[go: up one dir, main page]

WO2024220447A2 - Préparations cellulaires modifiées pour le traitement de la maladie de niemann-pick b - Google Patents

Préparations cellulaires modifiées pour le traitement de la maladie de niemann-pick b Download PDF

Info

Publication number
WO2024220447A2
WO2024220447A2 PCT/US2024/024830 US2024024830W WO2024220447A2 WO 2024220447 A2 WO2024220447 A2 WO 2024220447A2 US 2024024830 W US2024024830 W US 2024024830W WO 2024220447 A2 WO2024220447 A2 WO 2024220447A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
lineage
population
engineered
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/024830
Other languages
English (en)
Other versions
WO2024220447A3 (fr
Inventor
Timothy James MULLEN
Ishara Datta
Hanlan Liu
Huy Anh TU
Anja Fides Hohmann
Claudia FIORINI
Sean Philip Leary ARLAUCKAS
Richard A. Morgan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Be Biopharma Inc
Original Assignee
Be Biopharma Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Be Biopharma Inc filed Critical Be Biopharma Inc
Publication of WO2024220447A2 publication Critical patent/WO2024220447A2/fr
Publication of WO2024220447A3 publication Critical patent/WO2024220447A3/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/231—Interleukin-10 (IL-10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/52—CD40, CD40-ligand (CD154)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • Cell-based therapeutics are an emerging class of medicine that make use of innate cellular machinery to combat disease. Unlike 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-based therapies have potential applications for a broad range of diseases, including those that have proved intractable or difficult to manage with traditional treatment options.
  • the present disclosure encompasses, among other things, compositions and methods comprising modified immune cells (e.g., B lineage cells).
  • modified immune cells e.g., B lineage cells
  • the present disclosure also encompasses, among other things, nucleic acid constructs comprising one or more nucleic acid sequences encoding sphingomyelin phosphodiesterase 1 (SMPD1) described herein and methods of producing the same.
  • SMPD1 sphingomyelin phosphodiesterase 1
  • the present disclosure provides methods of preparing an engineered B lineage cell population that express sphingomyelin phosphodiesterase 1 (SMPD1), the method comprising the steps of: (a) isolating primary B cells so that a primary B cell population is obtained, (b) activating the primary B cell population, and (c) during or after the activating step, engineering the primary B cell population by integrating a transgene encoding SMPD1 at a site selected from a CCR5 site, a IgH site, and a JCHAIN site, thereby generating an engineered B lineage cell population.
  • the method further comprises a step of expanding the engineered B lineage cell population.
  • 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.
  • 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).
  • the present disclosure provides methods of engineering B lineage cell populations.
  • the method further comprises a step of transferring an engineered B lineage cell population to cell culture media without human or bovine serum for about 24 hours.
  • 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 a PAM site, and/or (iii) symmetrical or asymmetrical in length with a 5’ homology arm.
  • a 5’ homology arm has at least 95% identity with one or more of SEQ ID NOs: 1-5.
  • a 3’ homology arm has at least 95% identity with one or more of SEQ ID NOs: 6-9.
  • a donor construct comprises a sequence with at least 95% identity with one or more of SEQ ID NOs: 10-19.
  • a donor construct further comprises one or more of: (a) Kozak sequence of 5’-gccacc-3’, (b) a promoter sequence selected from SEQ ID NOs: 20-21, (c) a nucleic acid sequence encoding a SMPD1 selected from SEQ ID NOs: 22-26, and (c) a polyA sequence selected from SEQ ID NOs: 27-28.
  • a donor construct is or comprises an adcno-associatcd viral (AAV) vector.
  • a step of engineering comprises contacting a primary B cell population with a targeted nuclease capable of introducing double- stranded breaks.
  • the 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 is or comprises Cas9, Casl2a, or Casl3a, or a variant thereof.
  • a Cas protein is complexed with a guide RNA (gRNA).
  • a gRNA is a single guide RNA (sgRNA).
  • a gRNA comprises at least 95% identity with any one of SEQ ID NOs: 29-33.
  • a step of engineering further comprises electroporation of the primary B cell population; and/or 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 CCR5 site has at least 95% identity with SEQ ID NO: 29.
  • a JCHAIN site has at least 95% identity with one or more SEQ ID NOs: 30-32.
  • an IgH site has at least 95% identity with SEQ ID NO: 33.
  • a transgene has at least 95% identity with SEQ ID NO: 22-26.
  • a population of genetically modified B lineage cells comprises a transgene sequence encoding a SMPD1 protein, wherein a transgene is expressed from an endogenous CCR5 locus.
  • a population of genetically modified B lineage cells express SMPD1 protein and at least partially disrupt expression of endogenous CCR5.
  • a population of genetically modified B lineage cells comprises a transgene sequence encoding a SMPD1 protein, wherein a transgene is expressed from an endogenous JCHAIN locus.
  • a population of genetically modified B lineage cells express SMPD1 protein and at least partially disrupt expression of endogenous JCHAIN.
  • a population of genetically modified B lineage cells comprises a transgene sequence encoding a SMPD1 protein, wherein a transgene is expressed from an endogenous IgH locus.
  • a population of genetically modified B lineage cells express SMPD1 protein and at least partially disrupt expression of endogenous IgH.
  • a population of genetically modified B lineage cells is or comprises a population of genetically modified plasma cells.
  • a population of genetically modified B lineage cells is or comprises a population of plasmablasts.
  • a population of genetically modified B lineage cells is or comprises a population of plasma cell precursors.
  • the present disclosure provides a pharmaceutical composition comprising one or more B lineage cells selected from a population of genetically modified B lineage cells.
  • a pharmaceutical composition comprises one or more B lineage cells selected from a population of genetically modified B lineage cells and further comprises one or more pharmaceutically acceptable excipients.
  • a method of administering a pharmaceutical composition wherein the pharmaceutical composition comprises: (a) one or more B lineage cells selected from the population of genetically modified B lineage cells of any aspect or embodiment described herein, and (b) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition is administered to a subject.
  • the present disclosure provides methods of treating a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, thereby treating the disease, disorder, or condition in the 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.
  • a pharmaceutical composition is administered directly to the central nervous system.
  • a subject has Niemann Pick Disease type B.
  • the present disclosure provides methods of characterizing a population of genetically modified B lineage cells, the method 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/cell/day of IgG, (e) secretion of at least 2.5 pg/ccll/day of IgM, (f) secretion of at least 0.5 pg/ccll/day of IgD, and (g) secretion of at least 100-300 ng/10 6 cells/day of a SMPD1 protein.
  • 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), and enzyme-linked immunosorbent assay (ELISA).
  • FAC-sort fluorescence-activated cell sorting
  • ELISpot enzyme-linked immunosorbent spot assay
  • ELISA enzyme-linked immunosorbent assay
  • one or more B lineage cells selected from a population of genetically modified B lineage cells engraft within bone marrow of a subject.
  • a method of monitoring engraftment of genetically modified B lineage cells within a subject comprising one or more of: bioluminescence, ELISpot, flow cytometry, and enzyme-linked immunosorbent assay.
  • Figure 1A shows an AAV6 viral vector comprising an expression cassette
  • Construct p00009 comprises 5’ and 3’ homology arms (HAs) for integration into a CCR5 exon 2 target locus, an MND promoter, wild-type SMPD1 transgene (WT SMPD1), WPRE3 enhancer, BGH poly-adenylation (polyA) sequence.
  • Figure IB shows SMPD1 in B lineage cells treated with Cas9/guide RNA RNP alone or in combination with p00009.
  • Figure 2A shows AAV6 viral vectors comprising specific expression cassettes. Wild-type or codon optimized SMPD1 were used, along with other sequence elements.
  • Figure 2B shows SMPD1 expression in B lineage cells treated with Cas9/guide RNA RNP alone or in combination with indicated constructs.
  • Figure 3A demonstrates different expression cassettes developed and disclosed in the present application.
  • Figure 3B shows comparison of production of SMPD1 in B lineage cell populations engineered with different expression cassettes.
  • Figure 4A demonstrates the gating scheme through flow cytometry of engineered B cell lineage populations for plasma cells.
  • Figure 4B shows content of plasma cells in engineered B lineage cell populations with different expression cassettes and conditions disclosed in the present application.
  • Figure 5 shows graphs comparing percent homology directed repair (% HDR) that occurs within two donor B lineage cell populations engineered with expression cassettes disclosed herein.
  • Figure 6 shows SMPD1 production measured via ELISA assay of B lineage cell populations engineered with exemplary expression cassettes.
  • Figure 7 shows graphs of percentage CD38 + CD138 hlgh plasma cells gated by flow cytometry of B lineage cell populations engineered with either gCCR5 RNP alone, wild type SMPD1 expression cassette (WT SMPD1), SMPD1 C629S expression cassette (C629S-WT), fixed GeneArt codon optimized SMPD1 expression cassette (GeneArtFixed), fixed GeneArt codon optimized SMPD1 C629S expression cassette (C629S-GeneArtFixed), full GeneArt codon optimized SMPD1 expression cassette (GeneArtCO), or full GeneArt codon optimized SMPD1 C629S expression cassette (C629S-GeneArtCO).
  • Figure 8 demonstrates percent homology directed repair (% HDR) measured in B lineage cell populations engineered with exemplary expression cassettes at 7 days and 14 days after initiation of culturing methods disclosed herein.
  • Figure 9 demonstrates production of SMPD1 in B lineage cell populations engineered with exemplary expression cassettes, measured at 7 days and 1 days after initiation of culturing methods disclosed herein.
  • Figure 10A shows SMPD1 activity in wild type HAP1 cells (control) or SMPD1 knockout (KO) HAP1 cells cultured with either recombinant human SMPD1 (rhSMPDl) or supernatant from a B lineage cell population engineered with an exemplified SMPD1 -containing expression cassette.
  • Figures 10B and 10C show sphingomyelin content in wild type HAP1 cells (control) or SMPD1 knockout (KO) HAP1 cells cultured with either recombinant human SMPD1 (rhSMPDl) or supernatant from a B lineage cell population engineered with an exemplified SMPD1 -containing expression cassette.
  • Figure 11 shows an exemplary expression cassette comprising 5’ and 3’ homology arms (HAs), MND promoter, wild type SMPD1 transgene, and BGH poly-adenylation sequence.
  • This base expression cassette was tested with different homology arms and guide RNAs that direct potential integration of the SMPD1 transgene at CCR5 exon 2 (gCCR5_232), JCHAIN intronic region (gJCHAIN_2), JCHAIN exon 1 (gJCHAIN_31), or JCHAIN exon 4 (gJCHAIN_54).
  • Figure 12A shows content of plasma cells of B lineage cell populations engineered with either ribonucleoprotein (RNP) complexes alone or RNP complexes in conjunction with an adeno-associated viral (AAV) donor expression cassette using the culturing and engineered methods provided by the present disclosure.
  • Figure 12B shows percentage of HDR in these aforementioned engineered B lineage cell populations.
  • Figure 12C shows production of SMPD1 of these B lineage cell populations.
  • Figure 13A demonstrates percentage HDR as measured by droplet digital polymerase chain reaction (ddPCR) for B lineage cell populations engineered with expression cassettes as described herein targeting the CCR5 locus.
  • Figure 13B shows acid sphingomyelinase (ASM) secretion measured via ELISA in B lineage cell populations engineered with an exemplary ASM-encoding expression cassettes with a CCR5 -targeting guide RNA.
  • ASM acid sphingomyelinase
  • Figure 14 shows 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 15 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 16 shows fluorescence micrographs of sphingomyelin in HAP1 cells that were either wild-type untreated (top left), SMPD1 knockout (top right), or SMPD1-KO treated with supernatant from a B lineage cell population engineered with an exemplified SMPD1- containing expressing cassette (bottom left and right panels).
  • Figures 17A-17C show sphingomyelin content using different sphingomyelin lengths (18:1,2; 34:1,2; and 42:2,2, respectively) in wild type HAP1 cells (control) or SMPD1 knockout (KO) HAP1 cells cultured with supernatant of CCR5 RNP engineered B lineage cells (no transgcnc), GcncArt Codon Optimized 1 (also known as “SMPD1 GcncArt Partial Codon Optimized expression cassette”) engineered B lineage cells, GeneArt Codon Optimized 2 (also known as “SMPD1 GeneArt Full Codon Optimized expression cassette”) engineered B lineage cells, or recombinant human acid sphingomyelinase (rhASM).
  • rhASM human acid sphingomyelinase
  • Figure 18 shows AAV6 viral vectors comprising specific expression cassettes. Wild-type or codon optimized SMPD1, or variants thereof, that were used as described herein, along with other sequence elements.
  • FIG 19 shows expansion and percentage of antibody- secreting cells (ASCs) relative to culture time for engineered B lineage cells generated with methods described herein. Percentage of ASCs in engineered B lineage cell populations and non-engineered B lineage cell populations is also shown.
  • ASCs antibody- secreting cells
  • Figure 20A shows percentage of on-target integration of a pay load (e.g., SMPD1 or a variant thereof) at an endogenous CCR5 target locus as well as ASM secretion rate for Construct 1 (p00689), Construct 2 (p00690), Construct 3 (p00691), Construct 4 (p00695), and Construct 5 (p00696) as described herein.
  • Figure 20B demonstrates activity of cellular lysate from SMPDF HAP1 cells treated with engineered B lineage cells generated with indicated constructs. Treated HAP1 cells were lysed, and activity of lysate was analyzed through a fluorescence assay.
  • Figure 20C shows sphingomyelin content for different sphingomyelin lengths (18:1,2; 34:1,2; and 42:2,2) in wild type HAP1 cells (control) or SMPD1 knockout (KO) HAP1 cells cultured with supernatant of CCR5 RNP engineered B lineage cells (no transgene), Construct 1 engineered B lineage cells, Construct 4 engineered B lineage cells, or recombinant human acid sphingomyelinase (rhASM).
  • Figure 21 shows histological images of H&E stained sections of indicated organs from SMPD1 KO and WT controls. Images indicate that SMPD1-/- B-NDG/IL6 develop foam cell infiltration of visceral organs as well as in the central nervous system. Arrows indicate foam cells.
  • Figure 22A demonstrates percentage of indels generated by using methods and constructs described herein at the CCR5 locus.
  • Figure 22B demonstrates integration as measured by droplet digital polymerase chain reaction (ddPCR) for B lineage cell populations engineered with expression cassettes as described herein.
  • Figure 22C shows percentage of engineered B lineage cells that arc CD38+.
  • Figures 23A-23B demonstrate IgG and IgM production in mouse plasma after engraftment of engineered B lineage cells using constructs described herein.
  • Figure 24 shows measurements of IgG and IgM over time for mice after administration of indicated fresh engineered B lineage cell preparations or cryopreserved engineered B lineage cell preparations using the methods and expression cassettes disclosed herein.
  • Figure 25 A shows a schematic representation of tran swell activity assay involving
  • FIG. 25B shows activity measurements of wildtype HAP1 control, SMPD1 KO HAP1, and SMPD1 KO HAP1 cells treated with rhSMPDl compared to three sets of SMPD1 KO HAP1 cells that were cultured with the supernatant of B lineage cells engineered without a construct (engineering control) or with two different constructs targeting JCHAIN (p00177 and p00147).
  • Figure 26 shows a CRISPR schematic for knockout of SMPD 1 wild type allele in a mouse model.
  • Figure 27A shows a bar graph of ataxia composite score for SMPD1 wild type mice (first and third bar graphs) compared to SMPD1 KO mice (second and fourth bar graphs) assessing for grooming, motor function, kyphosis (spinal curvature), and ledge test (ability to walk on a ledge).
  • Figure 27B shows computerized tomography (CT) scan assessing the spinal curvature for SMPD1 wild type mouse compared with SMPD1 KO mouse. Arrows designating proximity for spinal curvature.
  • CT computerized tomography
  • Figure 28 shows measurements of sphingomyelin levels from tissue collected from either SMPD1-WT or SMPD 1 -KO mice.
  • Figure 29 shows images of DAPI and Bodipy stained peritoneal cells from either SMPD1-WT mice or SMPD1 knockout mice. Bai’ graph demonstrates percentage of foam cells counted within these peritoneal cell populations.
  • Figure 30 shows a Western Blot assessment for protein levels of LAMP1, LAMP2, LC3-II, and GAPDH (control) from liver samples collected from SMPD1-WT and SMPD1-KO mice. Bai’ graph demonstrates the protein levels as fold change against GAPDH.
  • Figure 31 shows measurements of IgG and IgM over time for mice after administration of indicated fresh engineered B lineage cell preparations or cryopreserved engineered B lineage cell preparations using the methods and expression cassettes disclosed herein.
  • Figure 32 shows measurements of IgG and IgM over time for mice after administration of indicated fresh engineered B lineage cell preparations or cryopreserved engineered B lineage cell preparations using the methods and expression cassettes disclosed herein.
  • Figure 33A shows images of DAPI and Bodipy stained peritoneal cells collected from SMPD1-KO mice after administration of indicated fresh engineered B lineage cell preparations or cryopreserved engineered B lineage cell preparations using the methods and expression cassettes disclosed herein.
  • Figure 33B shows a bar graph of percentage of foam cells of total cells from peritoneal cells collected from SMPD1-KO mice after administration of indicated fresh engineered B lineage cell preparations or cryopreserved engineered B lineage cell preparations using the methods and expression cassettes disclosed herein.
  • Figure 34 shows measurements of sphingomyelin levels from tissue collected from SMPD1-KO mice after administration of either plasmalyte (control), indicated fresh engineered B lineage cell preparations, or cryopreserved engineered B lineage cell preparations using the methods and expression cassettes disclosed herein.
  • Figure 35 shows ASM secretion measured via ELISA assay of B lineage cell populations engineered with exemplary expression cassettes.
  • Figure 36 demonstrates activity of cellular lysate from SMPDl" HAP1 cells treated with engineered B lineage cells generated with indicated constructs. Treated HAP1 cells were lysed, and activity of lysate was analyzed through a fluorescence assay.
  • Figure 37 shows fluorescence micrographs of sphingomyelin in HAP1 cells that were either wild-type untreated (top left), SMPDl knockout (bottom left), or SMPDl -KO treated with supernatant from a B lineage cell population engineered with an exemplified SMPDl - containing expressing cassette (top right and bottom right panels).
  • Figure 38 shows an exemplary schematic of SMPDl activity assay involving a fluorescently labeled sphingomyelin.
  • Cells unable to metabolize sphingomyelin, such as SMPDl KO cells, will lead to accumulation. Addition of sphingomyelinase will lead to a decrease of fluorescently labeled sphingomyelin congregates.
  • Figures 39A-C show sphingomyelin content using different sphingomyelin lengths (34:1,2; 42:2,2; and 18:1,2, respectively) in wild type HAP1 cells (control), SMPDl knockout (KO) HAP1 cells (“KO”), or SMPDl KO HAP1 cells cultured with supernatant of CCR5 RNP engineered B lineage cells (no transgene; “gCCR5 RNP only”), GeneArt Codon Optimized 1 (also known as “GeneArt Co-1”) engineered B lineage cells, GeneArt Codon Optimized 2 (also known as “GeneArt Co-2”) engineered B lineage cells, or recombinant human SMPDl (rhSMPDl).
  • rhSMPDl recombinant human SMPDl
  • Figure 40 shows histological images of H&E stained sections of indicated organs from SMPDl KO and WT controls. Images indicate that SMPDl-/- B-NDG/IL6 develop foam cell infiltration of visceral organs (Spleen, Kidney, Lung, and Liver) as well as in the central nervous system (Adrenal Gland and Cerebellum).
  • 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.e., 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 alloantigcn on the grafted tissue.
  • allorccognition involves T cell recognition of the alloantigen.
  • T cells recognize an alloantigcn 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 ait-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 biomarker 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 biomarkcr 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 art.
  • 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.
  • the term “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 arc expanded ex vivo increase in number relative to other cell types in a culture.
  • expansion may occur in vivo.
  • 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%, 35%, 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.
  • Gene product or 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 molecules When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules arc identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position.
  • 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 arc 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. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses.
  • 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. In some embodiments, presence or level of a particular marker may be characteristic of presence or stage of a disease, disorder, or condition. To give but one example, in some embodiments, 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, deoxythymidine, 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.
  • 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, 200, 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 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.
  • compositions 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.
  • pharmaceutical 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.
  • parenteral administration for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation
  • parenteral application for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity
  • intra vaginally or intrarectally for example, as a pessary, cream, or foam
  • sublingually ocularly
  • transdermally or nasally, pulmonary, and to other mucosal surfaces.
  • Polynucleotide refers to a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • 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.
  • 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 (e.g., amino acids) that are typically linked by peptide bonds.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • 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.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
  • a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids.
  • 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is lineal’. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, 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.
  • 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 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.
  • a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
  • a reference or control is a historical reference or control, optionally embodied in a tangible medium.
  • a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
  • 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).
  • 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 component(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 transccllular 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.
  • two variables e.g., sequence identity, protein production, spatiotemporal conditions, etc.
  • 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.
  • 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 does 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 are naturally associated in their natural state.
  • the cells are cultured in vitro. In other embodiments, the cells arc 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 As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “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. ., 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. ., 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 “valiant” 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 art 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 are not limited to, adenoviral vectors, adeno- associatcd virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • the present disclosure encompasses culturing and engineering of B lineage cell populations in order to express SMPD1.
  • Lipid biosynthesis, accumulation, and degradation are key mechanisms and pathways for maintaining cellular membrane integrity, metabolism, and homeostasis.
  • NPD Niemann Pick Disease
  • ASM sphingomyelin phosphodiesterase 1
  • NPD N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-
  • Type B NPD is described as a later-onset form of acid spingomyelinase deficiency, in which subjects may exhibit little or no neurological symptoms, but may have severe and progressive visceral organ abnormalities including hepatosplenomegaly, pulmonary insufficiency, and cardiovascular disease (Schuchman et al., 2001). Children with NPB may have increased life expectancy in comparison to Type A, but often still require supplemental oxygen due to lung impairment.
  • ERT enzyme replacement therapy
  • olipudase alfa Xenopyzyme
  • Bone marrow transplantation has been tested in a few individuals, with mixed results being reported (NIH, 2023).
  • ERT for NPB with olipudase alfa is limited by the need to dose relatively frequently (every two weeks) via intravenous administration and has numerous side effects.
  • the present disclosure provides methods and preparations of engineered B lineage cell populations that may be useful as a viable cell therapy option for treatment of NPD generally, and specifically for NPB.
  • engineered B lineage cell populations prepared via methods disclosed herein may express one or more therapeutic proteins, including, e.g., ASM.
  • 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., payload delivery, targeting, long-term payload 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 valiant 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 valiant 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 arc used as reference cell populations.
  • naive B cell populations express CD 19 (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 CD19 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 CD19 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 CD19 (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 pay load 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 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., SMPD1) through an endogenous repair pathway (e.g., homologous recombination, homology- directed repair, etc.).
  • a payload e.g., expression cassette comprising one or more transgenes
  • a transgene e.g., SMPD1
  • endogenous repair pathway e.g., homologous recombination, homology- directed repair, etc.
  • methods for integration of an expression cassette comprising a SMPD1 transgene comprise site-specific cleavage at a target locus (e.g., CCR5) with a guide RNA / Cas9 complex, followed by integration of SMPD1 at the target locus through homologous recombination.
  • a target locus 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., AAV2, 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 pay load 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 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 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) or variant thereof.
  • an expression cassette comprises a polynucleotide sequence encoding SMPD1 or a variant thereof (e.g., SMPD1 C629S).
  • an expression cassette comprises a polynucleotide sequence encoding a transgene (e.g., SMPD1) or variant thereof for expression of a peptide or polypeptide (e.g., ASM) or variant thereof.
  • 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 are 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. In some embodiments, a transgene is a gene that causes expression of sphingomyelin phosphodiesterase 1 (SMPD1) or a variant thereof.
  • SMPD1 sphingomyelin phosphodiesterase 1
  • 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 arms flank a polynucleotide sequence encoding a pay load (e.g., one homology arm is 5’ to a pay load (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 are 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 integration of a payload (e.g., a transgene) at a target locus (e.g., an endogenous gene).
  • a payload e.g., a transgene
  • compositions and constructs provided herein direct integration of a payload at a target locus in a specific cell type (e.g., naive B cells, B lineage cell populations, etc.).
  • compositions and constructs provided herein direct integration of a payload (e.g., expression cassette comprising a transgene encoding SPMD1) at a target locus in a specific cell type (e.g, naive B cells, B lineage cell populations, etc.).
  • a payload is or comprises a transgene or variant thereof (encoding SMPD1, or a variant thereof (e.g., SMPD1 C629S).
  • compositions and constructs provided herein direct 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 CD19, CD20, IGH, B2M, CCR5, JCHAIN, PAX5, IRF4, IRF8, BACH2, EZH2, XBP1, CARD11, PRDM1, and BAFF.
  • 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).
  • 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).
  • media comprising at least about 0.1 ug/mL, 0.2 ug/mL,
  • 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.
  • 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 are 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). 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 + / CD38 + / CD 138'.
  • cytokines e.g., IL-2, IL-6, IL- 10, and/or IL- 15
  • 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 100 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 are 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 100 ng/mL of one or more cytokines (e.g., IL-6, IL- 15,
  • 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 an SMPD1 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 an SMPD1 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 an SMPD1 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 an SMPD1 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 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 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 100 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 char acterization 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 (e.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 encoding SMPD1.
  • 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).
  • NPD e.g., NPD type 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.
  • the methods disclosed herein comprise measuring and/or monitoring treatment of NPD type B.
  • 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 SMPD1 are measured from sample collection.
  • 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. In some embodiments, levels of a transgene (e.g., SMPD1) are determined to measure engraftment.
  • a transgene e.g., SMPD1
  • 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).
  • cngraftmcnt 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 the clinical subject e.g., chemotherapy, immunosuppressive treatment, etc. 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.
  • the present application provides methods that result in engineered B lineage cell populations. These methods 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
  • B lineage cells are contacted with media lacking serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof) during gene editing.
  • 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 lacking serum (e.g., human serum, bovine serum, horse serum, newborn calf serum, goat serum, rabbit serum, porcine serum, chicken serum, or a combination thereof) and then incubated at 37°C for up to 24 hours.
  • B lineage cells are contacted with media substantially free 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) and then incubated at 37 °C for up to 24 hours.
  • 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 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 5 days.
  • cytokines and/or oligonucleotides e.g., CD40L, IL-2, IL-10, IL-15, IL-21 and/or CpG
  • 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 (rhSMPD 1 ) or with supernatant from the engineered B lineage cell population as presently disclosed. Next, cells were imaged on Cytation5 at 40X using a GFP fluorescent filter and bright field as a control. Images were processed using ImageJ ( Figure 38).
  • KO mice and wild type (WT) controls were sacrificed and the lung, liver, brain, cerebellum, kidney, spleen, and adrenal gland were harvested.
  • the organs were then rinsed in phosphate buffered saline (PBS) and fixed in 10% neutral buffered formalin for 24-48 hours at room temperature. Samples were washed for 5 minutes in PBS and then dehydrated by subsequent ethanol washes (25%, 50%, 75% and 100%) for 30 minutes each with gentle agitation at room temperature. The tissue was then cleared twice in xylene for one hour with gentle agitation at room temperature.
  • PBS phosphate buffered saline
  • mice Approximately 3-month-old mice were conditioned to an empty cage for approximately 5 minutes for 5 days in a row. The following week, the mice were assessed for grooming, motor function, kyphosis (spinal curvature), and ledge test (ability to walk on a ledge) and assigned a score of 0 to 3 for each. The scores for each assessment were averaged to generate a composite score for each mouse.
  • Example 2 Construct Development and Production of SMPD1 Engineered B Lineage Cell Population.
  • B lineage cell populations can be engineered to express and secrete active acid sphingomyelinase (ASM) protein.
  • B lineage cell populations are engineered with a payload.
  • a payload is or comprises one or more expression cassettes.
  • expression cassettes may or may not comprise of an enhancer (e.g., WPRE3).
  • expression cassettes may or may not comprise a transgene.
  • the transgene comprises SMPD1.
  • the transgene may or may not be a codon optimized SMPD1.
  • the transgene may or may not comprise a SMPD1 mutant variant (e.g., C629S).
  • Construct p00009 was prepared, comprising an expression cassette as outlined in Figure 1A (Hung et al, 2018). B lineage cells engineered with p00009 produced between 1 and 6 ng/mL of SMPD1 per 1 million cells over a 24-hour culture ( Figure IB).
  • AAV6 viral vector compositions were prepared, comprising expression cassettes with various polynucleotide sequence elements as outlined in Figure 2A. Each construct was administered to B lineage cell populations for homologous recombination to integrate specific expression cassettes into the CCR5 locus, as described herein, and then evaluated for effects on overall SMPD1 expression using the ELISA methods disclosed in Example 1 ( Figure 2B).
  • FIG. 3A Next, additional expression cassettes were generated and tested in B lineage cells as outlined in Figure 3A.
  • Various components of the expression cassettes were assessed for SMPD1 production in engineered B lineage cell populations, including different combinations of terminator sequences (e.g., BGH or SV40), enhancer elements (e.g., presence or absence of WPRE3), promoters (e.g., EF-la, MND), and SMPD1 transgene (e.g., wild type or GeneArt codon optimized SMPD1) (Figure 3B).
  • terminator sequences e.g., BGH or SV40
  • enhancer elements e.g., presence or absence of WPRE3
  • promoters e.g., EF-la, MND
  • SMPD1 transgene e.g., wild type or GeneArt codon optimized SMPD1
  • the present disclosure includes, but not limited to, expression cassettes that may or may not comprise a SMPD 1 transgene.
  • expression cassettes may or may not comprise of a SMPD1 mutant variant (e.g., cysteine 629 to serine mutant variant).
  • expression cassettes may or may not comprise of codon optimization (“Co”) of SMPD1.
  • codon optimization of SMPD1 may or may not comprise of alteration in specific regions (e.g., “GeneArt Fixed”).
  • codon optimization may or may not comprise the entire SMPD1 CDS (e.g.s, “GeneArt codon optimized 1,” and “GeneArt codon optimized 2”).
  • B lineage cells were isolated and engineered with an SMPD1 containing expression cassette, then underwent serum variation as described in Example 1 for 24 hours followed by expansion and differentiation. Percentage of indels and genetic integration was measured via ddPCR ( Figure 13 A, Figure 22A, and Figure 22B) and secretion of ASM was measured via ELISA ( Figure 13B). Locus cutting efficiency of greater than 90% gene editing was established at CCR5. Insertion efficiency of SMPD1 encoding constructs showed -50% HDR. Further, percentage of CD38 positive expressing engineered B lineage cells were assessed via flow cytometry (Figure 22C). Greater than 97% of engineered B lineage cells were CD38 demonstrating that these were well-differentiated antibody secreting cells.
  • B lineage cells engineered with the aforementioned expression cassettes may demonstrate SMPD1 production.
  • B lineage cell populations engineered with codon optimized SMPD1 expression cassette may or may not have reduced SMPD1 production.
  • engineered B lineage cells may comprise of plasma cells (>15% of B lineage cell population).
  • B lineage cells engineered as disclosed herein may have homology-directed repair (>10%).
  • B lineage cell populations engineered with an expression cassette lacking the WPRE3 enhancer may have increased SMPD1 production when compared to expression cassettes containing WPRE3.
  • the production of SMPD1 with the lack of an enhancer element e.g., WPRE3 is not necessarily expected by one skilled in the ait.
  • Example 3 Engineered B Lineage Cell Population Capable of Phenotypic Recovery of SMPD1 Activity.
  • the present disclosure seeks to provide novel methods and expression cassettes for engineering of B lineage cell population that secretes active SMPD1 for the treatment of Niemann Pick Disease Type B in a patient population.
  • SMPD1 knockout (KO) HAP1 cells populations were seeded in 12- well plate and treated with either recombinant human SMPD1 (rhSMPDl), wild type (non-engineered) B lineage cell population supernatant or engineered B lineage cell population supernatant.
  • Engineered B lineage cell populations were engineered with WPRE3-containing expression cassettes using the 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.
  • 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 Figures 10A and 14
  • liquid chromatography-mass spectrometry Figures 10B-C and Figure 15 were performed using the methods disclosed in Example 1. These results were compared to wild type HAP1 cells.
  • B lineage cell populations are engineered with one or more expression cassettes.
  • the one or more expression cassettes may or may not comprise of WPRE3.
  • the supernatant of engineered B lineage cell populations with the expression cassettes disclosed herewith may result in reduced sphingomyelin ( Figures 10B-C and 15).
  • Example 4 Optimization of Guide RNA for Engineering of B Lineage Cell Populations.
  • the present disclosure provides, inter alia, a combination of culturing methods and expression cassettes that can be engineered into a B lineage cell population allowing production of SMPD1. Furthermore, the present disclosure provides methods of which to engineered B lineage cell populations. In some embodiments, engineering of B lineage cell populations may or may not comprise a guide RNA.
  • B lineage cells were engineered with various guide RNAs as part of a CRISPR/Cas9 RNP complex either alone (Control and RNP only) or with an adeno-associated viral (AAV) expression cassette as outlined in Figure 11. 12 days after engineering, these B lineage cell populations were evaluated for plasma cell content via use of flow cytometry as disclosed above ( Figure 12A). Next, ddPCR analysis and ELISA was performed on the B lineage cell populations ( Figure 12B-C).
  • B lineage cells engineered with the various guide RNAs arc capable of producing a transgcnc (e.g., SMPD1). Further, the culturing and engineering methods disclosed by the present application results in development of a plasma cell population (>10%).
  • engineering of B lineage cells with the guide RNAs disclosed herein will be capable of homology-direct repair ( ⁇ >5%).
  • engineering of B lineage cells with the guide RNAs disclosed herein will be capable of transgene expression.
  • engineering of B lineage cells with the guide RNAs disclosed herein will result in production of SMPD1.
  • Example 5 Phenotypic Recovery of SMPD1 Activity of Engineered B Lineage Cell Populations Using Codon Optimized SMPD1 Expression Cassettes.
  • the present disclosure includes, but is not limited to, expression cassettes that may or may not comprise a SMPD1 transgene.
  • expression cassettes comprise a codon-optimized version of SMPD1.
  • a codon-optimized version of SMPD1 comprises a full length SMPD1 CDS e.g., “GeneArt codon optimized”).
  • Additional expression cassettes were generated comprising different codon- optimized SMPD1 transgenes, wherein one comprises only certain regions of the SMPD1 coding sequence being codon optimized (also referred to herein as “SMPD1 GeneArt Partial Codon Optimized” and “GeneArt Codon Optimized 1”) and the other comprising the full coding sequence of the SMPD1 being codon optimized (also referred to herein as “SMPD1 GeneArt Full Codon Optimized” and “GeneArt Codon Optimized 2”). Both expression cassettes were engineered in B lineage cell populations as disclosed herein.
  • SMPD1 knockout (KO) HAP1 cells populations were seeded in 12- well plates and treated with recombinant human SMPD1 (rhSMPDl), wild type (non-engineered) B lineage cell population supernatant, GeneArt Codon Optimized 1 engineered B lineage cell population supernatant, or GeneArt Codon Optimized 2 engineered B lineage cell population supernatant.
  • rhSMPDl recombinant human SMPD1
  • wild type (non-engineered) B lineage cell population supernatant wild type (non-engineered) B lineage cell population supernatant
  • GeneArt Codon Optimized 1 engineered B lineage cell population supernatant wild type (non-engineered) B lineage cell population supernatant
  • GeneArt Codon Optimized 2 engineered B lineage cell population supernatant GeneArt Codon Optimized 2 engineered B lineage cell population supernatant.
  • B lineage cell populations were engineered with one or more expression cassettes.
  • supernatant of engineered B lineage cell populations with codon optimized SMPD1 expression cassettes disclosed herein resulted in reduced sphingomyelin (SM) accumulation (e.g., reduced levels of one or more SM isoforms) in cells (e.g., SMPD1 KO HAP1 cells described herein) treated with supernatant ( Figures 17A-C, Figure 20C, and Figures 39A-C).
  • engineered B lineage cell populations described herein can produce functional SMPD1 , or variants thereof. In some embodiments, such engineered B lineage cell populations can produce functional SMPD1 in vivo.
  • the present disclosure includes, but is not limited to, B lineage cells engineered with SMPD1 expression cassettes as described herein.
  • engineered B lineage cells may be administered intravenously in a subject.
  • engineered B lineage cells may be administered through an intraperitoneal route to a subject.
  • a subject may include a mouse model.
  • a mouse model was developed with SMPD 1 knockout and knock-in of human intcrlcukin-6 (BNDG-hIL6 SMPD -/-) and compared to a wildtypc model ( Figure 26).
  • B lineage cells engineered using a SMPD1 expression cassette described herein were then administered intravenously to a SMPD1 knockout mouse model and mice were evaluated post engraftment.
  • Mouse plasma collected from treated mice demonstrated engraftment of engineered B lineage cells and persistent, long-term production of human IgG and IgM ( Figures 23A-B).
  • levels of IgG and IgM secretion indicate engraftment of engineered B lineage cells.
  • B lineage cells were engineered to express sphingomyelin phosphodiesterase 1 (SMPD1) from the endogenous CCR5 locus. After Ficoll clean-up and isolation, cell populations were transferred into a base cell culture media and split into 6 groups. The first four groups (fresh engineered cell preparations, “Fresh BCM-SMPD1”) was administered to mice immediately.
  • SMPD1 sphingomyelin phosphodiesterase 1
  • the second set of three groups (frozen B lineage preparations, “Cryo BCM-SMPD1”) was transferred into media comprising 50% CS10 and 50% PlasmaLyte supplemented with HSA at a concentration of 15 x 10 6 cells/mL and were frozen via controlled rate freezer before being transferred to storage at - 170°C in liquid nitrogen. These cells were then administered to mice after thawing. Plasma samples were collected, and levels of secreted IgG and IgM were quantified (Figure 24).
  • mice were assessed for ataxia using the methods described in Example 1. Both sets of mice were assessed for grooming, motor function, kyphosis (spinal curvature), and ledge ability (Figure 27 A). CT scans were done to analyze the spinal curvature of WT and SMPD1 KO mouse models ( Figure 27B). Western Blot analysis was performed from liver samples collected from SMPD1 KO mice and SMPD1 WT and assessed for protein levels of LAMP1, LAMP2, LC3-II, and were compared to the GAPDH control ( Figure 30).
  • SMPD1 KO and WT mice underwent peritoneal lavage.
  • the cell fraction was then isolated and plated in a 96 well plate. After 2-3 days in culture, peritoneal cells were washed with PBS, fixed with 4% PFA, and stained with F4/80, Bodipy, and DAPI. Images were captured using Cytation 5 and F4/80 positive (macrophage). Bodipy negative cells were counted. Three images per mouse were counted ( Figure 29).
  • SMPD1 KO mice treated with the indicated freshly prepared and cryoprcscrvcd engineered B lineage cell preparations were euthanized and tissues harvested from the liver, spleen, kidney, and lung. Lipids were isolated form cryo-pulverized tissues from these mice and assessed for sphingomyelin levels (Figure 34).
  • the present disclosure demonstrates that a subject treated with engineered B lineage cell populations described herein may exhibit reduced phenotypic characteristics of Niemann Pick Disease Type B relative to an untreated population.
  • subjects treated with engineered B lineage cell populations may exhibit persistent engraftment e.g., greater than or for at least 6 weeks).
  • engineered B lineage cell populations may be administered to a subject fresh or cooled.
  • cooled engineered B lineage cell populations may exhibit persistent engraftment.
  • subjects treated with engineered cell populations may exhibit improved motor function.
  • subjected treated with engineered B lineage cell populations may exhibit reduced foam cell formation.
  • subjects treated with engineered B lineage cell populations may exhibit reduced sphingomyelin accumulation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Diabetes (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente divulgation concerne des procédés et des compositions pour la culture, la modification et l'administration de populations de cellules de lignée B modifiées qui produisent de la sphingomyéline phosphodiestérase 1 (SMPD1).
PCT/US2024/024830 2023-04-17 2024-04-16 Préparations cellulaires modifiées pour le traitement de la maladie de niemann-pick b Pending WO2024220447A2 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US202363459983P 2023-04-17 2023-04-17
US63/459,983 2023-04-17
US202363502636P 2023-05-16 2023-05-16
US63/502,636 2023-05-16
US202363592497P 2023-10-23 2023-10-23
US63/592,497 2023-10-23
US202463549943P 2024-02-05 2024-02-05
US63/549,943 2024-02-05
US202463553522P 2024-02-14 2024-02-14
US63/553,522 2024-02-14

Publications (2)

Publication Number Publication Date
WO2024220447A2 true WO2024220447A2 (fr) 2024-10-24
WO2024220447A3 WO2024220447A3 (fr) 2025-02-13

Family

ID=93153463

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/024830 Pending WO2024220447A2 (fr) 2023-04-17 2024-04-16 Préparations cellulaires modifiées pour le traitement de la maladie de niemann-pick b

Country Status (1)

Country Link
WO (1) WO2024220447A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2018212652B2 (en) * 2017-01-26 2024-03-28 Sangamo Therapeutics, Inc. B-cell engineering
JP2020517692A (ja) * 2017-04-27 2020-06-18 イミュソフト コーポレーション 治療剤のｉｎ ｖｉｖｏ送達のためのＢ細胞およびその投薬量

Also Published As

Publication number Publication date
WO2024220447A3 (fr) 2025-02-13

Similar Documents

Publication Publication Date Title
JP6921797B2 (ja) ヒト疾患に関連する生物製剤およびタンパク質の産生のための修飾ポリヌクレオチド
JP6946384B2 (ja) 脂質ナノ粒子を含む医薬組成物
JP6396415B2 (ja) コレステロールレベルを変更する組成物および方法
KR102581977B1 (ko) Rna 분자의 번역 효율을 증가시키는 utr
US20170175143A1 (en) Method for editing a genetic sequence
CN103687957A (zh) 工程化核酸及其用于非人类脊椎动物的方法
US11279935B2 (en) Method for screening prophylactic or therapeutic agents for diseases caused by interleukin 6, interleukin 13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 and agent for the prevention or treatment of diseases caused by interleukin 6, interleukin 13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5
KR20180127319A (ko) 신경 질환의 치료를 위한 방법 및 조성물
US20240409603A1 (en) Modified stem cell compositions and methods for use
US20230072226A1 (en) Basic domain-deleted dnase1-like 3 and uses thereof
WO2024238828A2 (fr) Systèmes et compositions d'édition de gènes pour le traitement d'hémoglobinopathies et leurs méthodes d'utilisation
AU2024257819A1 (en) Engineered cell preparations for treatment of hemophilia
WO2024220447A2 (fr) Préparations cellulaires modifiées pour le traitement de la maladie de niemann-pick b
WO2025049959A2 (fr) Systèmes et compositions d'édition génique, et méthodes de traitement du syndrome vexas
WO2024238710A1 (fr) Compositions et procédés de production de cellules immunitaires modifiées
WO2024238707A2 (fr) Compositions et procédés de production de populations améliorées pouvant être greffées de cellules immunitaires modifiées
WO2024178069A1 (fr) Compositions et procédés d'édition génomique
WO2024238712A2 (fr) Compositions et procédés d'ingénierie multiplex de cellules immunitaires
KR20250175332A (ko) 혈우병 치료를 위한 조작된 세포 제제
WO2025096483A2 (fr) Préparations de cellules modifiées
JP2024527933A (ja) 改良されたｃｒｉｓｐｒ－ｃａｓ技術
WO2025097067A9 (fr) Préparations cellulaires modifiées pour le traitement de l'hypophosphatasie
WO2024036214A2 (fr) Compositions de cellules souches modifiées et procédés d'utilisation
US20230180725A1 (en) Production of Human Cells, Tissues, and Organs in a Growth Factor Receptor-Deficient Animal Host
WO2024173502A2 (fr) Procédés de conservation de cellules

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2024793341

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024793341

Country of ref document: EP

Effective date: 20251117

ENP Entry into the national phase

Ref document number: 2024793341

Country of ref document: EP

Effective date: 20251117

ENP Entry into the national phase

Ref document number: 2024793341

Country of ref document: EP

Effective date: 20251117

ENP Entry into the national phase

Ref document number: 2024793341

Country of ref document: EP

Effective date: 20251117