and/or longer-term treatment efficacy (e.g., enabling lower dosage frequency, single treatment options, etc.) are highly favored or necessary.
  • Cell therapies may employ a number of different cell types, which are typically modified to provide a therapeutic effect (e.g., transgene expression, reprogrammed cellular targeting, etc.). Although many cell types have potential to provide some form of therapeutic effect, recent therapies have heavily favored adaptive immune cells such as T lymphocytes and B lymphocytes (referred to interchangeably herein as T cells and B cells, respectively). For example, chimeric antigen receptor T (CAR-T) cells have been engineered to treat various cancers through recognition of one or more tumor cell markers, leading to cytotoxic destruction of tumor cells. CAR-T cells are also being adapted to treat infectious diseases (e.g., HIV) through recognition of other targeted antigens. Recent engineering efforts are focused on enhancing CAR-T receptor functionality, reducing innate immune response to CAR-T, and developing allogeneic therapies that make use of donor cells.
  • CAR-T chimeric antigen receptor T
  • Monoclonal antibodies can be produced to target antigens within the body, which is valuable for treatment of diseases where such antibodies cannot be induced through natural processes (e.g., self-antigens for cancer and/or autoimmune diseases, antigens that fail to elicit natural immune response through infection and/or vaccination, etc.).
  • Current antibody therapies require frequent administration and are costly to produce.
  • researchers have attempted to address these issues through use of gene therapy, which makes use of various techniques (e.g., viral vectors, CRISPR/Cas9 editing) to deliver antibody payloads to endogenous cells in the body, leading to persistent antibody production within a subject.
  • these approaches can result in low levels of antibody expression and a counter- active response by the subject’s own immune system.
  • B lineage cells are a highly desirable cell-based target to secrete other payloads including, but not limited to, enzymes, complement proteins, cytokines, cytokine receptors, chimeric antigen receptors (CARs), anti-fibrotic molecules, antithrombotic molecules, antigens, both wild type and variant proteins, coagulation factors, glucose response elements, and fragments of antibodies, antigens, and proteins.
  • CARs chimeric antigen receptors
  • B lineage cells are an attractive option for development of cell-based therapies and may potentially offer improved therapeutic effects (e.g., pay load delivery, targeting, long-term pay load expression, reduced auto-immune response, etc.) as compared to traditional therapies (e.g., antibody -based therapeutics, other cell therapies, etc.).
  • a B lineage cell is a cell that expresses one or more B cell receptors (BCR) on a cell membrane.
  • BCR B cell receptors
  • a B lineage cell is a modified version or variant of a cell that expresses one or more B cell receptors (BCRs) on a cell membrane.
  • a B lineage cell is a naive or memory B cell.
  • a B lineage cell is a cell derived from a naive B cell (e.g., activated B lineage cell, plasmablast, plasma cell) or a variant thereof.
  • a B lineage cell is an activated B lineage cell.
  • a B lineage cell is a plasmablast.
  • a B lineage cell is a plasma cell.
  • a B lineage cell population comprise naive B cells.
  • naive B cell populations are used as reference cell populations.
  • naive B cell populations express CD19 (CD19 + ).
  • expression of CD 19 in naive B cell populations is used as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express CD20 (CD20 + ).
  • expression of CD20 in naive B cell populations is used as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express low amounts of CD27 (CD27 10 ).
  • expression of CD27 in naive B cell populations are as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express low amounts of CD38 (CD38 10 ).
  • expression of CD38 in naive B cell populations are used as a reference to assist in characterization of other B lineage cell populations.
  • naive B cell populations express low amounts of CD138 (CD138 10 ).
  • expression of CD138 in naive B cell populations are used as a reference to assist in characterization of other B lineage cell population.
  • a B lineage cell population comprises activated B lineage cells.
  • activated B lineage cell populations are used as reference cell populations.
  • activated B lineage cell populations are compared to a reference cell population (e.g., naive B cell population).
  • activated B lineage cell populations express lower amounts of CD 19 (CD19 10 ) as compared to a reference cell population (e.g., naive B cell populations).
  • activated B lineage cell populations express different e.g., higher or lower) amounts of CD 19 as compared to a reference cell population (e.g., differentiated B cell populations, etc.).
  • activated B lineage cell populations express lower amounts of CD20 (CD20 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, activated B lineage cell populations express different (e.g., higher or lower) amounts of CD20 as compared to a reference cell population (e.g., differentiated B cell populations, etc.). In some embodiments, activated B lineage cell populations express higher amounts of CD27 (CD27 hl ) as compared to a reference cell population (e.g., naive B cell populations).
  • activated B lineage cell populations express different (e.g., higher or lower) amounts of CD27 as compared to a reference cell population (e.g., differentiated B cell populations, etc.). In some embodiments, activated B lineage cell populations express lower amounts of CD38 (CD38 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, activated B lineage cell populations express different (e.g., higher or lower) amounts of CD38 as compared to a reference cell population (e.g., differentiated B cell populations, etc.).
  • activated B lineage cell populations express lower amounts of CD138 (CD138 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, activated B lineage cell populations express different (e.g., higher or lower) amounts of CD138 as compared to a reference cell population (e.g., differentiated B cell populations, etc.).
  • a B lineage cell population may comprise plasmablast cells.
  • plasmablast cell populations are used as reference cell populations.
  • plasmablast cell populations are compared to a reference cell population (e.g., naive B cell population).
  • plasmablast cell populations express lower amounts of CD 19 (CD19 10 ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasmablast cell populations express different (e.g., higher or lower) amounts of CD 19 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • plasmablast cell populations express lower amounts of CD20 (CD2O 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasmablast cell populations express different (e.g., higher or lower) amounts of CD20 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.). In some embodiments, plasmablast cell populations express higher amounts of CD27 (CD27 111 ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasmablast cell populations express different (e.g., higher or lower) amounts of CD27 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • plasmablast cell populations express higher amounts of CD38 (CD38 hl ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasmablast cell populations express different (e.g., higher or lower) amounts of CD38 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • plasmablast cell populations express lower amounts of CD138 (CD138 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasmablast cell populations express different (e.g., higher or lower) amounts of CD138 as compared to a reference cell population (e.g., activated cell populations, plasma cell populations, etc.).
  • a B lineage cell population comprises plasma cells.
  • plasma cell populations are used as reference cell populations.
  • plasma cell populations are compared to a reference cell population (e.g., naive B cell population).
  • plasma cell populations express lower amounts of CD 19 (CD19 10 ) as compared to a reference cell population (e.g., naive B cell populations).
  • plasma cell populations express different (e.g., higher or lower) amounts of CD 19 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.).
  • plasma cell populations express lower amounts of CD20 (CD20 10 ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasma cell populations express different (e.g., higher or lower) amounts of CD20 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.). In some embodiments, plasma cell populations express higher amounts of CD27 (CD27 hl ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasma cell populations express different (e.g., higher or lower) amounts of CD27 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.).
  • a reference cell population e.g., activated cell populations, plasmablast populations, etc.
  • plasma cell populations express higher amounts of CD38 (CD38 hl ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasma cell populations express different (e.g., higher or lower) amounts of CD38 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.). In some embodiments, plasma cell populations express higher amounts of CD138 (CD138 hl ) as compared to a reference cell population (e.g., naive B cell populations). In some embodiments, plasma cell populations express different (e.g., higher or lower) amounts of CD138 as compared to a reference cell population (e.g., activated cell populations, plasmablast populations, etc.). Cell engineering
  • engineered cells for various uses including, e.g., cell therapies is an active area of development. Genomic and cpigcnomic modifications, synthetic biology, and application of biomaterials may be employed to generate engineered cells with desired properties for therapeutic applications. Selection of an appropriate method for generating engineered cells is typically dependent upon the desired output and effects of cell therapy and requires optimization for different cell types, transgenes of interest, etc. It is understood in the art that engineering methods that are effective for a particular cell type may be less effective (or not viable) for another cell type. Furthermore, engineering methods may vary depending upon whether a therapy requires cellular localization, expression of an endogenous or exogenous protein, removal of an endogenous protein, etc.
  • Genome editing tools can alter a cellular genome to produce a desired therapeutic effect, e.g., expression of a therapeutic protein.
  • Targeted nucleases e.g., Cas proteins, TALENs, ZFNs, etc.
  • viral vectors e.g., AAV, lentiviral, adenoviral, etc.
  • recombinases e.g., Cre recombinase, Flp recombinase, PhiC31 integrase, etc.
  • Gene editing efficiency may vary depending on, e.g., cell type, desired function, and ease of delivery. Accordingly, editing methods often require extensive optimization to provide engineered cells with intended functionality for therapeutic applications.
  • Various B lineage cell engineering techniques are described in the art, including, e.g., CRISPR/Cas9, AAV, lentiviral, and recombinase-based methods. These methods are generally employed to introduce a payload (e.g., expression cassette, transgcnc, etc.) into naive B cells in order to express a protein of interest.
  • a payload may be designed for episomal expression, integration into a specific target locus (e.g., endogenous target gene locus), or integration into a non-specific locus (e.g., endogenous random or non-target gene locus).
  • Payloads may be designed such that an endogenous target gene locus continues to produce functional protein and/or fulfill its natural function (non-disruptivc integration). Pay loads may also be designed to intentionally disrupt an endogenous target gene locus to produce lowered or non-detectable levels of functional protein and/or some amount of non-functional protein.
  • Methods for integration of a payload (e.g., expression cassette comprising one or more transgenes) into an endogenous target locus may comprise site-specific cleavage with a targeted nuclease (e.g., Cas protein, including Cas9), followed by integration of a transgene (e.g., encoding SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.) through an endogenous repair pathway (e.g., homologous recombination, homology-directed repair, etc.).
  • a transgene e.g., encoding SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.
  • endogenous repair pathway e.g., homologous recombination, homology-directed repair, etc.
  • methods for integration of an expression cassette comprising a transgene comprise site-specific cleavage at a target locus (e.g., CCR5) with a guide RNA I Cas9 complex, followed by integration of a transgene at the target locus through homologous recombination.
  • a target locus e.g., CCR5
  • a guide RNA I Cas9 complex e.g., CCR5
  • a method of B lineage cell engineering is or comprises administration of an ribonucleoprotein (RNP) to a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a Cas protein complexed with guide RNA (gRNA) to a cell population.
  • gRNA guide RNA
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a Cas9/guide RNA complex a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a Cas9/guide RNA complex a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a payload (e.g., expression cassette comprising one or more transgenes) of interest to a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a payload (e.g., expression cassette comprising one or more transgenes) to a cell population through use of a viral vector.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a payload (e.g., expression cassette comprising one or more transgenes) encapsulated within an AAV capsid (e.g., AAN2, AAV3, AAV5, AAV6, AAV8, etc.) to a cell population.
  • a method of B lineage cell engineering is or comprises administration of a composition comprising a transgene encapsulated within an AAV capsid (e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.) in combination with or in addition to administration of a composition comprising a Cas9/gRNA complex.
  • a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of one or more engineering components. In some embodiments, a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex. In some embodiments, a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex and a payload.
  • a method of B lineage cell engineering may comprise a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex and a payload (e.g., expression cassette comprising one or more transgenes) encapsulated in an AAV capsid (AAV2, AAV3, AAV5, AAV6, AAV8, etc.).
  • a method of B lineage cell engineering comprises a step of electroporation to facilitate cellular uptake of a Cas9/gRNA complex and a payload (e.g., expression cassette comprising one or more transgenes) encapsulated in an AAV6 capsid.
  • a method of B lineage cell engineering comprises a step of viral transduction to facilitate cellular uptake of a payload (e.g., expression cassette comprising one or more transgenes).
  • a method of B lineage cell engineering comprises a step of viral transduction to facilitate cellular uptake of a payload (e.g., transgene) encapsulated in an AAV capsid (AAV2, AAV3, AAV5, AAV6, AAV8, etc.).
  • a method of B lineage cell engineering comprises a step of viral transduction to facilitate cellular uptake of a payload (e.g., expression cassette comprising one or more transgenes) encapsulated in an AAV6 capsid.
  • a method of B lineage cell engineering comprises one or more steps of: (i) electroporation to facilitate cellular uptake of a Cas9/gRNA complex; and (ii) viral transduction to facilitate cellular uptake of a payload encapsulated in an AAV capsid.
  • a method of cell engineering is particularly effective for one type of cell (e.g., T cell) and less effective for another type of cell (e.g., B cell).
  • a method of B lineage cell engineering provides improved genome editing efficiency in B lineage cells as compared to other cell types (e.g., T cell).
  • a method of B cell engineering provides at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% editing efficiency.
  • a method of B lineage cell engineering provides at least about 1E6, 1.5E6, 2E6, 2.5E6, 3E6, 3.5E6, 4E6, 4.5E6, 5E6, 5.5E6, 6E6, 6.5E6, 7E6, 7.5E6, 8E6, 8.5E6, 9E6, 9.5E6, 1E7, 1.5E7, 2E7, 2.5E7, 3E7, 3.5E7, 4E7, 4.5E7, or 5E7 edited B lineage cells.
  • a method of B lineage cell engineering comprises a step of editing activated B cells.
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of about 1, 2, 3, 4, or 5 day(s).
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of 2 days.
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of about 1 , 2, 3, 4, or 5 day(s) and expanding edited B lineage cells in the activation media for an additional period of about 1, 2, 3, 4, 5, 6, 7, or 8 days.
  • a method of B lineage cell engineering comprises a step of editing B lineage cells after an activation step of 2 days and expanding edited B lineage cells in the activation media for an additional 6 days.
  • engineered cells comprising one or more payloads.
  • engineered cells e.g., B lineage cell populations, etc.
  • any of a variety of payloads may be used (e.g., those with a therapeutic or monitoring purpose), alone or in combination.
  • a payload is or comprises a polynucleotide sequence encoding a peptide or polypeptide.
  • a payload is or comprises one or more transgenes.
  • a payload is or comprises one or more homology arm sequences.
  • a pay load is or comprises a transgene flanked by one or more homology sequences.
  • a payload may be or comprise a polynucleotide sequence, which comprises an expression cassette.
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.).
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.) in a particular configuration and/or combination.
  • polynucleotide sequence elements e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.) in a particular configuration and/or combination in order to promote expression of a transgene in a cell population.
  • polynucleotide sequence elements e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.
  • an expression cassette comprises one or more polynucleotide sequence elements (e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.) in a particular configuration and/or combination in order to promote expression of a transgene in an engineered B lineage cell population.
  • polynucleotide sequence elements e.g., promoters, enhancers, transgenes, termination elements, homology arms, biomarkers, signal peptide sequences, internal ribosome entry site elements, self-cleaving peptide sequences, ubiquitous chromatin opening element, etc.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more promoters (e.g., MND, CMV, SFFV, FEEK I, EF-la, etc.). In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous promoters. In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more endogenous promoters. In some embodiments, an expression cassette does not comprise one or more promoters. In some embodiments, an expression cassette does not comprise one or more exogenous promoters. In some embodiments, an expression cassette does not comprise one or more endogenous promoters.
  • promoters e.g., MND, CMV, SFFV, FEEK I, EF-la, etc.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous promoters.
  • an expression cassette comprises one or
  • an expression cassette comprises one or more polynucleotide sequences encoding a translation initiation site (e.g., Kozak consensus sequence, ribosomal binding site, etc.). In some embodiments, an expression cassette comprises one or more exogenous polynucleotide sequences encoding a translation initiation site. In some embodiments, an expression cassette comprises one or more endogenous polynucleotide sequences encoding a translation initiation site. In some embodiments, an expression cassette docs not comprise one or more polynucleotide sequences encoding a translation initiation site.
  • an expression cassette does not comprise one or more exogenous polynucleotide sequences encoding a translation initiation site. In some embodiments, an expression cassette does not comprise one or more endogenous polynucleotide sequences encoding a translation initiation site.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more enhancers (e.g., WPRE, beta-globin etc.). In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous enhancers. In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more endogenous enhancers. In some embodiments, an enhancer may be viral (e.g., WPRE, etc.) or non-viral. In some embodiments, an expression cassette does not comprise one or more enhancers. In some embodiments, an expression cassette does not comprise one or more exogenous enhancers. In some embodiments, an expression cassette does not comprise one or more endogenous enhancers.
  • enhancers e.g., WPRE, beta-globin etc.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous enhancers. In some
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more terminators (e.g., polyA, including, e.g., BGH polyA, SV40 polyA, etc.). In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous terminators. In some embodiments, an expression cassette comprises one or more polynucleotide sequences encoding one or more endogenous terminators. In some embodiments, an expression cassette does not comprise one or more terminators. In some embodiments, an expression cassette does not comprise one or more exogenous terminators. In some embodiments, an expression cassette does not comprise one or more endogenous terminators.
  • terminators e.g., polyA, including, e.g., BGH polyA, SV40 polyA, etc.
  • an expression cassette comprises one or more polynucleotide sequences encoding one or more exogenous terminators.
  • an expression cassette does not comprise one or more termin
  • an expression cassette comprises a polynucleotide sequence encoding a transgene (e.g., SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.) or variant thereof.
  • a transgene e.g., SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.
  • an expression cassette comprises a polynucleotide sequence encoding a transgene (e.g., SMPD1, Factor IX, BiTE (e.g., blinatumomab), etc.) or variant thereof for expression of a peptide or polypeptide (e.g., acid sphingomyelinase, Factor IX, BiTE (blinatumomab), etc.) or variant thereof.
  • a transgene e.g., SMPD1, Factor IX, BiTE (e.g., blinatumomab), etc.
  • a transgene e.g., SMPD1, Factor IX, BiTE (e.g., blinatumomab), etc.
  • a transgene e.g., SMPD1, Factor IX, BiTE (e.g., blinatumomab), etc.
  • a peptide or polypeptide e.g., acid
  • a transgene is a corrective gene chosen to improve one or more signs and/or symptoms of a disease, disorder, or condition.
  • transgenes are functional versions of disease associated genes (i.e., gene isoform(s) which are associated with the manifestation or worsening of a disease, disorder or condition) found in a subject.
  • one or more transgenes arc optimized versions of disease- associated genes found in a subject (e.g., codon optimized or expression-optimized variants).
  • transgenes are variants of disease-associated genes found in a subject (e.g., a functional gene fragment or variant thereof).
  • a transgene is a gene that causes expression of a peptide that is normally expressed in one or more healthy tissues.
  • a transgene is a gene that causes expression of an altered protein with a gain- or loss-of-function mutation. In some embodiments, a transgene is a gene that causes expression of a fusion protein. In some embodiments, a transgene is a gene that causes expression of an antibody agent. In some embodiments, a transgene is a gene that causes expression of a multispecific antibody. In some embodiments, a transgene is a fragment of an antibody, antigen, or protein. In some embodiments, a transgene is a gene that causes expression an enzyme (e.g., for enzyme replacement therapy). In some embodiments, a transgene is a gene that causes expression of a cytokine.
  • a transgene is a gene that causes expression of a cytokine receptor. In some embodiments, a transgene is a gene that causes expression of a chimeric antigen receptor (CAR). In some embodiments, a transgene is a gene that causes expression of an anti-thrombotic molecule. In some embodiments, a transgene is a gene that causes expression of a coagulation factor. In some embodiments, a transgene is a gene that causes expression of a glucose response element. In some embodiments, a transgene is a gene that causes expression of a nanobody.
  • CAR chimeric antigen receptor
  • a transgene is a gene that causes expression of an anti-thrombotic molecule.
  • a transgene is a gene that causes expression of a coagulation factor.
  • a transgene is a gene that causes expression of a glucose response element. In some embodiments, a transgene is a gene that causes expression of a nanobody.
  • a transgene is a gene that causes expression of sphingomyelin phosphodiesterase 1 (SMPD1), acid sphingomyelinase (ASM), bispecific T-cell engager (BiTE; e.g., blinatumomab), or a variant thereof.
  • SMPD1 sphingomyelin phosphodiesterase 1
  • ASM acid sphingomyelinase
  • BiTE bispecific T-cell engager
  • a transgene is or comprises a gene encoding a functional nucleic acid.
  • a therapeutic agent is or comprises an agent that has a therapeutic effect upon a host cell or subject (including, e.g., a ribozyme, guide RNA (gRNA), antisense oligonucleotide (ASO), miRNA, siRNA, and/or shRNA).
  • a therapeutic agent promotes a biological process to treat a medical condition, e.g., at least one symptom of a disease, disorder, or condition such as Niemann Pick Disease Type B.
  • transgene expression in a subject results substantially from integration at a target locus.
  • 75% or more e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more
  • 25% or less e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less
  • of total transgene expression in a subject is from a source other than transgene integration at a target locus (e.g., episomal expression, integration at a non-target locus).
  • a transgene is or comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, a transgene is or comprises a sequence having a least 80%, 85%, 90%, 95%, 99%, or 100% identity to a portion of a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence).
  • a payload may comprise one or more flanking polynucleotide sequences with significant sequence homology to a target locus (e.g., homology arms).
  • homology anus flank a polynucleotide sequence encoding a payload (e.g., one homology arm is 5’ to a payload (also referred to herein as a 5’ homology arm) and one homology arm is 3’ to a pay load (also referred to herein as a 3’ homology arm).
  • homology arms direct site- specific integration of a payload.
  • homology arms are between 400 and 1000 nt in length. In some embodiments, homology arms are between 800 and 1500 nt in length. In some embodiments, homology arms are at least 500 nt in length. In some embodiments, homology arms are less than 1500 nt in length. In some embodiments, homology arms are 1000 nt in length. In some embodiments, homology arms are at least 50 nt in length. In some embodiments, homology arms are at least 600 nt in length. In some embodiments, homology arms are of the same length. In some embodiments, homology arms arc not of the same length. In some embodiments, homology arms have at least 70% sequence homology to a target locus.
  • homology arms have at least 80% sequence homology to a target locus. In some embodiments, homology arms have at least 90% sequence homology to a target locus. In some embodiments, homology arms have at least 95% sequence homology to a target locus. In some embodiments, homology arms have at least 99% sequence homology to a target locus. In some embodiments, homology arms have 100% sequence homology to a target locus. In some embodiments, homology arms have at least 70% sequence identity to a target locus. In some embodiments, homology arms have at least 80% sequence identity to a target locus. In some embodiments, homology arms have at least 90% sequence identity to a target locus. In some embodiments, homology arms have at least 95% sequence identity to a target locus. In some embodiments, homology arms have at least 99% sequence identity to a target locus. In some embodiments, homology arms have 100% sequence identity to a target locus.
  • constructs comprising homology arms provide rates of target site integration of at least 5%. In some embodiments, constructs comprising homology arms provide rates of target site integration of 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. In some embodiments, constructs comprising homology arms provide rates of target site integration of 30% or more. In some embodiments, constructs comprising homology arms provide rates of target site integration of 35% or more.
  • compositions disclosed herein direct increased genetic integration of a payload (e.g., a transgene) at a target locus (e.g., an endogenous gene).
  • a target locus e.g., an endogenous gene.
  • compositions provided herein direct increased genetic integration of a payload at a target locus in a specific cell type (e.g., naive B cells, B lineage cell populations, etc.).
  • methods provided herein direct increased genetic integration of a payload (e.g., expression cassette comprising a transgene encoding SPMD1, Factor IX, or bispecific T-cell engager (BiTE; e.g., blinatumomab)) at a target locus in a specific cell type (e.g, naive B cells, B lineage cell populations, etc.).
  • a payload e.g., expression cassette comprising a transgene encoding SPMD1, Factor IX, or bispecific T-cell engager (BiTE; e.g., blinatumomab)
  • a payload is or comprises a transgcnc or variant thereof.
  • methods and compositions provided herein direct increased genetic integration of a payload at a target locus that is considered a safe-harbor site (e.g., CCR5, AAVS1).
  • a target locus is selected from any genomic site appropriate for use with methods and compositions provided herein.
  • a target locus encodes a polypeptide.
  • a target locus encodes a polypeptide that is highly expressed in a subject (e.g., a subject not suffering from a disease, disorder, or condition, or a subject suffering from a disease, disorder, or condition).
  • a target locus is selected from one or more of CD 19, CD20, IGH, B2M, CCR5, JCHAIN, PAX5, IRF4, IRF8, BACH2, EZH2, XBP1, CARD11, PRDM1, and BAFF.
  • methods and compositions disclosed herein direct increased genetic integration of a payload at a target locus at a higher rate when compared to methods and compositions not including method described herein.
  • methods and compositions that lead to increased integration include, but are not limited to, serum variation, in particular, transferring and maintaining a B lineage cell population (e.g., an engineered B lineage cell population) in a cell culture media that varies in serum from initial media conditions for a first period of time followed by a step of changing back to the initial medium conditions for a second period of time.
  • a B lineage cell population e.g., an engineered B lineage cell population
  • methods and compositions described herein provide rates of target site integration of 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.
  • methods and compositions disclosed herein direct increased genetic integration of a payload, which provides higher rates of engraftment of an engineered B lineage cell population when administered to a subject.
  • a subject is a clinical species (e.g., human).
  • a subject is a non-clinical species (e.g., mouse model).
  • methods disclosed herein comprise measuring and/or monitoring engraftment of B lineage cells engineered with methods and compositions described herein.
  • a sample is collected from a subject administered with engineered B lineage cell population described herein.
  • sample collection is or comprises venipuncture.
  • sample collection is or comprises tissue collection.
  • levels of protein e.g., Factor IX, SMPD1, BiTE (e.g., blinatumomab), etc.
  • lipids are assessed from a collected sample.
  • engraftment of one or more B lineage cells of an engineered B lineage cell population described herein is measured in a non-clinical species (e.g., hlL6-/NOG mice). In some embodiments, engraftment is measured by use of bioluminescence. In some embodiments, engraftment is measured by use of ELISpot. In some embodiments, levels of plasma IgG are determined to measure engraftment. In some embodiments, levels of plasma IgM are determined to measure engraftment.
  • levels of a transgene are determined to measure engraftment.
  • engraftment of one or more B lineage cells of an engineered B lineage cell population described herein occurs in a non-clinical species (e.g., hIL6-/NOG mice) with or without preconditioning of a non-clinical subject (e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein).
  • engraftment of one or more B lineage cells of an engineered B lineage cell population described herein occurs in a clinical species (e.g., human subject) with or without preconditioning of a clinical subject (e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein).
  • a clinical species e.g., human subject
  • preconditioning of a clinical subject e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein.
  • B lineage cell culturing conditions can significantly affect normal human B lineage development and for production of mature Ig- secreting B cells.
  • B lineage cell activation and proliferation in vitro employed CD40L-expressing feeder cell layer systems. Such feeder cell systems were described as difficult to standardize and often unreliable for providing consistent levels of activation and proliferation of B lineage cells. Recent advances have shifted to protocols for in vitro activation and proliferation of B lineage cells in specific culture systems comprising cytokines or other components in the absence of feeder cells (See Jourdan et al., 2009 and Hartweger et al., 2019, each of which is incorporated herein by reference in its entirety).
  • methods for B lineage cell activation comprise contacting cells with media comprising one or more components of the present disclosure.
  • methods for B lineage cell activation comprise contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG).
  • cytokines and/or oligonucleotides e.g., multimeric human CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG.
  • methods for B cell activation comprise contacting cells with media comprising at least about 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 150 ng/mL, 200 ng/mL, or 500 ng/mL of one or more cytokines and/or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) .
  • cytokines and/or oligonucleotides e.
  • methods for B lineage cell activation comprise contacting cells with media comprising at least about 0.1 ug/mL, 0.2 ug/mL, 0.3 ug/mL, 0.4 ug/mL, 0.5 ug/mL, 0.6 ug/mL, 0.7 ug/mL, 0.8 ug/mL, 0.9 ug/mL, 1 ug/mL, 1.5 ug/mL, 2 ug/mL, 2.5 ug/mL, 3 ug/mL, 3.5 ug/mL, 4 ug/mL, 4.5 ug/mL, or 5 ug/mL of one or more cytokines and/or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG).
  • cytokines and/or oligonucleotides e.g., multimeric human
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, 4, or 5 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least about 1 , 2, 3, 4, or 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion.
  • media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion for at least about 1, 2, 3, 4, 5, 6, 7, or 8 days.
  • media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days, followed by a step of B lineage cell expansion for at least about 1, 2, 3, 4, 5, 6, 7, or 8 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • methods described herein result in an activated B lineage cell population.
  • the present application provides, amongst other things, methods that result in enhanced genetic integration in B lineage cell populations.
  • the application of these methods may further lead to increased engraftment of an engineered B lineage cell population in a subject (e.g., a human subject).
  • Increased engraftment of an engineered B lineage cell population in a subject has larger ramification for cell therapy, such as treatment of disorders including, without limitation, hemophilia B and Niemann Pick Disease type B.
  • Such methods to enhance genetic integration and increase engraftment include, but are not limited to, serum variation methods described herein.
  • B lineage cells are isolated and cultured in media comprising one or more cytokines and/or oligonucleotides (e.g.,CD40L, IL-2, IL- 10, IL- 15, IL- 21 and/or CpG) for at least about 1, 2, 3, 4, or 5 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG
  • a B lineage cell population is contacted with cell culture media that varies in serum (e.g., lacks serum or is substantially free of serum) from initial media conditions during or after gene engineering.
  • cell culture medium varies in (e.g., lacks or is substantially free of) one or more of: human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, or chicken serum.
  • a B lineage cell population undergoes a wash in base media that varies in serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof) from initial media conditions and lacks any added cytokines and/or oligonucleotides during or immediately after gene engineering.
  • serum e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof
  • media that varies in serum e.g., lacks or is substantially free of serum
  • a B lineage cell population, post-gene engineering is contacted with media that varies in serum (e.g., lacks or is substantially free of serum) from initial media conditions, and then incubated for at least 1 day.
  • a B lineage cell population, post-gene engineering is contacted with media that varies in serum (e.g., lacks or is substantially free of serum) from initial media conditions and comprises one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG).
  • a B lineage cell population after incubation, are spun down, supernatant removed, and re-plated in media with initial serum conditions (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof).
  • initial serum conditions e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof.
  • B lineage cells are replated in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) and expanded for at least 1, 2, 3, 4, or 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • B lineage cells are contacted with cell culture media that varies in plasma (e.g., lacks plasma or is substantially free of plasma) from initial media conditions during gene editing.
  • B lineage cells are contacted with cell culture media that varies in plasma (e.g., lacks plasma or is substantially free of plasma).
  • plasma comprises or is human plasma, bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma, or a combination thereof.
  • B lineage cells are contacted with cell culture media that varies in plasma derived from bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma (e.g., lacks plasma or is substantially free of plasma).
  • a B lineage cell population after incubation, is spun down, supernatant removed, and re-plated in media with initial plasma conditions (e.g., human plasma, bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma, or a combination thereof).
  • initial plasma conditions e.g., human plasma, bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma, or a combination thereof.
  • B lineage cells are replated in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) and expanded for at least I, 2, 3, 4, or 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • B lineage cells are contacted with cell culture media that varies in one or more plasma or serum components (e.g., proteins).
  • B lineage cells are contacted with cell culture media that lacks one or more proteins or is substantially free of one or more proteins from plasma or serum (e.g., human plasma or human serum) from initial media conditions during gene editing.
  • B lineage cells are contacted with cell culture media that varies in one or more recombinant proteins (e.g., lacks one or more recombinant proteins or is substantially free of one or more recombinant proteins).
  • recombinant proteins are or comprise recombinant proteins from plasma (e.g., human plasma, bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma, or a combination thereof).
  • recombinant proteins are or comprise recombinant proteins made from Chinese Hamster Ovary (CHO) cells or E. coli.
  • recombinant proteins include, but are not limited to insulin, transferrin, and albumin. In some embodiments, recombinant proteins are derived from proteins present in human plasma or human serum.
  • B lineage cells are contacted with cell culture media that comprises one or more recombinant proteins made from CHO cells or E. coli, while lacking other recombinant proteins derived from human plasma or human serum (e.g., substantially similar to or identical to proteins found in human plasma or human serum).
  • B lineage cells are contacted with cell culture media that comprises one or more recombinant proteins made from CHO cells or E.coli, rather than from whole human plasma or human serum.
  • B lineage cells are contacted with cell culture media that comprises one or more recombinant proteins purified from human plasma.
  • Plasmablasts are short-lived, rapidly produced effector cells that are primarily present in an early antibody response and are one potential product of terminal B cell differentiation.
  • Plasmablasts arc capable of secreting antibodies, including IgM subtype antibodies, in order to mount an immediate response to certain antigens in the body.
  • Differentiation of B cells into plasmablasts in vitro may be promoted through use of certain signaling molecules, including one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15).
  • cytokines e.g., IL-2, IL-6, IL- 10, and/or IL- 15.
  • a variety of methods for differentiation of B cells into plasmablasts are known, but not limited to, those outlined in WO/2018/170150, incorporated herein by reference in its entirety. Plasmablasts produced by such methods may be characterized as cells that are CD27 + 1 CD38 + / CD138'.
  • methods for B cell differentiation into plasmablasts comprise contacting cells with media comprising one or more components.
  • methods for B cell differentiation into plasmablasts comprise contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15).
  • cytokines e.g., IL-2, IL-6, IL- 10, and/or IL- 15.
  • methods for B cell differentiation into plasmablasts comprise contacting activated B lineage cells with media comprising at least about 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 3.5 ng/mL, 4 ng/mL, 4.5 ng/mL, 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, or ng/mL of one or more cytokines (e.g., IL-2,
  • methods for B cell differentiation into plasmablasts comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days.
  • methods for B cell differentiation into plasmablasts comprise a step of contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15) for at least about 1, 2, 3, or 4 days.
  • cytokines e.g., IL-2, IL-6, IL- 10, and/or IL- 15
  • methods for B cell differentiation into plasmablasts comprise a step of contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL- 10, and/or IL- 15) for at least 3 days.
  • methods for B cell differentiation into plasmablasts comprise a step of contacting activated B lineage cells with media comprising one or more cytokines (e.g., IL-2, IL-6, IL-10, and/or IL-15) for at least 3 days, followed by a step of plasmablast expansion.
  • Plasmablasts secrete more antibodies than naive B cells, they arc shorter-lived and secrete fewer antibodies than plasma cells (PCs).
  • Long-lived plasma cells (LLPCs, used interchangeably throughout with plasma cells) localize to bone marrow in the body and are capable of secreting high levels of antibodies and surviving for decades in the absence of proliferation (See, Hammerland et al., 2017 and Khodadadi et al., 2019, both of which incorporated herewith in their entirety).
  • Differentiation of plasmablasts to long-lived plasma cells can be triggered by certain events in the body, including, e.g., activity of transcription factors Blimp- 1/PRDM1 and IRF4.
  • Plasmablasts to plasma cells in vitro may be promoted through use of certain signaling molecules, including one or more cytokines (e.g., IL- 6, IL-15, and/or IFNa-2P).
  • cytokines e.g., IL- 6, IL-15, and/or IFNa-2P.
  • a variety of methods for differentiation of plasmablasts into plasma cells are known, but not limited to, those outlined in Jourdan et al. 2019 and WO/2018/170150 (each of which incorporated herein by reference in its entirety).
  • Plasma cells produced by such methods may be characterized as cells that are CD27 + / CD38 + / CD138 + .
  • methods for plasmablast differentiation into plasma cells comprise contacting cells with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-20). In some embodiments, methods for plasmablast differentiation into plasma cells comprise contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-20).
  • cytokines e.g., IL-6, IL- 15, and/or IFNa-20.
  • methods for plasmablast differentiation into plasma cells comprise contacting plasmablasts with media comprising at least about 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 3.5 ng/mL, 4 ng/mL, 4.5 ng/mL, 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, or ng/mL of one or more cytokines (e.g., IL-6, IL-15, and
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL-15, and/or IFNa-2 ) for at least about 1, 2, 3, or 4 days.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-20) for at least 3 days.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P) for at least 3 days, followed by a step of cell isolation.
  • methods for plasmablast differentiation into plasma cells comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P) for at least 3 days, followed by a step of administration to a subject.
  • engineered cell preparations comprising populations of cells modified to perform one or more desired functions.
  • engineered cell preparations are compositions comprising genetically modified immune cell populations (e.g., B cell, T cell).
  • engineered cell preparations are compositions comprising genetically modified B lineage cell populations (e.g., B cell, plasmablast, plasma cell).
  • engineered cell preparations are compositions comprising genetically modified plasmablast cell populations.
  • engineered cell preparations are compositions comprising genetically modified plasma cell populations.
  • engineered cell preparations are genetically modified to express a payload (e.g., transgcnc) of interest.
  • engineered cell preparations are genetically modified to express a transgene from an expression cassette (e.g., comprising additional polynucleotide sequence elements).
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.). In some embodiments, engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus. In some embodiments, engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus under control of an endogenous promoter.
  • a transgene of interest e.g., therapeutic protein, antibody, etc.
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus under control of an exogenous promoter.
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus without disrupting endogenous gene expression and/or function.
  • engineered cell preparations comprise genetically modified cells that express a transgene of interest (e.g., therapeutic protein, antibody, etc.) from an endogenous gene locus and partially or fully disrupt endogenous gene expression and/or function.
  • engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more promoters. In some embodiments, engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more enhancers. In some embodiments, engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more terminators.
  • engineered cell preparations are genetically modified to express a transgene from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more homology arms. In some embodiments, engineered cell preparations are genetically modified to express a transgcnc from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding one or more promoters, one or more enhancers, one or more terminators, and/or one or more homology arms.
  • engineered cell preparations are genetically modified to express a transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a WPRE enhancer, a BGH poly A, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express a transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a BGH poly A, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express a transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a WPRE enhancer, a SV40 polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express a transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a MND promoter, a SV40 polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express a transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a EF- la promoter, a WPRE enhancer, a BGH polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an SMPD1 transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a EF-la promoter, a BGH polyA, a 5’ homology arm, and a 3’ homology arm.
  • engineered cell preparations are genetically modified to express an SMPD1 transgene, or variant thereof, from an expression cassette, wherein the expression cassette further comprises one or more polynucleotide sequences encoding a EF-la promoter, a SV40 polyA, a 5’ homology arm, and a 3’ homology arm.
  • a B lineage cell preparation comprises both engineered and non-engineered cells.
  • an engineered B lineage cell population comprises plasmablasts.
  • an engineered B lineage cell population comprises plasma cells.
  • an engineered B lineage cell population comprises long-lived plasma cells.
  • an engineered B lineage cell population comprises plasmablasts, plasma cells, and/or long-lived plasma cells and/or any mixtures or combinations thereof disclosed herein.
  • an engineered B lineage cell preparation comprises plasmablasts.
  • plasmablasts are rapidly produced and short-lived effector cells of the early antibody response.
  • Plasmablasts can be generated from activated B lineage cells using methods described herein.
  • engineered plasmablasts may be generated from engineered activated B lineage cells.
  • plasmablast cell populations may be contacted with media comprising one or more components of the present disclosure (e.g., IL-6, IL-15, and/or IFNa-2P) in order to initiate differentiation into plasma cell populations.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-20).
  • methods for a plasmablast cell population differentiation into a plasma cell population comprise contacting plasmablast cell population with media comprising at least about 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 3.5 ng/mL, 4 ng/mL, 4.5 ng/mL, 5 ng/mL, 10 ng /mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, or ng/mL of one or more cytokines (e.g.,
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting cells with media comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL-15, and/or IFNa-2P) for at least about 1, 2, 3, or 4 days.
  • cytokines e.g., IL-6, IL-15, and/or IFNa-2P
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL-15, and/or IFNa-20) for at least 3 days.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablast cell population with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-20) for at least 3 days, followed by a step of cell isolation.
  • methods for plasmablast cell population differentiation into a plasma cell population comprise a step of contacting plasmablasts with media comprising one or more cytokines (e.g., IL-6, IL- 15, and/or IFNa-2P) for at least 3 days, followed by a step of administration to a subject.
  • cytokines e.g., IL-6, IL- 15, and/or IFNa-2P
  • an engineered B lineage cell preparation comprises a plasma cell population.
  • a plasma cell population comprises a predetermined engineered plasma cell precursors (e.g., plasma blasts) that, upon administration to a subject, further differentiates into a mature plasma cell population.
  • a mature plasma cell population comprises quiescent, non-dividing cells of the humoral immune response that are capable of secreting large amounts of antibodies.
  • a mature plasma cell population can comprise short-lived plasma cells and/or long-lived plasma cells (LLPCs) and/or any combination thereof.
  • the present disclosure provides various methods for characterization of engineered cell populations (e.g., B lineage cell populations).
  • methods disclosed herein are used to segregate and/or characterize naive B cell subpopulations within a B lineage cell population.
  • naive B cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic- activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic- activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing naive B cell subpopulations are employed during each step of a controlled cooling method.
  • methods of characterization are employed to segregate and/or characterize engineered naive B cell subpopulations.
  • engineered naive B cell subpopulations are characterized through one or more of flow cytometry, fluorescence- activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence- activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing engineered naive B cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize activated B lineage cell subpopulations within a B lineage cell population.
  • activated B lineage cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing activated B lineage cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered activated B lineage cell subpopulations.
  • engineered activated B lineage cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered activated B lineage cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize plasmablast cell subpopulations within a B lineage cell population.
  • plasmablast cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography e.g., affinity chromatography.
  • one or more methods of characterizing plasmablast cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered plasmablast cell subpopulations.
  • engineered plasmablast cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered plasmablast cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize plasma cell precursor subpopulations within a B lineage cell population.
  • plasma cell precursor subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography e.g., affinity chromatography.
  • one or more methods of characterizing plasma cell precursor subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered plasma cell precursor subpopulations.
  • engineered plasma cell precursor subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACs), magnetic-activated cell sorting (MACs), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered plasma cell precursor subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize plasma cell subpopulations within a B lineage cell population.
  • plasma cell subpopulations may be characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography chromatography
  • one or more methods of characterizing plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered plasma cell subpopulations.
  • engineered plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize short-lived plasma cell populations within a B lineage cell population.
  • short-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing short-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered short-lived plasma cell subpopulations.
  • engineered short-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography. In some embodiments, one or more methods of characterizing engineered short-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein may be used to segregate and/or characterize long-lived plasma cell subpopulations with a B lineage cell population.
  • long-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography affinity chromatography
  • one or more methods of characterizing long-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • methods disclosed herein are used to segregate and/or characterize engineered long-lived plasma cell subpopulations.
  • engineered long-lived plasma cell subpopulations are characterized through one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and/or affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • affinity chromatography e.g., affinity chromatography
  • one or more methods of characterizing engineered long-lived plasma cell subpopulations are employed during each step of a controlled cooling method.
  • the present disclosure provides methods of treating a disease, disorder, or condition (c.g., a disease, disorder, or condition described herein) in a subject comprising administering a pharmaceutical composition described herein.
  • a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder.
  • Pharmaceutical compositions described herein can be for use in the manufacture of a medicament for treating a disease, disorder, or condition (e.g., a disease, disorder, or condition described herein) in a subject.
  • compositions described herein can comprise one or more B lineage cells selected from a population of genetically modified B lineage cells described herein.
  • B lineage cell populations are engineered to express or comprise a payload.
  • a payload is or comprises an expression cassette.
  • an expression cassette comprises a transgene (e.g., encoding SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.).
  • a subject to be treated with methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having, or at risk of having, a disease, disorder, or condition described herein. ).
  • a subject has NPD (e.g., NPD type B).
  • a subject has Hemophilia B.
  • a subject can be an adult subject.
  • engineered B lineage cells are administered to a pediatric subject.
  • compositions described herein may be carried out in any convenient manner (e.g., injection, ingestion, transfusion, inhalation, implantation, or transplantation).
  • a pharmaceutical composition described herein is administered by injection or infusion.
  • Pharmaceutical compositions described herein may be administered to a subject intravenously transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intraperitoneally.
  • a pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly).
  • a pharmaceutical composition described herein is administered by intravenous infusion or injection.
  • a pharmaceutical composition described herein is administered by intramuscular or subcutaneous injection.
  • a pharmaceutical composition described herein is administered directly to central nervous system (CNS) tissue.
  • CNS central nervous system
  • a pharmaceutical composition described herein is administered at a pharmaceutically suitable dosage to a subject. In some embodiments, a pharmaceutical composition described herein is administered monthly. In some embodiments, a pharmaceutical composition described herein is administered once every other month. In some embodiments, a pharmaceutical composition described herein is administered once every three months. In some embodiments, a pharmaceutical composition described herein is administered once every six months. In some embodiments, a pharmaceutical composition described herein is administered once a year. In some embodiments, a pharmaceutical composition described herein is administered to a subject having Niemann Pick Disease type B. In some embodiments, a pharmaceutical composition described herein is administered to a subject with mild-to-severe Niemann Pick Disease type B. In some embodiments, a pharmaceutical composition described herein is administered to a subject with acid sphingomyelinase deficiency. In some embodiments, a pharmaceutical composition described herein is administered to a subject having Hemophilia B .
  • the methods disclosed herein comprise measuring and/or monitoring treatment.
  • a sample is collected from a subject treated with a pharmaceutical composition described herein.
  • sample collection is or comprises venipuncture.
  • sample collection is or comprises tissue collection.
  • levels of protein e.g., SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.
  • protein e.g., SMPD1, Factor IX, bispecific T-cell engager (BiTE; e.g., blinatumomab), etc.
  • lipids are assessed from a collected sample.
  • engraftment of one or more B lineage cells of a population of genetically modified B lineage cells described herein is measured in a non-clinical species (e.g., hIL6-/NOG mice). In some embodiments, engraftment is measured by use of bioluminescence. In some embodiments, engraftment is measured by use of ELISpot. In some embodiments, levels of plasma IgG are determined to measure engraftment. In some embodiments, levels of plasma IgM are determined to measure engraftment.
  • levels of a transgene arc determined to measure engraftment.
  • engraftment of one or more B lineage cells of an engineered B lineage cell population described herein occurs in a non-clinical species (e.g., hIL6-/NOG mice) with or without preconditioning (e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein).
  • engraftment of one or more B lineage cells of an engineered B lineage cell population described herein occurs in a clinical species (e.g., human subject) with or without preconditioning (e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein).
  • preconditioning e.g., chemotherapy, immunosuppressive treatment, etc. of a subject prior to administering B lineage cells as described herein.
  • B lineage cell populations can comprise or be B lineage cell populations.
  • B lineage cell populations comprise or be naive B cells.
  • B lineage cell populations comprise or be activated B lineage cells.
  • B lineage cell populations may comprise or be plasmablast.
  • B lineage cell populations may comprise or be plasma cells.
  • B lineage cells are isolated and cultured in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL- 21 and/or CpG) for at least about 1, 2, 3, 4, or 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL- 21 and/or CpG
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days.
  • methods for B lineage cell activation comprise a step of contacting cells with media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL- 10, IL- 15, IL-21 and/or CpG) for at least 2 days, followed by a step of gene editing.
  • B lineage cells are contacted with cell culture media that varies in serum (e.g., lacks serum or is substantially free of serum) from initial media conditions during gene editing.
  • serum comprises or is human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof.
  • B lineage cells are contacted with cell culture media that is substantially free of fetal bovine serum.
  • B lineage cells are contacted with cell culture media that is substantially free of human serum.
  • B lineage cells are contacted with cell culture media that is substantially free of human plasma.
  • B lineage cells are contacted with cell culture media that comprises one or more recombinant proteins (e.g., transferrin, insulin, albumin, etc.).
  • B lineage cells arc contacted with cell culture media that comprises one or more recombinant proteins, and also lacks (e.g., whole, purified, etc.) human plasma or human serum.
  • B lineage cells are contracted with cell culture media that comprises one or more recombinant proteins purified from human plasma in combination with one or more recombinant proteins made from CHO cells or E. coli.
  • B lineage cells subsequently undergo a wash step with base media (e.g., Excellerate) with or without cytokines.
  • base media e.g., Excellerate
  • B lineage cells are contacted with media that varies in serum (e.g., lacks serum or is substantially free of serum) and then incubated at 37°C for up to 24 hours.
  • B lineage cells after incubation arc spun down, supernatant removed, and replated in media comprising initial conditions of serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof).
  • B lineage cells arc rc-platcd in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) and expanded for at least 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • B lineage cells subsequently undergo a wash step with base media e.g., Excellerate) with or without cytokines.
  • base media e.g., Excellerate
  • B lineage cells are contacted with media that varies in plasma (e.g., lacks human plasma or is substantially free of plasma) and then incubated at 37°C for up to 24 hours.
  • B lineage cells after incubation arc spun down, supernatant removed, and replated in media comprising initial conditions of plasma (e.g., human plasma, bovine plasma, horse plasma, newborn calf plasma, goat plasma, rabbit plasma, porcine plasma, chicken plasma, or a combination thereof).
  • B lineage cells are re-plated in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL- 21 and/or CpG) and expanded for at least 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL- 21 and/or CpG
  • B lineage cells subsequently undergo a wash step with base media e.g., Excellerate) with or without cytokines.
  • base media e.g., Excellerate
  • B lineage cells are contacted with media that varies in recombinant proteins (e.g., lacks recombinant proteins or is substantially free of recombinant proteins) and then incubated at 37°C for up to 24 hours.
  • B lineage cells after incubation are spun down, supernatant removed, and re-plated in media comprising initial conditions of recombinant proteins (e.g., human recombinant proteins, bovine recombinant proteins, horse recombinant proteins, newborn calf recombinant proteins, goat recombinant proteins, rabbit recombinant proteins, porcine recombinant proteins, chicken recombinant proteins, or a combination thereof).
  • recombinant proteins e.g., human recombinant proteins, bovine recombinant proteins, horse recombinant proteins, newborn calf recombinant proteins, goat recombinant proteins, rabbit recombinant proteins, porcine recombinant proteins, chicken recombinant proteins, or a combination thereof.
  • B lineage cells are re -plated in media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) and expanded for at least 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • One such example of culturing methods described herein, but not limited to, involves isolation of B lineage cells followed by contacting them in activation media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days followed by gene editing.
  • activation media comprising one or more cytokines and/or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG) for at least 2 days followed by gene editing.
  • B lineage cells undergo a wash step and are further contacted with media that varies in bovine serum (e.g., lacks serum or is substantially free of scrum). Then, B lineage cells arc incubated at 37°C for up to 24 hours.
  • B lineage cells are spun down, supernatant removed, and re-plated in media comprising initial conditions of serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof) for at least 5 days.
  • serum e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof
  • ICE Forward primer GCAGCAAACCTTCCCTTCACTAC (SEQ ID NO: 1)
  • ICE Reverse primer AGGATTCCCGAGTAGCAGATGAC (SEQ ID NO: 2)
  • ICE Sequencing primer GGGTGGAACAAGATGGATTATC (SEQ ID NO: 3)
  • B2M Forward primer AGGACCTTCTCTGAGCTGTC (SEQ ID NO: 10)
  • B2M Reverse/Sequencing primer GCCCTAAACTTTGTCCCGAC (SEQ ID NO: 11)
  • JCHAIN_2 Forward/Sequencing Primer CATGACAAGTGTCAGATGTCTG (SEQ ID NO: 12)
  • JCHAIN_2 Reverse Primer CCAATGCAITCCATGTTGTAC (SEQ ID NO: 13)
  • JCHAIN_31 Forward/Sequencing Primer TTCAGACTTCAACCACAGTTG (SEQ ID NO: 14)
  • JCHAIN_31 Reverse Primer AACC ACAGACTCAGTGATAGG (SEQ ID NO:
  • JCHAIN_54 Forward Primer TTGGCA1 TCAI GAAAGGCC (SEQ ID NO:
  • JCHAIN Reverse/Sequencing Primer AAAAGCCCTGCHTTCAAATTCATTG (SEQ ID NO: 17)
  • % Targeted integration was then calculated using the above equation based on the adjusted thresholding and the copies/uL of FAM and HEX.
  • CCR5 ddPCR Forward primer catcgcattgtctgagtagg (SEQ ID NO: 4)
  • CCR5 ddPCR Reverse primer CAGTGGATCGGGTGTAAAC (SEQ ID NO: 5)
  • CCR5_ddPCR Probe FAM: TCGGGAGCCTCTTGCTGGAAAATAGAA (SEQ ID NO: 6)
  • CCRL2_ddPCR Forward primer CCACATCAGAAGGAAGACTAC (SEQ ID NO: 7)
  • CCRL2_ddPCR Reverse primer GCTGTATGAATCCAGGTCC (SEQ ID NO: 8)
  • CCRL2_ddPCR Probe (HEX): TGTTTCCTCCAGGATAAGGCAGCTGT (SEQ ID NO: 9)
  • JCHAIN_2 reference gene primers and probes were used:
  • JCHAIN_2 ddPCR Forward primer CATCGCATTGTCTGAGTAGG (SEQ ID NO: 18)
  • JCHAIN_2 ddPCR Reverse primer GGCCTATTCTTAATCACATTTCTG (SEQ ID NO: 19)
  • JCHAIN_2 Probe TCATCTTGAAATAAGAACCACAGACTCAGTGA (SEQ ID NO: 20) Cell count and viability
  • Cells were sampled for the CellacaMx counter after thorough resuspension by gentle pipetting. Cells were gently mixed with the appropriate diluent of AOPI stain, loaded into a Nexcelom count plate, and analyzed for count and viability based on the acquired image and dilution factor.
  • IgM/lgG profiling intracellular staining was performed using cell fixation and permeabilization. Following the last wash from viability staining, cells were suspended in fixation buffer (4% paraformaldehyde) and incubated at room temperature for 20 minutes. Cells were then washed in Permeabilization Buffer (eBioscience) three times, then resuspended in Ig profiling antibody master mix (dilutions used were manufacturer’s recommendation). Samples were incubated 20 minutes at room temperature protected from light, then washed 2 times with permeabilization buffer. Pellets were suspended in staining buffer as for fresh cell staining and run with forward/sidc-scattcr gating adjusted for cell shrinkage after fixation.
  • fixation buffer 4% paraformaldehyde
  • 96 well multiscreen filter plates were briefly washed with 35% ethanol for 40-60 seconds until the membrane turned gray from white. Plates were then washed 3 times with Dulbecco’s PBS (dPBS), and subsequently coated with the capture antibody (GMA102) at 30 pg/mL in PBS overnight at 4°C. The next day, plates were washed 3 times with an automated plate washer. The antibody coated plates were then blocked against non-specific binding with Iscovc's Modified Dulbccco's Medium (IMDM) with 1% FBS for 2 hours at 37°C. Next, cells were counted and added to the wells of the plate. The plates were incubated in an incubator at 37°C for 16-24 hours.
  • IMDM Iscovc's Modified Dulbccco's Medium
  • Immunoglobulin G IgG
  • Immunoglobulin M IgM
  • Plate-bound hlgG or hlgM was then captured by secondary antibody, goat anti-IgG polyclonal antibody HPR or goat anti-IgM polyclonal antibody HPR, respectively, at 1:5000 dilution for 1 hour at room temperature.
  • TMB two-part substrate was added for 1 min (development time to be determined empirically) and then the reaction was stopped.
  • the plate absorbance was read at 450 nm and 570 nm by a Cytation5 Plate Reader. Sample values were interpolated using the standards at known human IgG or human IgM concentrations.
  • Engineered B lineage cell population supernatant was harvested from relevant experiments and frozen at -80°C prior to testing. Samples were then tested for concentrations of human Factor IX (hFIX) in the 96 well FIX ELISA (Abcam-ablO8831). Briefly, all kit components, samples, and standards were thawed and allowed to equilibrate to room temperature. 50 pL of sample or standard were added per well of pre-coated plates and then incubated at room temperature for 2 hours. After incubation, plates were washed 5 times with 200 pL of lx Wash Buffer.
  • hFIX human Factor IX
  • Sandwich ELISA with commercially available antibodies and solutions was used for analysis with a specialized protocol as described herein.
  • primary antibody being mouse monoclonal anti-human SMPD1, R&D cat#MAB5348; secondary antibody being goat polyclonal biotinylated anti-SMPDl, R&D AF5348, poly Streptavidin HRP Pierce N200
  • secondary antibody being goat polyclonal biotinylated anti-SMPDl, R&D AF5348, poly Streptavidin HRP Pierce N200
  • wild type (WT) and SMPD1 knockout (KO) HAP1 cell were seeded in 12-well plate and treated with engineered B lineage cell population supernatants for 48 hours while incubated at 37 °C, 5% CO2. Cells were lysed using frcczc-thaw cycles and the protein concentration was normalized across samples using a BCA assay (Pierce Rapid Gold BCA assay). Substrate (6-Hexadecanoylamino-4-methylumbelliferyl phosphorylcholine) was added to the proteins for 18 hours at 37°C. Next, the reaction was stopped and the plate was read on CytationS fluorescence spectrophotometer with excitation at 350; 20 nm and emission at 450; 20 nm.
  • HAP1 SMPD1 KO and WT cells were incubated with BODIPY-Sphingomyelin for 30 minutes at 4°C, and then were washed once with HEPES-HBSS (Gibco). HAP1 SMPD1 KO and WT cells were then incubated for 24 hours with either recombinant human SMPD1 (rhSMPDl) or with supernatant from the engineered B lineage cell population as presently disclosed. Next, cells were imaged on CytationS at 40X using a GFP fluorescent filter and bright field as a control. Images were processed using ImageJ.
  • SM cytosolic and lysosomal sphingomyelin
  • HAP1 cells 1.5 x 10 6
  • rhSMPDl recombinant human SMPD1
  • supernatant from engineered B lineage cell populations for 24 hours. Cells were then collected and frozen down in liquid nitrogen before undergoing liquid chromatography-mass spectrometry.
  • anti-tumor activity of engineered B lineage cell populations were analyzed.
  • B lineage cells were isolated, cultured, edited, expanded, and differentiated using methods described herein. Supernatant was collected from both engineered and non-engineered B lineage cell populations. Supernatant was then added to plates containing both CD8 + T cells and GFP stably expressing Raji (human B lymphoblastoid) cells. After 72 hours of incubation, the Raji cells analyzed by flow cytometry for amount of cellular death (percent loss of GFP expressing population).
  • B lineage cell populations were cultured and engineered as described above. On Day 13 of culture, lOOng/ml of menadione sodium bisulfite was added to B lineage cells over a time course of about 24 hours. Harvested supernatant was purified using an FIX affinity resin. Purified FIX was then spiked into FIX-deficient plasma. Clotting times were generated on the Stage Satellite, recorded, and interpolated against an activity curve generated with normal pooled plasma and the same FIX-deficient plasma. Corresponding clotting times were converted to percent activity using a log-log regression. This results in % activity and lU/mL and lU/mg which was then recorded accordingly.
  • Example 2 Engineered B lineage cell populations are able to expand, differentiate, and retain functionality
  • B lineage cell populations can be engineered with one or more expression cassettes (e.g., luciferase containing expression cassette) and may integrate a polynucleotide sequence encoding for a transgene and express said transgene (e.g., luciferase). Further, using methods described herein, these engineered B lineage cell populations may differentiate and comprise antibody- secreting cells (ASCs, e.g., plasma cells, plasmablasts, activated B lineage cells) ( Figure 1).
  • ASCs antibody- secreting cells
  • B lineage cells were isolated, expanded, and differentiated using the culturing methods described herein and were analyzed for antibody-secreting cell growth (e.g., plasma cells, plasma blasts, activated B lineage cells) over time using flow cytometry ( Figures 2A-B) at days 1, 8, 11, and 14. Furthermore, B lineage cell populations from six blood donors were isolated and engineered with or without a luciferase expression cassette with a CCR5-targeting guide RNA. These B lineage cell populations were further expanded and differentiated as described above. On day 13, both non-engineered (control) and engineered B lineage cell populations were assessed by flow cytometry for percentage of living antibody- secreting cells (e.g., plasma cells).
  • antibody-secreting cell growth e.g., plasma cells, plasma blasts, activated B lineage cells
  • Figures 2A-B flow cytometry
  • B lineage cell populations described herein can be engineered and differentiated into an engineered B lineage cell population that comprises plasma cells (interchangeable referred to herein as “ASCs”).
  • engineered B lineage cell populations may express and/or secrete one or more transgenes (e.g., luciferase).
  • transgenes e.g., luciferase
  • engineered B lineage cell populations described herein comprise increased plasma cell content when compared to a reference condition.
  • engineered B lineage cell populations produced using engineering methods described herein have relatively high incidence of transgene integration (e.g., percentage HDR, etc.).
  • engineered B lineage cell populations as described herein produce and secrete a product (e.g., polypeptide, protein, cytokine, etc.) encoded by a transgene (e.g., luciferase).
  • a product e.g., polypeptide, protein, cytokine, etc.
  • a transgene e.g., luciferase
  • culturing and engineering of B lineage cells as described herein results in significant plasma cell content (>40% of total B lineage cell population).
  • Example 3 Engineered B cell populations can engraft and persist over a relatively long period of time
  • B lineage cell populations can be engineered with one or more expression cassettes (e.g., luciferase containing expression cassette) and may integrate a polynucleotide sequence encoding for a transgene and express said transgene (e.g., luciferase) at a desired locus (e.g., CCR5).
  • these engineered B lineage cell populations may differentiate and comprise antibody- secreting cells (ASCs, e.g., plasma cells, plasmablasts, activated B lineage cells).
  • ASCs antibody- secreting cells
  • Subjects treated with engineered cell populations described herein may demonstrate persistent levels of transgene expression (e.g., luciferase) over an extended period of time (e.g., one or more weeks, one or more months, etc.)
  • B lineage cells were engineered with a luciferase expression cassette and with culturing conditions described herein (e.g., including a step of serum variation). Next, B lineage cell populations were expanded and differentiated using methods described herein with a CCR5 guide RNA. Droplet digital polymerase chain reaction (ddPCR) was performed on these engineered B lineage cells to assess for percentage indels and homology-directed repair using luciferase expression at CCR5 ( Figures 3A-B).
  • ddPCR Droplet digital polymerase chain reaction
  • Non-engineered, PBS control, or engineered B lineage cells using methods described above were administered to an immunocompromised mouse model (hlL6-/NOG mice). Mice were imaged (Figure 4A) and assessed for bioluminescence at 8, 15, 22, 29, 35, 44, 58, 93, 104, and 125 days post administration ( Figure 4B).
  • B lineage cell populations engineered and cultured using methods disclosed herein may provide enhanced engraftment and/or transgene expression (e.g., luciferase) that is comparable or not significantly reduced as compared to a reference condition after administration to a subject.
  • engineered B lineage cell populations described herein may provide improved engraftment and/or transgene expression as compared to a reference condition.
  • engineered B lineage cell populations described herein may engraft and/or express transgene for extended durations (e.g., greater than about 44 days).
  • Example 4 Engineered B cell populations using methods described herein have genetic integration and secrete functional protein [0249]
  • the present disclosure provides, inter alia, culturing methods (e.g, including a scrum variation step) that can be used to produce an engineered B lineage cell population with increased genetic integration of a transgene at a desired locus when compared to a reference condition (e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a serum variation step).
  • a reference condition e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a serum variation step.
  • the present disclosure provides methods that result in engineered B lineage cell populations that can express and/or secrete functional protein.
  • the present example provides B lineage cells produced using methods disclosed herein and engineered with a variety of different transgenes.
  • transgene products include, but are not limited to, an anti-CD19/anti-CD3 bispecific T-cell engager (BiTE), Factor IX, and acid sphingomyelinase (interchangeably referred to herein as “‘sphingomyelin phosphodiesterase 1” and“SMPDl”).
  • B lineage cells were isolated, activated, and engineered with an anti-CD19/anti- CD3 BiTE (Blinatumomab) encoding expression cassette and CCR5 targeting RNP.
  • B lineage cells then underwent serum variation (e.g., included a step of serum variation) for 24 hours, then were expanded and differentiated as described supra.
  • On-target integration frequency was measured by ddPCR ( Figure 5 A), and production of an anti-CD19/CD3 BiTE was measured via sandwich ELISA and Jurkat T cell reporter assay ( Figure 5B).
  • B lineage cells were isolated, activated, engineered with human SMPD1 containing expression cassette (interchangeable also referred to as acid sphingomyelinase, “ASM”) and CCR5 targeting RNP.
  • B lineage cells underwent serum variation for 24 hours, then were expanded and differentiated using methods described herein.
  • on-target integration frequency at CCR5 was measured by ddPCR ( Figure 6A) and SMPD1 secretion was measured by ELISA ( Figure 6B).
  • SMPD1 knockout (KO) HAP1 cells populations were seeded in 12- well plate and treated with either recombinant human SMPD1 (500 ng/ml or 1000 ng/ml rhSMPDl), nonengineered B lineage cell population supernatant (gCCR5 RNP only, no donor expression cassette) or engineered B lineage cell population supernatant.
  • Engineered B lineage cell populations were engineered with SMPD1 containing expression cassettes using methods disclosed herein.
  • cells were lysed using freeze-thaw cycles and the protein concentration was normalized across samples using a BCA assay (Pierce Rapid Gold BCA assay).
  • Substrate (6-Hexadecanoylamino-4-methylumbelliferyl phosphorylcholine) was added to the proteins for 18 hours at 37°C. Next, the reaction was stopped, and the plate was read on Cytation5 fluorescence spectrophotometer with excitation at 350; 20 nm and emission at 450; 20 nm.
  • Activity assays ( Figure 7) and liquid chromatography-mass spectrometry ( Figure 8) were performed using methods disclosed in Example 1. These results were compared to wild type HAP1 cells.
  • B lineage cells were isolated, activated, and engineered with a human Factor IX (FIX) containing expression cassette and CCR5 targeting RNP.
  • B lineage cells underwent serum variation for 24 hours, then were expanded and differentiated as described supra.
  • FIX transgene insertion frequency was assessed in engineered B lineage cell populations by using a ddPCR method as described in Example 1.
  • FIX protein secretion was measured via ELISA ( Figure 10B) as described in Example 1.
  • B lineage cells engineered with FIX containing expression cassettes were cultured with or without soluble vitamin KI.
  • FIX- activity of these engineered B lineage cells was assessed via FIX immunocapture assay (Figure 10C) and activated partial thromboplastin clotting time (aPPT) assay ( Figure 10D) as described in Example 1.
  • B lineage cell populations described herein can be engineered to express and/or secrete one or more transgene products of interest (e.g., FIX) using methods described herein.
  • engineered B lineage cell populations express and/or secrete one or more transgene products compared to a reference (e.g., non-engineered B lineage cell population, alternative engineered B lineage cell population, etc.).
  • engineered B lineage cell populations described herein comprise increased plasma cell content when compared to a reference condition.
  • engineered B lineage cell populations produced using methods described herein exhibit enhanced transgene integration (e.g., percentage of HDR, FIX expression, etc.).
  • engineered B lineage cell populations as described herein produce and secrete a product (e.g., polypeptide, protein, cytokine, etc.) encoded by a transgene (e.g., FIX, SMPD1, anti-CD19/CD3 BiTE).
  • a transgene e.g., FIX, SMPD1, anti-CD19/CD3 BiTE.
  • culturing and engineering of B lineage cells as described herein results in increased genetic integration (>30% HDR of total B lineage cell population) relative to a comparator (e.g., an otherwise comparable engineered B lineage cell population that is not maintained in cell culture media that varies in scrum from initial media conditions).
  • B lineage cell populations using methods described herein may demonstrate engraftment and/or transgene expression (e.g., FIX) in a subject.
  • Subjects treated with engineered B lineage cell populations described herein may demonstrate persistent levels of transgene expression over an extended period of time (e.g., one or more weeks, one or more months, etc.).
  • B lineages cells were isolated from two different donors, activated, engineered with the human FIX expression cassette, expanded, and differentiated as described above.
  • Engineered B lineage cells were administered into an immunodeficient mouse model (NOG- hIL6 mouse).
  • Mouse plasma samples were collected at 2, 4, 6, 8, and 10 weeks post-dosing. The collected mouse plasma samples from these two donors were assessed for hFIX (interchangeably, “huFIX”, Figure 11 A) as well as human IgG (hlgG) and hlgM (hlgM) ( Figure 11B).
  • the present Example demonstrates that using methods and compositions disclosed herein can provide for increased genetic integration in B lineage cell populations.
  • Methods and compositions exemplified herein are applicable to higher integration for numerous transgenes.
  • These transgenes include, but are not limited to, FIX, alpha-galactosidase (GLA), SMPD1, and human leukocyte antigen-E (HLA-E) single-chain fusion (e.g., HLA-E single-chain dimers, HLA-E trimers, B2M/HLA-E single-chain fusion).
  • B lineage cells engineered with methods and compositions disclosed herein exhibit higher rates of genetic integration when compared to a reference (e.g., non-engineered B lineage cells, alternative engineered B lineage cell population, etc.). Further, this increased genetic integration can produce an enhanced constituency of engineered B lineage cells that have increased expression and/or secretion of a transgene (e.g., FIX, alpha-galactosidase, SMPD1, etc.).
  • a transgene e.g., FIX, alpha-galactosidase, SMPD1, etc.
  • B lineage cells were isolated from two different donors (Donor 73 and Donor 80), activated, and engineered with a human FIX (hFIX) encoding expression cassette with either JCHAIN or CCR5 guide RNA.
  • hFIX human FIX
  • Two different sets of culture conditions during and after engineering were tested with each donor group and guide RNA subset.
  • a first culture condition involved a serum variation step (referred herein and Figures 12A-B as “+ Serum Variation”) as described in Example 1 for about 24 hours post-editing, whereas a second culture condition used base media (e.g., Excellerate) with fetal bovine serum (FBS; referred herein and Figures 12A-B as Serum Variation”).
  • B lineage cells were then expanded and differentiated using methods disclosed herein. Percentage homology-directed repair (% HDR) was measured using droplet digital PCR (ddPCR) as described in Example 1 and secretion of hFIX was measured by ELTS A as described in Example 1
  • B lineage cells were isolated, activated, and engineered with a GLA containing expression cassette with either JCHAIN or CCR5 guide RNA.
  • activated B lineage cells were either then subjected to serum variation as described in Example 1 or cultured in a base media with FBS for about 24 hours post-engineering. Percentage of HDR was measured by ddPCR as described in Example 1 ( Figure 13 A), and GLA secretion was measured by ELISA as described in Example 1 ( Figure 13B).
  • B lineage cells were also isolated, activated, and engineered with a FIX encoding expression cassette with two different JCHAIN guide RNAs. Conditions and methods including, but not limited to, serum variation were tested on these engineered B lineage cells. Engineered B lineage cells were either subjected to serum variation as described in Example 1 or cultured in base media with FBS. Percentage HDR was measured using ddPCR as described in Example 1 ( Figure 14A), and production of hFIX was measured by ELISA as described in Example 1 ( Figure 14B).
  • B lineage cells were also isolated, activated, and engineered with a HLA-E singlechain fusion (e.g., HLA-E single-chain dimers, HLA-E trimers, B2M/HLA-E single-chain fusion) encoding expression cassette with a B2M guide RNA.
  • HLA-E singlechain fusion e.g., HLA-E single-chain dimers, HLA-E trimers, B2M/HLA-E single-chain fusion
  • B2M/HLA-E single-chain fusion encoding expression cassette with a B2M guide RNA.
  • activated B lineage cells were either then subjected to serum variation as described in Example 1 or cultured in a base media with FBS for about 24 hours post-engineering. Percent of HLA-E integration was measured by flow cytometry as described in Example 1 ( Figure 15).
  • B lineage cell populations described herein can be engineered to have increased genetic integration and/or secretion of one or more transgene products of interest (e.g., FIX, GLA, SMPD1, HLA-E singlechain fusion etc.) using methods described herein.
  • engineered B lineage cell populations express and/or secrete one or more transgenes compared to a reference (e.g., non-engineered B lineage cell population, alternative engineered B lineage cell population, etc.).
  • engineered B lineage cell populations produced using methods described herein have enhanced transgene integration (e.g., percentage HDR, etc.).
  • engineered B lineage cell populations described herein exhibit increased genetic integration of one or more transgenes of interest when compared to a reference condition (e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a scrum variation step).
  • engineered B lineage cell populations as described herein produce and secrete a product (e.g., polypeptide, protein, cytokine, etc.) encoded by a transgene described herein.
  • culturing and engineering of B lineage cells as described herein may result in increased secretion and/or production of a product when compared to a reference condition.
  • Example 7 Engineered B cell populations using methods described herein have sustained viability and homology directed repair.
  • compositions disclosed herein can provide for increased homology directed repair and sustained viability in B lineage cell populations.
  • methods and compositions exemplified herein can provide increased integration of a transgene into a target locus in a B lineage cell.
  • use of the compositions described herein can provide increased levels of viable plasmablasts and plasma cells produced through expansion and differentiation of naive B lineage cell population.
  • culture media compositions comprise serum, plasma, or human recombinant proteins (e.g., insulin, transferrin, albumin, and combinations thereof). In some embodiments, culture media compositions do not comprise serum (e.g., fetal bovine serum, human serum).
  • culture media compositions do not comprise plasma (e.g., fetal bovine plasma, human plasma).
  • culture media compositions comprise one or more recombinant proteins from plasma (e.g., human plasma), but not isolated (e.g., purified, whole, etc.) plasma.
  • culture media compositions comprise one or more recombinant proteins made from CHO cells or E. coli.
  • B lineage cells were isolated and then cultured in base media for at least two days before undergoing transduction with an AAV vector comprising Factor IX.
  • Various culture conditions for AAV transduction over a 24-hour period are outlined in Table 1, Table 2, Table 3, and Table 4 below. Table 1 .
  • Figure 16 Exemplified B Lineage Cell Culturing Compositions
  • B lineage cells were then expanded and differentiated using methods disclosed herein. Percentage homology-directed repair (% HDR) was measured using droplet digital PCR (ddPCR) as described in Example 1 ( Figures 16-19).
  • B lineage cell populations engineered using methods disclosed herein can exhibit increased levels of HDR to integrate one or more payloads (e.g., transgenes).
  • B lineage cell populations cultured with compositions disclosed herein e.g., media compositions lacking serum
  • an engineering step e.g., AAV transduction step
  • can provide increased levels of HDR as compared to a reference e.g., media comprising serum.
  • B lineage cell populations cultured with compositions disclosed herein can provide increased levels of HDR as compared to a reference (e.g., media comprising fetal bovine serum).
  • B lineage cell populations cultured with compositions disclosed herein e.g., media compositions lacking human serum
  • an engineering step e.g., AAV transduction step
  • B lineage cell populations cultured with compositions disclosed herein can provide comparable or increased levels of HDR as compared to a reference (e.g., media comprising human serum).
  • B lineage cell populations cultured with compositions disclosed herein can provide comparable or increased levels of HDR as compared to a reference (e.g., media comprising human plasma).
  • B lineage cell populations cultured with compositions disclosed herein e.g., media compositions comprising one or more proteins derived from human plasma or made from CHO cells or E.
  • coli while lacking isolated (e.g., whole, purified, etc.) human plasma or human serum) during an engineering step can provide comparable or increased levels of HDR as compared to a reference (e.g., media comprising isolated (e.g., whole, purified, etc.) human plasma or human serum).
  • a reference e.g., media comprising isolated (e.g., whole, purified, etc.) human plasma or human serum).
  • engineered B lineage cell populations described herein exhibit sustained viability and increased fold expansion over a duration of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, etc.) when compared to a reference condition (e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a scrum, plasma, or recombinant protein variation step).
  • a reference condition e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a scrum, plasma, or recombinant protein variation step.
  • engineered B lineage cell populations described herein exhibit sustained populations of CD27+CD138+ and CD38+CD138- cells when compared to a reference condition (e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a serum, plasma, or recombinant protein variation step).
  • a reference condition e.g., an otherwise comparable engineered B lineage cell population that is not cultured with a serum, plasma, or recombinant protein variation step.

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Abstract

La présente divulgation propose des procédés et des compositions pour la culture, l'ingénierie et l'administration de populations de cellules de lignée B modifiées qui conduisent à une intégration génétique accrue et à une prise de greffe améliorée.
PCT/US2024/029546 2023-05-16 2024-05-15 Compositions et procédés de production de cellules immunitaires modifiées Pending WO2024238710A1 (fr)

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