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WO2025096931A1 - Technologies de production - Google Patents

Technologies de production Download PDF

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Publication number
WO2025096931A1
WO2025096931A1 PCT/US2024/054104 US2024054104W WO2025096931A1 WO 2025096931 A1 WO2025096931 A1 WO 2025096931A1 US 2024054104 W US2024054104 W US 2024054104W WO 2025096931 A1 WO2025096931 A1 WO 2025096931A1
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Prior art keywords
polypeptide
cells
capsid
cell
peptone
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Inventor
Seyyedhassan PAYLAKHI
Seung Baek HAN
Jungeun Lee
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64 X Inc
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64 X Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C12N2500/30—Organic 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
    • C12N2511/00—Cells for large scale production
    • 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
    • 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
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • Protein production technologies and specifically viral capsid (e.g., AAV) production technologies are critical to the burgeoning biotechnology industry-.
  • AAV viral capsid
  • the present disclosure provides certain technologies useful in such production, including specifically- for production of viral (e.g.. AAV) capsid polypeptides, which, in many embodiments, are part of viral capsids, e.g.. in viral vectors.
  • viral e.g.. AAV
  • capsid polypeptides which, in many embodiments, are part of viral capsids, e.g.. in viral vectors.
  • a cell culture system comprising: a population of mammalian cells; one or more transfection reagents; one or more nucleic acids sufficient to engineer mammalian cells to express a heterologous polypeptide (e g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload); and a peptone at a concentration in a range of about 0.05% to about 2%.
  • a heterologous polypeptide e g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a peptone at a concentration in a range of about 0.05% to about 2%.
  • a cell preparation engineered to express a heterologous polypeptide e.g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a cell preparation comprises: a population of mammalian cells; one or more transfection reagents; one or more nucleic acids sufficient to engineer mammalian cells to express a heterologous polypeptide; and a peptone at a concentration in a range about 0.05% to about 2%.
  • a method of expressing a heterologous polypeptide e.g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a heterologous polypeptide e.g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a heterologous polypeptide e.g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a heterologous polypeptide e.g., recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a population of mammalian cells is contacted with (iii) prior to contacting with (i) and/or (ii).
  • a population of mammalian cells is contacted with (iii) at least 5 minutes prior to contacting with (i) and/or (ii).
  • a population of mammalian cells is contacted with (iii) for substantially all of the time a population of mammalian cells is contacted with (i) and/or (ii).
  • contacting a population of mammalian cells with (ii) prior to contacting with (i) and/or (ii) increases expression of a heterologous polypeptide by at least 5% as compared to an otherwise similar cell that has not been contacted with (iii) or that is contacted with (iii) after (i) and/or (ii).
  • This disclosure provides, a method of manufacturing a pharmaceutical composition
  • a method of manufacturing a pharmaceutical composition comprising, formulating an expressed heterologous polypeptide preparation (e.g., a preparation of recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload) with one or more pharmaceutically acceptable excipients.
  • an expressed heterologous polypeptide preparation e.g., a preparation of recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload
  • a method comprising delivering: (1) a heterologous polypeptide produced from a cell culture system disclosed herein; (2) a heterologous polypeptide made by any of the methods disclosed herein; (3) a heterologous polypeptide made with a cell preparation disclosed herein; or (4) a pharmaceutical composition comprising a heterologous polypeptide disclosed herein, to a cell, tissue or subject.
  • a heterologous polypeptide is or comprises recombinant AAV particles comprising a capsid and a nucleic acid encoding a payload.
  • delivering comprises administering.
  • a method disclosed herein is a treatment method.
  • a method disclosed herein is a prevention method.
  • a subject is a mammal. In some embodiments, a mammal is a human.
  • a peptone comprises one or more of: tryptone N1 (TNI), Tryptone Plus, Casein Peptone El, Gelatin Peptone N2, C-Cell Pl 12, C-Cell G115, C-Cell S146B, C-Cell S204.
  • a peptone is or comprises Casein El .
  • a peptone is or comprises Gelatin N2.
  • a peptone is or comprises C-Cell S146B.
  • a peptone is not TNI .
  • a peptone is provided or otherwise present at a concentration of about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5% about, 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.1%. about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%.
  • a peptone is provided or otherwise present at a concentration of about 0.05% to about 1.9%, about 0.05% to about 1.8%, about 0.05% to about 1.7%, about 0.05% to about 1.6%, about 0.05% to about 1.5%, about 0.05% to about 1.4%, about 0.05% to about 1.3%, about 0.05% to about 1.2%, about 0.05% to about 1.1%.
  • a population of mammalian cells is contacted with a peptone and one or more transfection reagents at substantially the same time.
  • a population of mammalian cells is contacted with a peptone prior to contacting with one or more transfection reagents.
  • a population of mammalian cells is contacted with a peptone at least 5 minutes prior to contacting with one or more transfection reagents.
  • a population of mammalian cells comprises: HEK cells, CHO cells, HeLa cells, PER.C6 cells, HKB11 cells. CAP cells, BHK cells, mouse myeloma cells, green African monkey kidney cells, or A549 cells.
  • a population of mammalian cells comprises HEK cells.
  • HEK cells comprise HEK293 cells.
  • a heterologous polypeptide is or comprises a viral capsid polypeptide, an antibody polypeptide, a cytokine polypeptide, a chemokine polypeptide, an enzyme polypeptide, or a hormone polypeptide.
  • a heterologous polypeptide is or comprises a viral capsid polypeptide.
  • a viral capsid polypeptide is part of a viral capsid.
  • a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • a viral capsid polypeptide is an AAV capsid polypeptide.
  • an AAV capsid polypeptide is chosen from an AAV1; AAV2; AAV3; AAV4; AAV5; AAV6; AAV7; AAV8; AAV9; AAV10; AAV11; AAV12; or an AAV13 capsid polypeptide, or a variant or derivative of any of the foregoing.
  • an AAV capsid polypeptide is or comprises an AAV8 capsid polypeptide or a variant or derivative thereof.
  • an AAV capsid polypeptide is or comprises an AAV1 capsid polypeptide or a variant or derivative thereof. In some embodiments, an AAV capsid polypeptide is or comprises an AAV2 capsid polypeptide or a variant or derivative thereof. In some embodiments, an AAV capsid is or comprises an AAV3 capsid polypeptide or a variant or derivative thereof. In some embodiments, an AAV capsid polypeptide is or comprises an AAV4 capsid polypeptide or a variant or derivative thereof. In some embodiments, an AAV capsid polypeptide is or comprises an AAV5 capsid polypeptide or a variant or derivative thereof.
  • an AAV capsid polypeptide is or comprises an AAV6 capsid polypeptide or a variant or derivative thereof. In some embodiments, an AAV capsid polypeptide is or comprises an AAV9 capsid polypeptide or a variant or derivative thereof.
  • a heterologous polypeptide is or comprises a recombinant AAV particle comprising an AAV capsid polypeptide and a nucleic acid encoding a payload.
  • a viral capsid polypeptide is or comprises a retrovirus capsid polypeptide.
  • a viral capsid polypeptide is or comprises a lentivirus capsid polypeptide.
  • a viral capsid polypeptide is or comprises a HSV capsid polypeptide.
  • a heterologous polypeptide is an antibody polypeptide.
  • a heterologous polypeptide is a cytokine polypeptide.
  • a heterologous polypeptide is a chemokine polypeptide.
  • a heterologous polypeptide is a hormone polypeptide.
  • a heterologous polypeptide is an enzyme polypeptide.
  • FIG. 1 presents viral genome (titer) results of a small-scale AAV8 production (24 DWP) study that compared impact of eight peptones at two different concentrations, 0.5% and 1.0%. An asterisk indicates a significant difference, with a p-value of ⁇ 0.05.
  • FIG. 2 presents viral genome (titer) results of a small-scale AAV8 production (24 DWP) study that used three selected high-performing peptones at three different concentrations, 0.25%, 0.5%, and 1.0%.
  • An asterisk indicates a significant difference, with a p-value of ⁇ 0.05.
  • FIG. 3 presents viral genome (titer) results of a small-scale AAV8 production (30 ml shake flask) study that used three selected high-performing peptones at a concentration of 0.5%.
  • the asterisk indicates a significant difference, with a p-value of ⁇ 0.05.
  • FIG. 4 presents viral genome (titer) results of a small-scale AAV production (24 DWP) study that used three selected high-performing peptones at a concentration of 0.5%.
  • FIG. 5 presents viral genome (titer) results from an exemplary large scale AAV production (2L bioreactor) study that used selected high-performing peptones at a concentration of 0.25%.
  • FIG. 6 presents viral genome (titer) results from an exemplary large scale AAV production (2L bioreactor) study that used selected high-performing peptones at a concentration of 0.25% with two different transfection reagents.
  • the left-side Y-axis represents AAV titer as measured by vector genomes per L (vg/L) using a PCR assay as described herein.
  • the right-side Y-axis represents % of full capsid / empty capsid measured by calculating the percentage of full capsid to empty capsid using the formula: (vg titer/capsid titer)*100.
  • Capsid titer was measured using an assay (Gator Bio) that detects AAV capsid particles (irrespective of whether the capsid contains an AAV genome [e.g., a full capsid] or a portion of an AAV genome [e.g., a partially full capsid] or no AAV genome [e.g., an empty capsid]).
  • 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.
  • Antibody refers to an immunoglobulin molecule, or fragment thereof, that binds specifically to an epitope (e.g., of an antigen).
  • Naturally-occurring human antibodies typically include two identical heavy chains and two identical light chains, each of which includes a variable domain and a constant domain.
  • the constant domain defines the antibody isotype (IgG, IgA, IgM, IgE, etc).
  • camelid antibodies are single chain antibodies.
  • antibody variable domains are typically characterized by framework region (FR) sequences and complement-determining-region (CDR) sequences.
  • Each variable domain typically includes three CDRs - CDR1, CDR2, and CDR3, which together contribute to specificity and/or affinity of epitope binding.
  • antibodies may be produced in or by organisms. In some embodiments, antibodies may be produced in or by cells in vitro (e.g., by hybridomas and/or by engineered cells). In some embodiments, antibody fragment(s) (e.g., as may be produced by cleavage or recombinantly) may be generated and/or utilized in accordance with the present disclosure.
  • binding features e.g., one or more CDRs and/or FR sequences, and in particular sets of 3 CDRs, optionally together with FR sequences
  • CDR sequences may be maintained and other elements changed - e.g., as is done in humanization.
  • variable regions may be associated with alternative constant regions (e.g., with constant regions from a different organism and/or that include one or more particular sequence features or elements desired, for example, to impart a particular attribute to the antibody agent.
  • antibody agents i.e., agents that incorporate antibody binding sequences such as one or more CDRs, or a full set of CDRs
  • antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof: single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE
  • Characteristic sequence element refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer.
  • presence of a characteristic sequence element correlates with presence or level of a particular activity or property of the polymer.
  • presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers.
  • a characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides).
  • a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., contiguously linked monomers).
  • a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share the sequence element.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • corresponding to refers to a relationship between two or more entities.
  • corresponding to may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition).
  • a monomeric residue in a polymer may be identified as “corresponding to” a residue in an appropriate reference polymer.
  • residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids.
  • sequence alignment strategies including software programs such as, for example, BLAST. CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER. HHpred/HHsearch, IDF. Infernal. KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify 7 “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
  • software programs such as, for example, BLAST. CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER. HHpred/HHsearch, IDF. Infernal. KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, Scal
  • corresponding to may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity).
  • a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identify or homology, or shares a particular characteristic sequence element.
  • Dosage form or unit dosage form may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject.
  • an active agent e.g., a therapeutic or diagnostic agent
  • each such unit contains a predetermined quantity of active agent.
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
  • individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • 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 the 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 the 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.
  • the 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 w as performed on a prior entity.
  • Heterologous is used herein to refer to an agent introduced into or otherwise present in or produced by a cell - at all, or at a particular level, or in a particular form - that would not be e.g., produced by or present in such cell (e.g., at all, at such level, or in such form), if not for engineering of the cell.
  • a heterologous agent may have been physically introduced into a cell (as, for example, a heterologous nucleic acid may have been transformed or transfected into a cell), or may have been expressed from an agent (e.g., a nucleic acid) so introduced.
  • a heterologous agent may be “heterologous” in that it is present at a different level (e.g., over- or -under expressed) relative to that at which it would be present absent engineering (e.g., genetic engineering of such cell.
  • homologous refers to 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 “substantially 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% homologous, meaning that identical or homologous (e.g., representing conservative substitutions) residues are present in corresponding positions of both molecules.
  • Calculation of percent homology of two nucleic acid or polypeptide sequences can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • a 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 length of a reference sequence; residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g..).
  • the two molecules i.e., first and second are identical at that position.
  • a position in the first sequence is occupied by the same residue or by a structurally and/or functionally related residue (as will be understood by those skilled in the art, in context)
  • the two molecules are considered "‘homologous’ 7 at that position.
  • Percent homology between two sequences is a function of the number of homologous positions shared by the two sequences being compared, 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. Comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
  • percent homology between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety ), which has been incorporated into the ALIGN program (version 2.0).
  • Host cell refers to a cell into which an exogenous nucleic acid (e.g., an engineered nucleic acid has been introduced and/or which otherwise expresses or comprises an engineered or otherwise heterologous polypeptide.
  • exogenous nucleic acid e.g., an engineered nucleic acid has been introduced and/or which otherwise expresses or comprises an engineered or otherwise heterologous polypeptide.
  • progeny refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progenymay not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • Nucleic acid As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
  • a "nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
  • a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
  • a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine).
  • adenosine thymidine, guanosine, cytidine
  • uridine deoxyadenosine
  • deoxythymidine deoxy guanosine
  • deoxy cytidine deoxy cytidine
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (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.
  • a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and 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, 1 10, 120, 130, 140, 150, 160, 170. 180, 190, 20, 225, 250, 275, 300, 325. 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900. 1000, 1500, 2000, 2500.
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
  • Polypeptide As used herein refers to a polymeric chain of amino acids.
  • 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 non-natural amino acids.
  • a polypeptide may comprise D-amino acids. L-amino acids, or both.
  • 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. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion.
  • a polypeptide is not cyclic and/or does not comprise any cyclic portion.
  • a polypeptide is linear.
  • a polypeptide may be or comprise a stapled polypeptide.
  • the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • 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 relevant 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.
  • Reference As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
  • treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject know n to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.
  • 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 chemical moi eties as compared to the reference entity. In some embodiments, a variant also differs functionally from 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. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a polypeptide may have a characteristic sequence element comprised of a plurality' of amino acids having designated positions relative to one another in linear or three- dimensional space and/or contributing to a particular structural motif and/or biological function;
  • a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three- dimensional space.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • moieties e.g., carbohydrates, lipids, phosphate groups
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
  • a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference.
  • a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference.
  • a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference.
  • a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19. about 18, about 17, about 16, about 15, about 14, about 13, about 10.
  • a reference polypeptide or nucleic acid is one found in nature. In some embodiments, a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.
  • Vector typically refers to an agent used to deliver genetic material (e.g., a genetic payload, in many embodiments encoding a polypeptide of interest) into cells.
  • a vector utilized in accordance with the present disclosure is a viral vector.
  • a viral vector includes a nucleic acid with features characteristic of a viral genome.
  • a viral vector is or comprises a viral capsid.
  • a viral vector is an enveloped vector.
  • a vector, e.g., a viral vector is not replication-competent absent one or more helper polypeptides.
  • a viral vector may be or comprise an adenovirus vector, and adeno-associated virus vector, a baculovirus vector, a herpes simplex virus vector, a lentivirus vector, a poxvirus vector, or a retrovirus vector.
  • AAV vectors are particularly useful.
  • the present disclosure relates, among other things, to production of heterologous polypeptides in mammalian cells in vitro (i.e.. in cultured mammalian cells).
  • relevant cell cultures are adherent cell cultures.
  • relevant cell cultures are suspension cell cultures.
  • useful cell lines may be rodent cells, for example Chinese hamster ovary (CHO) cells.
  • CHO Chinese hamster ovary
  • CHO cells have been demonstrated to be useful for the production of a variety of heterologous polypeptides including, for example, antibody polypeptides, enzyme polypeptides, and/or other therapeutic polypeptides may be produced in any of the culture systems disclosed herein.
  • useful cell lines may be mammalian cell lines, such as for example human embryonic kidney (HEK) cells (e.g., HEK293F, HEK293FT, HEK293T, HEK293S, HEK293FTM, HEK293SG, HEK293SGGD, HEK 293H, HEK293A, HEK293E, HEK293 EBNA1-6E, or HEK293MSR, Expi293 cells).
  • HEK human embryonic kidney
  • CHO Chinese hamster ovary
  • HeLa cells e.g., HeLa S3 cells
  • PER.C6 cells HKB11 cells
  • CAP cells Baby Hamster Kidney fibroblasts (BHK cells) (e.g., BHK-21 cells)
  • mouse myeloma cells e.g., Sp2/0 cells and NSO cells
  • green African monkey kidney cells e.g., COS cells and Vero cells
  • A549 cells rhesus fetal lung cells (e.g., FRhL-2 cells), and any derivatives thereof.
  • certain mammalian cells and particularly certain human cells, such as for example, HEK293 cells, have been demonstrated to be particularly useful for production of certain viral capsid polypeptides, including specifically AAV capsid polypeptides (and, moreover, AAV capsids).
  • provided technologies are particularly useful for the production of AAV capsids from HEK293 cells.
  • AAV particles comprising an AAV capsid (e.g., as described herein) and a nucleic acid sequence encoding a payload (e.g., as described herein).
  • mammalian cell lines disclosed herein are suitable for adherent cell culture.
  • mammalian cells are cultured in an adherent cell culture medium.
  • mammalian cells can be grown under serum-free conditions.
  • mammalian cell lines disclosed herein are suitable for suspension cell culture.
  • mammalian cells suitable for suspension cell culture are CHO cells (e.g., CHO-K1, CHO-S, CHO-DG44, and/or CHO-DXB11 cells).
  • HEK 293 cells e.g., 293SF, 3F6, 293T
  • HeLa cells and derivatives thereof.
  • mammalian cells can be cultured in suspension under serum-free conditions.
  • HEK293 cells have the ability to grow in suspension under serum-free conditions.
  • cell lines disclosed herein are cultured in suspension cell culture.
  • mammalian cells for suspension cell culture as suitable for culturing in large quantities e.g., > 1 L capacity, > 2 L capacity, > 3 L capacity, > 4 L capacity 7 , > 5 L capacity 7 , > 10 L capacity, > 20 L capacity, > 30 L capacity 7 , > 40 L capacity, > 50 L capacity, > 60 L capacity, > 70 L capacity, > 80 L capacity, > 90 L capacity, > 100 L capacity, > 200 L capacity. > 300 L capacity, > 400 L capacity, or > 500 L capacity).
  • mammalian cell lines disclosed herein are suitable for manufacturing of biologies (e.g., heterologous polypeptides as described herein, which in many embodiments may be or comprise viral capsid polypeptides, particularly as viral capsids, which in many embodiments may be enveloped or otherwise may be viral vectors).
  • biologies e.g., heterologous polypeptides as described herein, which in many embodiments may be or comprise viral capsid polypeptides, particularly as viral capsids, which in many embodiments may be enveloped or otherwise may be viral vectors.
  • a mammalian cell line is suitable for use in industrialscale manufacturing of a biologic product.
  • a mammalian cell line is suitable for use in a method of manufacture that conforms with local regulatory 7 standards (e.g., FDA and/or EMA regulatory 7 standards).
  • a mammalian cell line is suitable for manufacturing of biologies (e.g., heterologous polypeptides as described herein, and in particular viral capsid polypeptides, particularly as part of viral capsids and/or viral vectors) using current good manufacturing practices (cGMP).
  • biologies e.g., heterologous polypeptides as described herein, and in particular viral capsid polypeptides, particularly as part of viral capsids and/or viral vectors
  • a mammalian cell line is suitable for manufacturing of biologies (e.g., heterologous polypeptides as described herein, and in particular viral capsid polypeptides, particularly as part of viral capsids and/or viral vectors) using good manufacturing practices (GMP).
  • a mammalian cell line is suitable for manufacturing of biologies (e.g.. heterologous polypeptides as described herein, and in particular viral capsid polypeptides, particularly as part of viral capsids and/or viral vectors) using non-good manufacturing practices (non-GMP).
  • the present disclosure relates to production of heterologous polypeptides in mammalian cells in vitro (e.g., in cultured mammalian cells).
  • a produced polypeptide may be or comprise a therapeutic polypeptide.
  • a produced polypeptide may be or comprise, for example, an antibody polypeptide, a cytokine polypeptide, an enzyme polypeptide, a hormone polypeptide, etc.
  • a produced polypeptide may be or comprise a fusion polypeptide.
  • a produced polypeptide may be or comprise a viral capsid polypeptide; in some particular embodiments, a produced polypeptide may be or comprise a viral capsid polypeptide or component thereof, e.g., a viral capsid polypeptide (which, in some embodiments, may encapsulate a viral genome). In some embodiments, a capsid polypeptide is part of a viral capsid. In some embodiments, a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • a produced polypeptide is or comprises a viral capsid polypeptide which is part of a viral capsid which is enveloped.
  • a viral capsid polypeptide is part of a viral vector (e.g.. that may include a viral genome comprising a payload nucleic acid - e.g., a gene of interest).
  • a produced polypeptide may be or comprise an antibody polypeptide.
  • an antibody polypeptide comprises Muromonab, Nebacumab, Abciximab, Edrecolomab, Daclizumab, Rituximab, Infliximab, Trastuzumab, Palivizumab, Basiliximab, Etanercept, Gemtuzumab ozogamicin, Alemtuzumab, Adalimumab, Ibritumomab tiuxetan.
  • Omalizumab Efalizumab. Alefacept, Tositumomab, Cetuximab.
  • Bevacizumab Natalizumab. Tocilizumab. Abatacept.
  • Panitumumab, Derlotuximab biotin Metuximab, Nimotuzumab, Ranibizumab, Eculizumab, Racotumomab, Rilonacept, Certolizumab pegol, Romiplostim, Canakinumab, Catumaxomab, Ustekinumab, Ofatumumab, Golimumab, Demosumab, Brentuximab vedotin, Belatacept, Aflibercept, Belimumab. Iplimumab, Pertuzumab, Raxibacumab.
  • Mogamulizumab Trastuzumab emtansine, Obnutuzumab, Conbercept, Itolizumab, Blinatumomab, Pembrolizumab, Alprolix, Vedolizumab, Secukinumab, Ramucirumab, Efmoroctocog alfa, situxumab.
  • Ibalizumab Galcanezumab, Tildrakizumab.
  • Emapalumab Cemipilimab, Toripalimab, Caplacizumab, Lanadelumab, Burosumab, Tislelizumab, Risankizumab, Trastuzumab deruxtecan, Brolucizumab, Crizanlizumab, Enfortumab vedotin, Romosozumab, Efgartigimod alfa, Camrelizumab, Polatuzumab vedotin, Luspatercept, Ineblizumab, Teprotumumab, Sacituzumab govitecan, Isatuximab, Levlilmab, Margetuximab.
  • Satralizumab Tafasitamab, Naxitamb, Ansuvimab.
  • Epitinezumab Prolgolimab, Olokizumab, Penpulimab, Dostralimab, Evinacumab, Sugemalimab, Envafolimab, Telitacicept, Regdanvimab, Amivantamb, Bimekizumab, Tralokinumab, Tezepelumab, Aducanumab, Sortovimab, Anifrolumab, Ormutivimab, Faricimab, Sutimilimab.
  • Mosunetuzumab Cadonilimab, Nemolizumab, Serplulima, Tebentafsup, or a fragment (e.g., an antigen binding fragment) or a variant of any of the foregoing.
  • a produced polypeptide may be or comprise a cytokine polypeptide.
  • a cytokine is a pro-inflammatory cytokine.
  • a cytokine is chosen from: IL-1, IL-2, IL-3, IL -4, IL-5, IL-6, IL7-, IL-12, IL- 13, IL-15, IL-17, IL-18, IL-21, IL-23, TNFa, TNF , IFNa, IFN
  • a produced polypeptide may be or comprise a chemokine polypeptide.
  • a chemokine is chosen from: CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, CCL2, CCL8, CCL12, CCL13, CCL19, CCL21, CX3CL1, CXCR3, CCR1, CCR5, CCR2 or CX3CR1.
  • a produced polypeptide may be or comprise an enzyme polypeptide.
  • an enzy me polypeptide is an enzyme or a fragment or variant thereof that is useful as enzyme replacement therapy.
  • an enzyme is Alphal -Antitrypsin, (3-Glucocerebrosidase.
  • Adenosine Deaminase Alpha- Galactosidase A, Acid alpha-Glucosidase, a-L-Iduronidase, Iduronate-2-Sulfatase, N- Acetylgalactosamine-6 Sulfatase, N-Acetylgalactosamine-4 Sulfatase, or Lysosomal Acid Lipase, or fragments or variants of any of the foregoing.
  • a produced polypeptide may be or comprise a hormone polypeptide.
  • a hormone is Adrenaline. Melatonin. Noradrenaline.
  • Parathyroid hormone Pituitary adenylate cyclase-activating peptide, Prolactin, Prolactin-releasing hormone, Relaxin, Renin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulating hormone, Thyrotropin-releasing hormone.
  • Vasoactive intestinal peptide Guanylin. Uroguanylin. androgen, estrogen, progesterone.
  • Vitamin D e.g., 1.25-dihydroxy vitamin D3. or 25-hydroxyvitamin D3.
  • Adeno-associated Virus (AA V) Capsid polypeptides and AA V Vectors
  • a produced polypeptide may be or compnse an AAV capsid polypeptide, or component thereof; in some embodiments, such AAV capsid polypeptide or component thereof is or comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7. AAV8, AAV9, AAV10, or AAV11 capsid polypeptide or component thereof.
  • an AAV capsid is an AAV5 capsid polypeptide.
  • an AAV capsid is and AAV8 capsid polypeptide.
  • an AAV capsid is a hybrid capsid polypeptide (e.g., an AAV-DJ capsid, such as an AAV-DJ8 capsid), or other engineered capsid polypeptide (e.g., an engineered capsid variant).
  • a hybrid capsid polypeptide e.g., an AAV-DJ capsid, such as an AAV-DJ8 capsid
  • other engineered capsid polypeptide e.g., an engineered capsid variant
  • an AAV capsid polypeptide is part of a viral capsid.
  • a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • an AAV capsid polypeptide is part of a viral vector.
  • a produced polypeptide is or comprises a recombinant AAV particle comprising an AAV capsid polypeptide and a nucleic acid encoding a payload.
  • viral vectors disclosed herein comprise adeno- associated virus (AAV) vectors.
  • AAVs are commonly used viral vectors for gene delivery.
  • an AAV vector has low immunogenicity (e.g., in humans).
  • an AAV vector is compatible with a broad range of host cells.
  • an AAV vector can transduce both dividing and quiescent cells.
  • a mammalian cell of the present disclosure produces an AAV vector as described herein.
  • the present disclosure provides nucleic acid sequences encoding one or more elements for production of an AAV vector.
  • Elements for an AAV vector can include Rep proteins and/or capsid (Cap) proteins (e.g., VP1, VP2 and VP3, which form an AAV capsid).
  • elements for an AAV vector can be encoded on one or more constructs (e.g., that may be integrated or present episomally within a mammalian cell).
  • nucleic acids encoding one or more elements essential for production of an AAV vector are integrated into the genome of a mammalian cell.
  • nucleic acids encoding one or more elements essential for production of an AAV vector are present episomally in a mammalian cell.
  • an AAV vector that include a capsid and a nucleic acid comprising a payload.
  • an AAV vector has an icosahedral protein capsid that encompasses a linear, single stranded DNA nucleic acid.
  • an AAV vector is also referred to as an AAV particle.
  • a viral vector produced by mammalian cells and/or methods of the present disclosure will comprise an AAV capsid and a nucleic acid, wherein the nucleic acid comprises (i) a payload, (ii) an identifier (e.g., comprising a barcode and/or library variant), and (iii) two ITR sequences (e.g.. derived from AAV).
  • the nucleic acid comprises (i) a payload, (ii) an identifier (e.g., comprising a barcode and/or library variant), and (iii) two ITR sequences (e.g.. derived from AAV).
  • an AAV vector is derived from (e.g.. is a variant of, such as an engineered variant of) a human AAV I ; AAV2; AAV3b; AAV4; AAV5; AAV6; AAV7; AAV8; AAV9; AAV 10; AAV11; AAV 12; AAV 13, or any variant, e.g., any engineered variant thereof.
  • an AAV vector is a synthetic and/or hybrid human AAV vector.
  • an AAV vector is derived from a bovine AAV (b-AAV); canine AAV (CAAV); mouse AAV1; caprine AAV; rat AAV; or avian AAV (AAAV).
  • AAV vectors can be described as having a serotype, which is a description of the capsid strain and the strain of certain sequences of the nucleic acid (e.g., ITRs).
  • an AAV vector may be described as AAV2, wherein the vector has an AAV2 capsid and a nucleic acid that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs).
  • ITRs Inverted Terminal Repeats
  • an AAV vector may be described as a pseudotype, wherein the capsid and ITRs are derived from different AAV strains, for example, AAV 2/9 would refer to an AAV vector that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid.
  • an AAV vector does not have a serotype and/or pseudotype.
  • an AAV vector comprises engineered AAV capsid and/or ITRs (e.g., that do not have significant homolog ⁇ ' to that of a known AAV serotype).
  • AAV vectors that may be produced or otherwise utilized in accordance with the present disclosure comprise an AAV capsid.
  • an AAV capsid is from or derived from an AAV capsid of an AAV2, 3. 4. 5, 6, 7, 8, 9, 10, rh8, rhlO, rh39, rh43 or Anc80 serotype, or one or more hybrids thereof.
  • an AAV capsid is from an AAV ancestral serotype.
  • an AAV capsid is an ancestral (Anc) AAV capsid.
  • An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence.
  • an AAV capsid/construct sequence is not known to have existed in nature.
  • an AAV capsid is an artificially engineered sequence (e.g., that does not have significant homology to a known AAV serotype capsid).
  • AAV vectors that may be produced or otherwise utilized in accordance with present disclosure may include any combination of AAV capsids and AAV nucleic acids (e.g., comprising a payload and/or AAV ITRs).
  • AAV capsids e.g., comprising a payload and/or AAV ITRs
  • wild type or variant AAV capsid that encapsulates an AAV nucleic acid comprising an identifier and/or a payload flanked by AAV-derived ITRs.
  • an AAV nucleic acid is comprised of single-stranded deoxyribonucleic acid (ssDNA).
  • an AAV nucleic acid comprises one or more components derived from or modified from a naturally occurring AAV genome.
  • an AAV nucleic acid comprises inverted terminal repeats (ITRs) sequences that have been derived from or modified from an AAV.
  • ITRs inverted terminal repeats
  • an AAV nucleic acid comprises a payload sequence and two ITRs.
  • an AAV vector comprises a capsid and a ssDNA comprising a payload sequence and two viral repeat sequences, e.g., ITR sequences, one at each end of the DNA strand (5‘ and 3’).
  • provided AAV nucleic acids comprise a payload that includes a coding sequence and one or more regulatory and/or control sequences, and optionally 5’ and 3’ AAV derived inverted terminal repeats (ITRs).
  • provided AAV nucleic acids are packaged into an AAV capsid to form an AAV vector.
  • a viral vector comprises a nucleic acid comprising an identifier and/or a payload sequence and associated regulatory elements that are flanked by 5’ or “left” and 3‘ or “right” AAV ITR sequences.
  • 5’ and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
  • One of ordinary skill in the art understands how to modify a given ITR sequence for use as either a 571eft or 37right ITR, or an antisense version thereof.
  • AAV nucleic acids included in AAV vectors that may be produced or otherwise utilized in accordance with the present disclosure include ⁇ cis- acting 5’ and 3’ ITR sequences (see, e.g., B. J. Carter, in “Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp. 155-168, 1990, which is incorporated herein by reference in its entirety).
  • at least 80% of a typical ITR sequence e.g., at least 85%, at least 90%, or at least 95%) is incorporated into a construct provided herein.
  • Those skilled in the art are familiar with a variety of strategies that may usefully be employed to modify ITR sequence.
  • an identifier and/or a payload sequence is flanked by 7 5’ and 3’ AAV ITR sequences.
  • an AAV nucleic acid comprises an identifier and a pay load flanked by 5’ and 3‘ AAV ITR sequences.
  • the AAV ITR sequences may be obtained from any known AAV. including presently identified AAV types.
  • an AAV vector nucleic acid comprises a payload, an identifier, and two AAV ITRs.
  • an AAV vector comprises a capsid and a dsDNA comprising (i) a payload and/or an identifier, and (ii) two AAV ITR sequences, one at each end of the DNA strand (5’ and 3 ? ).
  • ITRs are able to form a hairpin.
  • the ability to form a hairpin can contribute to an ITR’s ability' to self-prime, allowing primase-independent synthesis of a second DNA strand.
  • TTRs can also aid in efficient encapsulation of an AAV construct in an AAV vector.
  • An AAV ITR sequence may be obtained from any known AAV, including mammalian AAV types.
  • an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, of a naturally occurring ITR sequence.
  • modifications e.g., truncations, deletions, substitutions or insertions.
  • the ability to modify these ITR sequences is within the skill of the art. (See, e.g.. texts such as Sambrook et al. ‘'Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory 7 , New York (1989); and K. Fisher et al., J Virol., 70:520-532 (1996), each of which is incorporated in its entirety 7 herein by reference).
  • AAV2-derived ITR sequences are about 145 nucleotides in length.
  • an ITR comprises fewer than 145 nucleotides, e.g., 127, 130, 134 or 141 nucleotides.
  • an ITR comprises 110, 111, 112, 113, 114, 1 15, 116, 117, 118, 119, 120, 121, 122, 123 ,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141. 142, 143 144, or 145 nucleotides.
  • an AAV vector payload also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription, translation and/or expression in a cell transfected with a construct or infected with the viral vector produced by the disclosure.
  • an AAV vector payload optionally comprises a promoter, an enhancer, an untranslated region (e.g., a 5’ UTR, 3’ UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g.. an acceptor site, a donor site), a polyadenylation site, and/or any combination thereof.
  • an AAV vector payload is less than 4 kb.
  • an AAV vector payload can include a sequence that is at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb.
  • an AAV vector payload can include a sequence that is at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb.
  • an AAV vector payload can include a sequence that is about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb. about 2 kb to about 4 kb, about 2 kb to about 5kb. about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
  • an AAV vector can direct long-term expression of a payload. Tn some embodiments, an AAV vector can direct transient expression of a payload.
  • an AAV vector produced by mammalian cells and/or methods of the present disclosure will comprise an AAV capsid and a nucleic acid, wherein the nucleic acid comprises (i) a pay load, (ii) an identifier (e.g., a barcode and/or a library' variant), and (iii) two viral repeat sequences (e.g., ITRs derived from AAV).
  • the nucleic acid comprises (i) a pay load, (ii) an identifier (e.g., a barcode and/or a library' variant), and (iii) two viral repeat sequences (e.g., ITRs derived from AAV).
  • mammalian cells of a mammalian cell library' are genetically modified to comprise one or more nucleic acid sequences essential for production of an AAV vector.
  • mammalian cells of a mammalian cell library may have one or more AAV vector components provided such as, e.g., rep sequences, cap sequences, and helper functions required for producing an AAV vector.
  • one or more components of an AAV vector e.g., an AAV Rep gene, an AAV Cap gene, one or more helper genes, or a combination thereof
  • an inducible transcriptional control element e.g., promoter and/or enhancer.
  • a mammalian cell (e.g., of a population of mammalian cells) comprises: (i) an identifier positioned between two viral repeat sequences, and (ii) one or more polynucleotides comprising an AAV Rep gene, an AAV Cap gene, one or more helper genes, and/or a combination thereof, wherein the mammalian cell(s) produce an AAV vector comprising the identifier.
  • the AAV vector comprises a payload.
  • an identifier and/or pay load or a portion of thereof is later removed from the AAV vector.
  • the payload or a portion thereof is replaced with a different payload or a portion thereof.
  • a produced polypeptide may be or compnse an adenovirus capsid polypeptide, or component thereof.
  • an adenovirus capsid polypeptide is part of a viral capsid.
  • a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • viral vectors produced by methods and mammalian cells of the present disclosure are adenovirus vectors.
  • Adenoviruses are non-enveloped viruses that are commonly used as vaccines because of the strong immunogenic response they induce. Some adenoviruses are utilized for cancer therapy because of their abi 1 i ty to preferentially infect and kill cancer cells.
  • an adenovirus vector is derived from a human adenovirus.
  • Human adenoviruses encompass a family of at least 51 serotypes that are classified into several subgroups.
  • subgroup A includes adenovirus serotypes 12, 18, and 31.
  • Subgroup B includes adenovirus serotypes 3, 7, 1 la, l ip, 14, 16, 21, 34, 35 and 50.
  • Subgroup C includes adenovirus serotypes 1, 2, 5, and 6.
  • Subgroup D includes adenovirus seroty pes 8, 9, 10, 13, 15, 17, 19, 19p, 20, 22-30, 32, 33, 36-39, 42-49 and 51.
  • Subgroup E includes adenovirus serotype 4.
  • Subgroup F includes adenovirus serotypes 40 and 41.
  • An adenovirus vector of the present disclosure can be of any adenovirus group, subgroup, and/or serotype.
  • an adenovirus vector is derived from any serotype, such as a serotype 1 to a serotype 51 (e.g. 1, 2, 4, 5 . . . 51).
  • a serotype 1 e.g. 1, 2, 4, 5 . . . 51
  • an adenovirus is an adenovirus type 2 or adenovirus type 5.
  • an adenovirus vector is derived, at least in part, from adenovirus type 5.
  • an adenovirus vector is replication-defective (e.g., certain essential viral genes are deleted and/or replaced with a payload sequence). Any of the adenovirus vectors used in methods described herein can include a deletion in any one or more of the El, E2a, E2b, E3, or E4 coding regions. In some embodiments, an adenovirus vector is replication-defective and lacks the E4 locus (e.g., E4 coding region is deleted). In some embodiments, a replication-defective adenovirus vector is useful as a vaccine, for cancer therapy and/or for gene therapy.
  • replication-defective adenovirus vector is useful as a vaccine, for cancer therapy and/or for gene therapy.
  • adenoviruses are characterized by high transduction efficiency and direct high transgene expression.
  • an adenovirus vector can direct transient expression of a payload.
  • an adenovirus vector directs transient expression of a payload in a target cell.
  • an adenovirus vector is replication-competent.
  • replication-competent adenovirus vectors e g., oncolytic vectors
  • the present disclosure provides adenovirus vectors that include a capsid and a nucleic acid comprising a payload.
  • an adenovirus vector has an icosahedral protein capsid that encompasses a linear duplex nucleic acid.
  • an adenovirus vector is about 90-100 nm in diameter.
  • an adenovirus vector has an icosahedral protein capsid that encompasses a linear, double stranded DNA.
  • an adenovirus vector nucleic acid is comprised of double-stranded DNA (dsDNA) and has one or more components derived from or modified from a naturally occurring adenovirus genome.
  • an adenovirus vector nucleic acid comprises inverted terminal repeats (ITRs) sequences that have been derived from or modified from an adenovirus of any serotj pe (e g., adenovirus type 5).
  • ITRs inverted terminal repeats
  • an adenovirus vector nucleic acid comprises a payload and two viral repeat sequences, such as ITRs.
  • an adenovirus vector nucleic acid comprises an identifier and two viral repeat sequences, such as ITRs.
  • an adenovirus vector nucleic acid comprises a payload, an identifier, and two viral repeat sequences, such as ITRs.
  • an adenovirus vector comprises a capsid and a dsDNA comprising (i) a payload and/or an identifier, and (ii) two 1TR sequences, one at each end of the DNA strand (5’ and 3’).
  • adenovirus ITRs have a sequence that has a length about 40 bp to about 200 bp.
  • ITRs of human adenovirus type 5 are 103 bp.
  • an adenovirus ITR comprises a length of about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110 bp, about 120 bp. about 130 bp, about 140 bp. about 150 bp, about 160 bp, about 170 bp.
  • an adenovirus ITR sequence is about 50 to about 250 bp.
  • provided adenovirus vectors comprise an adenoviral- derived capsid and a nucleic acid that comprises a payload and one or more adenovirus- derived sequences (e.g., such as ITRs).
  • provided adenovirus vectors comprise an adenoviral-derived capsid and a nucleic acid that comprises an identifier, a payload, and at least two viral repeat sequences.
  • an adenoviral vector comprises an identifier that comprises a barcode and/or a library variant.
  • an adenoviral vector payload includes a coding sequence and one or more regulatory and/or control sequences.
  • an adenoviral vector comprises 5’ and 3’ adenovirus-derived inverted terminal repeats (ITRs).
  • an adenovirus vector comprises a capsid and an engineered adenovirus genome, wherein the engineered genome comprises a deletion of certain sequences (e.g.. an El deletion and/or E3 deletion) and an insertion of a payload and/or an identifier.
  • an adenovirus vector payload is less than 7.5 kb.
  • an adenovirus vector pay load can include a sequence that is at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb. at least 7.5 kb.
  • an adenovirus vector payload can include a sequence that is at most 10 kb, at most 9.5 kb, at most 9 kb, at most 8.5 kb, at most 8 kb, at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, or at most 4 kb.
  • an adenovirus vector payload can include a sequence that is about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb.
  • an adenovirus vector produced by mammalian cells and/or methods of the present disclosure will comprise an adenovirus capsid and a nucleic acid, wherein the nucleic acid comprises (i) a payload, (ii) an identifier (e.g., a barcode and/or a library variant), and (iii) two viral repeat sequences (e.g., ITRs derived from adenovirus).
  • an identifier e.g., a barcode and/or a library variant
  • two viral repeat sequences e.g., ITRs derived from adenovirus
  • mammalian cells of a mammalian cell library are genetically modified to comprise one or more nucleic acid sequences essential for production of an adenovirus vector.
  • mammalian cells of a mammalian cell library may have one or more adenovirus vector components provided such as. e.g., rep sequences, cap sequences, and helper functions required for producing an adenovirus vector.
  • one or more components of an adenovirus vector are under the control of an inducible transcriptional control element (e.g., promoter and/or enhancer).
  • an inducible transcriptional control element e.g., promoter and/or enhancer
  • a mammalian cell (e.g., of a population of mammalian cells) comprises: (i) an identifier positioned between two viral repeat sequences, and (ii) one or more polynucleotides comprising an adenovirus Rep gene, an adenovirus Cap gene, one or more helper genes, and/or a combination thereof, wherein the mammalian cell(s) produce adenovirus vector comprising the identifier.
  • the adenovirus vector comprises a payload.
  • an identifier and/or pay load or a portion of thereof is later removed from the adenovirus vector.
  • the pay load or a portion thereof is replaced with a different pay load or a portion thereof.
  • Retroviral and Lentiviral Capsid Polypeptides and Retrovirus and Lentivirus
  • a produced polypeptide may be or comprise a retroviral capsid polypeptide, or component thereof.
  • a retroviral capsid polypeptide is part of a viral capsid.
  • a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • retroviral vectors are enveloped viruses that replicate in a host cell by using a viral reverse transcriptase enzyme to transcribe its RNA into DNA.
  • the retroviral DNA replicates as part of the host genome and is referred to as a provirus.
  • Retroviral vectors may include, but are not limited to, those based upon or derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.
  • a produced polypeptide may be or comprise a lentivirus capsid polypeptide, or component thereof.
  • a lentivirus capsid polypeptide is part of a viral capsid.
  • a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • viral vectors produced by methods and mammalian cells of the present disclosure are lentivirus vectors.
  • Lentiviral vectors are versatile tools because of their ability to transduce non-dividing cells.
  • a lentivirus vector is capable of infecting infect both dividing and non-dividing cells.
  • lenti viral vectors enable long-term and/or stable gene expression and are integrated into a host cell genome.
  • Lentiviruses are enveloped particles that are about 80 to about 120 nm in diameter.
  • the present disclosure provides lentiviral vectors have a diameter within a range of about 50 nm to about 200 nm in diameter.
  • the present disclosure provides lentiviral vectors have a diameter within a range of about 80 nm to about 120 nm in diameter.
  • Lentiviruses may contain several structural proteins, including matrix, capsid, nucleocapsid, envelope, and reverse transcriptase proteins.
  • the present disclosure provides lentiviral vectors that include a capsid, an envelope.
  • the present disclosure provides lentiviral vectors that include a capsid, an envelope, and a nucleic acid.
  • the present disclosure provides lentiviral vectors that include a capsid, an envelope, and a nucleic acid that comprises a payload and/or an identifier.
  • a lentivirus vector produced by mammalian cells and/or methods of the present disclosure will comprise a lentivirus capsid (or a derivative thereof), an envelope, and a nucleic acid, where the nucleic acid comprises (i) a payload, (ii) an identifier, and (iii) two long terminal repeat (LTR) sequences (e.g., derived from lentivirus).
  • the two LTR sequences are capable packaging a nucleic acid into a lentiviral vector.
  • a lentivirus vector comprises a lentiviral Psi sequence (or a derivative or engineered variant thereof).
  • lentiviral vectors include those based on Human Immunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, Simian Immunodeficiency Virus (SIV), and maedi-visna virus (MVV).
  • HIV-1 Human Immunodeficiency Virus
  • HIV-2 feline immunodeficiency virus
  • FIV feline immunodeficiency virus
  • equine infectious anemia virus HIV
  • Simian Immunodeficiency Virus (SIV) Simian Immunodeficiency Virus
  • MVV maedi-visna virus
  • a lentiviral nucleic acid (i.e., engineered genome) and envelope glycoproteins will be based on different viruses, such that the resulting viral vector is pseudotyped.
  • a lentiviral vector is derived from HIV-1.
  • a lentiviral vector is derived from HIV-1 and comprises capsid protein and nucleic acid comprising (i) a payload, (ii) an identifier, and (iii) two long terminal repeat (LTR) sequences (e.g.. derived from lentivirus).
  • LTR long terminal repeat
  • a lentiviral vector is a HIV vector and wherein the mammalian cell comprises two viral repeat sequences comprising HIV LTR sequences.
  • a lentiviral vector is a SIV vector and wherein the mammalian cell comprises two viral repeat sequences comprising SIV LTR sequences.
  • a lentiviral vector is an equine infectious anemia viral vector and wherein the mammalian cell comprises two viral repeat sequences comprising equine infectious anemia viral LTR sequences.
  • a lentiviral vector is a FIV vector and wherein the mammalian cell comprises two viral repeat sequences comprising FIV LTR sequences.
  • a lentiviral vector is a visna viral vector and wherein the mammalian cell comprises two viral repeat sequences comprising visna viral LTR sequences.
  • a lentiviral vector comprises a gag protein or a fragment thereof.
  • a lentiviral vector comprises a gag protein that comprises one or more domains selected from a matrix (MA), capsid (CA). and nucleocapsid (NC) domain.
  • a lentiviral vector comprises an envelope protein or a fragment thereof.
  • a lentiviral vector is a pseudotyped lentiviral vector, wherein the gag protein and the envelope protein are derived from different viruses.
  • a lentiviral vector comprises a gag protein and/or an env protein derived from a human immunodeficiency virus (HIV) vector, a simian immunodeficiency virus (SIV) vector, an equine infectious anemia virus vector, a feline immunodeficiency virus vector, a visna virus vector or a derivative thereof.
  • HAV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • equine infectious anemia virus vector a feline immunodeficiency virus vector
  • a visna virus vector or a derivative thereof.
  • a lentiviral vector comprises (i) a lentiviral gag gene, (ii) a lentiviral env gene, (iii) a lentiviral pol gene, or (iv) a combination thereof.
  • mammalian cells that express a lentiviral vector comprise one or more of: (i) a lentiviral gag gene, (ii) a lentiviral env gene, and (iii) a lentiviral pol gene.
  • safety features are incorporated into a lentivirus vector, which can include, e g., self-inactivating long terminal repeat (LTR) and integration deficiency.
  • LTR long terminal repeat
  • integration deficiency may be conferred by elements of the vector genome but may also derive from elements of the packaging system (e.g., a nonfunctional integrase protein that may not be part of the vector genome but supplied in trans).
  • Lentiviruses have a single stranded RNA (ssRNA) genome.
  • a lentivirus vector comprises a nucleic acid that is ssRNA, and comprises a payload and sequences derived from a lentivirus, such as HIV-1 and/or SIV.
  • a payload is flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome.
  • LTR long terminal repeat
  • a lentivirus vector comprises a nucleic acid that is ssRNA, and comprises an identifier and viral repeat sequences (e.g. HIV-1 and/or SIV LTRs).
  • a lentivirus vector comprises a nucleic acid that is ssRNA, and comprises a payload, an identifier, and viral repeat sequences (e.g. HIV-1 and/or SIV LTRs).
  • a lentiviral vector nucleic acid may comprise sequences from the 5' and 3' LTRs of a lentivirus, and in particular may comprise the R and U5 sequences from the 5' LTR of a lentivirus and an inactivated or self-inactivating 3' LTR from a lentivirus.
  • LTR sequences may be LTR sequences from any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. In some embodiments, LTR sequences are HIV LTR sequences.
  • a lentivirus vector payload is less than 8 kb.
  • a lentivirus vector payload can include a sequence that is at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, or at least 8 kb.
  • a lentivirus vector payload can include a sequence that is at most 10 kb. at most 9.5 kb. at most 9 kb. at most 8.5 kb, at most 8 kb, at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, or at most 4 kb.
  • a lentivirus vector payload can include a sequence that is about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb.
  • mammalian cells of a mammalian cell library are genetically modified to comprise one or more nucleic acid sequences essential for production of a lentivirus vector.
  • mammalian cells of a mammalian cell library' may have one or more lentivirus vector components provided.
  • one or more components of a lentivirus vector are under the control of an inducible transcriptional control element (e.g., promoter and/or enhancer).
  • a mammalian cell (e.g., of a population of mammalian cells) comprises: (i) an identifier positioned between two viral repeat sequences, and (ii) one or more polynucleotides essential for production of the lentivirus vector, wherein the mammalian cell(s) produce a lentivirus vector comprising the identifier.
  • the lentivirus vector comprises a payload.
  • an identifier and/or payload or a portion thereof is later removed from the lentivirus vector.
  • the pay load or a portion thereof is replaced with a different payload or a portion thereof.
  • a produced polypeptide may be or comprise a herpes simplex virus (HSV) capsid polypeptide, or component thereof.
  • HSV capsid polypeptide is part of a viral capsid.
  • a viral capsid is enveloped.
  • an enveloped viral capsid comprises a nucleic acid construct encoding a pay load.
  • viral vectors produced by methods and mammalian cells of the present disclosure are herpes simplex viruses (HSV) vectors.
  • HSV is a large, enveloped virus with an icosadeltahedral capsid containing a toroidal dsDNA genome.
  • HSV is characterized in vivo by life-long latent infection of neurons. This characteristic makes HSV vectors useful for long-term transgene expression.
  • an HSV vector is a replication-competent attenuated vector, a replication-incompetent recombinant vector, or a replication-defective helper-dependent vector.
  • an HSV vector has a diameter that is within a range that is between about 120 nm to about 200 nm. In some embodiments, an HSV vector is an enveloped particle that is about 120 to about 200 nm in diameter. In some embodiments, an HSV vector has a diameter that is within a range that is between about 100 nm to about 200 nm.
  • an HSV vector is from or derived from herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV). Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 6 and/or human herpesvirus 7, and/or a derivative thereof.
  • an HSV vector is from or derived from HSV-1, HSV-2, or a combination thereof (e.g., include capsid from HSV-1 and include nucleic acid sequences derived from HSV-2).
  • a viral vector is an HSV-AAV hybrid vector.
  • a native HSV-1 genome consists of two stretches of genomic coding regions, referred to as long and short unique segments (UL and US), which are each flanked by inverted repeated sequences (TRL/IRL and IRS/TRS. respectively).
  • an HSV vector is engineered to defect or delete ICPO, ICP4, ICP22, ICP27 and/or ICP47 to reduce toxicity. Methods and considerations for designing HSV vectors are known in the art, for example, Manservigi, et al., Open Virol J. 2010; 4: 123-156.
  • an HSV vector that include capsid protein and a nucleic acid comprising a payload.
  • an HSV vector comprises a capsid comprising VP5, VP19C. VP23, pre-VP22a and/or the maturational protease (UL26 gene product).
  • an HSV nucleic acid further comprises a sequence obtained or derived from an HSV virus (e.g., TRL/IRL and/or IRS/TRS sequences).
  • the present disclosure provides HSV vectors that include capsid protein and a nucleic acid comprising HSV viral repeat sequences (e.g., TRL/IRL and/or IRS/TRS sequences).
  • the present disclosure provides HSV vectors that include a capsid, an envelope, and a nucleic acid comprising a payload.
  • the present disclosure provides HSV vectors that include a capsid, an envelope, and a nucleic acid comprising a payload and HSV viral repeat sequences (e.g., TRL/IRL and/or IRS/TRS sequences).
  • HSV viral repeat sequences e.g., TRL/IRL and/or IRS/TRS sequences.
  • a HSV vector payload is less than 100 kb.
  • a HSV vector payload can include a sequence that is at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 40 kb, or at least 50 kb.
  • a HSV vector payload can include a sequence that is at most 150 kb.
  • a lentivirus vector payload can include a sequence that is about 1 kb to about 150 kb.
  • the present disclosure provides HSV vectors that include capsid protein and a nucleic acid comprising an identifier. In some embodiments, the present disclosure provides HSV vectors that include capsid protein and a nucleic acid comprising an identifier and HSV viral repeat sequences (e.g., TRL/IRL and/or IRS/TRS sequences). In some embodiments, the present disclosure provides HSV vectors that include a capsid, an envelope, and a nucleic acid comprising an identifier.
  • HSV viral repeat sequences e.g., TRL/IRL and/or IRS/TRS sequences
  • the present disclosure provides HSV vectors that include a capsid, an envelope, and a nucleic acid comprising an identifier and HSV viral repeat sequences (e.g., TRL/IRL and/or IRS/TRS sequences).
  • a HSV vector comprises the two viral repeat sequences comprising a terminal a sequence.
  • an HSV nucleic acid comprises a payload, an identifier, and one or more sequences obtained or derived from an HSV virus.
  • HSV vectors produced by methods and/or mammalian cells of the present disclosure include an HSV capsid, an envelope, and a nucleic acid comprising a payload, an identifier, and one or more sequences obtained or derived from an HSV virus (e.g., TRL/IRL and/or IRS/TRS sequences).
  • an HSV vector comprises an HSV capsid, an envelope, and a nucleic acid comprising a payload and/or an identifier, flanked by HSV viral repeat sequences (e.g., TRL/IRL and/or IRS/TRS sequences).
  • HSV viral repeat sequences e.g., TRL/IRL and/or IRS/TRS sequences.
  • mammalian cells of a mammalian cell library are genetically modified to comprise one or more nucleic acid sequences essential for production of an HSV vector.
  • mammalian cells of a mammalian cell library may have one or more HSV vector components provided.
  • one or more components of an HSV vector are under the control of an inducible transcriptional control element (e.g., promoter and/or enhancer).
  • a mammalian cell (e.g., of a population of mammalian cells) comprises: (i) an identifier positioned between two viral repeat sequences, and (ii) one or more polynucleotides essential for production of the HSV vector, wherein the mammalian cell(s) produce an HSV vector comprising the identifier.
  • the HSV vector comprises a payload.
  • an identifier and/or pay load or a portion thereof is later removed from the HSV vector.
  • the payload or a portion thereof is replaced with a different payload or a portion thereof.
  • Peptones are a protein decomposition product, typically generated by partial hydrolysis of a protein source such as, for example, an animal protein source such as meat, milk, or other body part(s) (e.g., blood, bones, connective tissue, skin, etc., and/or another collagen source) or animal-produced protein source such as silk, a microbial protein source, and/or a plant protein source.
  • a protein source such as, for example, an animal protein source such as meat, milk, or other body part(s) (e.g., blood, bones, connective tissue, skin, etc., and/or another collagen source) or animal-produced protein source such as silk, a microbial protein source, and/or a plant protein source.
  • an animal protein source may be or comprise one or more proteins (or fragments thereof) such as casein (and/or tryptone, which is fragmented casein), fibrin, gelatin, etc.
  • an animal protein source may be or comprise a powder such as fish powder, milk powder, shrimp powder, etc.
  • an animal protein source may be or comprise silk pupae.
  • an animal protein source may be or comprise blood.
  • a plant protein source may be or comprise, for example, beans, com, peas, potato, soy, wheat, etc.
  • plant peptones may have higher sugar content than that found with peptones derived from other protein sources.
  • Microbial peptones are often relatively simple, and often have relatively high protein content.
  • peptone products are powder products, commonly light yellow to brown-yellow in color.
  • a peptone preparation may have an average molecular weight within a range of about 200 to about 3000 daltons.
  • peptones may be prepared, for example, by hydrolysis (e.g.. acid, alkali, and/or protease-mediated hydrolysis) of a protein source.
  • peptones may contain multiple proteins (or fragments thereof).
  • a peptone preparation useful in accordance with the present disclosure may be characterized, for example, by one or more of total nitrogen content, amino nitrogen content, sugar content, nitrite content, phosphorous content, sodium chloride content, ash content, moisture content, vitamin content, free amino acid content, or combination(s) thereof.
  • a peptone preparation used in accordance with the present disclosure may have a total nitrogen content if at least about 8- 15%, for example as measured by the Kjeldahl method.
  • bacterial peptones often have a nitrogen content above 14.5%; plant peptones often have a somewhat lower nitrogen content (e.g., around 8.5%); meat peptones may often be in between (e.g., around 12%, for example, for many chicken peptones).
  • Amino nitrogen content The content of amino nitrogen can reflect the degree of protein hydrolysis present in a particular peptone preparation.
  • a peptone utilized in accordance with the present disclosure will have an amino nitrogen content, as may be determined, for example, by formaldehyde titration, of no less than 2.5%.
  • a peptone preparation utilized in accordance with the present disclosure is characterized by a sugar content within a range of about 0.5 to about 1.5. as may be measured, for example, by phenol sulfuric acid method.
  • sugar content of vegetable peptones is usually higher than that of animal peptones.
  • sugar content of casein peptone is typically around 0.5-0.7%
  • of chicken feather peptone is ty pically around 0.7- 0.8%
  • offish peptone is around 1.2-1.5%.
  • Sodium chloride content In some embodiments, it may be desirable to assess sodium chloride content of a peptone utilized in accordance with the present disclosure. In some embodiments, sodium chloride content can be determined by titration.
  • Ash content In some embodiments, it may be desirable to assess ash content of a peptone utilized in accordance with the present disclosure.
  • peptone ash is mainly derived from raw materials and inorganic salt substances such as phosphate, silicate and sodium chloride used in the production process.
  • the ash content is commonly relatively high, and for example can reach more than 20%. Enzyme hydrolysis can usually reduce or avoid high ash content.
  • a peptone preparation utilized in accordance with the present disclosure will have an ash content of no more than about 15.0%. in some embodiments, ash content can be measured according to GB 5009.4-2016.
  • Moisture content In some embodiments, it may be desirable to assess moisture content of a peptone utilized in accordance w ith the present disclosure. In many embodiments, moisture content of a peptone preparation will be less than about 5%, and often will be less than about 4%. for example when determined according to GB 5009.3-2016.
  • Vitamin content In some embodiments, it may be desirable to assess vitamin content of a peptone utilized in accordance with the present disclosure.
  • Free amino acid content it may be desirable to assess free amino acid content of a peptone utilized in accordance with the present disclosure.
  • a particularly useful peptone preparation may be characterized by a particular level of one or more free amino acids (e g., above or below a specified threshold, and/or within a particular range).
  • a particularly useful peptone preparation may be characterized by relative amounts of (e.g., ratio(s) between) two or more free amino acids.
  • peptones include those provided by commercial sources such as, for example, Lab Associates, Organotechnie, Sigma-Aldrich, ThermoFisher Scientific (see. for example, available on the world wide web at assets.thermofisher.com/TFS-Assets/BPD/brochures/peptones- supplements-feeds-technical-reference-guide.pdf, incorporated herein by reference), etc.
  • the present disclosure identifies the source of a problem with prior assessments of peptone impact on heterologous protein production in mammalian cells, including specifically on AAV capsid protein production.
  • the present disclosure demonstrates that certain peptones are surprisingly useful to enhance production levels of various AAV seroty pes, in some embodiments specifically including one or more notoriously low titer serotypes. Furthermore, the present disclosure teaches that, in some embodiments, peptone supplementation can be useful prior to and/or during transfection, notwithstanding established teachings in the art that presence of peptones cane be detrimental during these periods.
  • virus vectors e.g., of one or more of adenovirus vectors, baculovirus vectors, herpes simplex virus vectors, lentivirus vectors, poxvirus vectors, and/or retrovirus vectors and/or to production of other (i.e., other than viral capsid) heterologous polypeptides (e.g., as described herein).
  • Various technologies and/or insights provided by the present disclosure may be applied, for example, to the identification and/or characterization of useful peptone component(s), and/or concentration(s) and/or timing of use. of such peptone component(s) to enhance production of heterologous proteins from mammalian (or other, e.g., insect) cells in culture.
  • findings provided by the present disclosure can be utilized to map biological pathways that contribute to production of heterologous proteins from such cultured cells.
  • a technique for reducing the production of heterologous proteins from such cultured cells e.g., HEK293 cells.
  • provided teachings can be utilized to assess differences between such cells in the presence vs absence of such peptone preparation(s), thus revealing biological pathways whose activation or inhibition can benefit heterologous protein production when the cells are cultured.
  • the present disclosure provides teachings that permit successful production of certain heterologous polypeptide products, including specifically viral capsid polypeptide products, which may in some embodiments, be part of viral capsid products and/or viral vector products.
  • certain heterologous polypeptide products including specifically viral capsid polypeptide products, which may in some embodiments, be part of viral capsid products and/or viral vector products.
  • viral capsid polypeptide products may in some embodiments, be part of viral capsid products and/or viral vector products.
  • an expression system is a transient expression system.
  • an expression system is a packaging expression system, e.g., a system that comprises one or more nucleic acid sequences encoding rep, cap, and/or helper polypeptides.
  • rep, cap, and/or helper polypeptides have been stably integration into the genome of cells.
  • an expression system is a producer cell line where rep, cap, helper and/or a gene of interest (GOI) have been integrated into the genome of the cell ..
  • GOI gene of interest
  • Technologies provided herein can be useful to produce AAV particles at a desired titer and/or quality'.
  • the present disclosure provides the insight that peptone supplementation of a mammalian cell culture system (e.g., in a large scale, [e.g., in a bioreactor]) produces AAV particles at a titer and/or quality sufficient for use in one or more applications, e.g., therapeutic applications.
  • AAV particles produced with peptone supplementation are produced at a higher titer and/or a higher quality 7 compared to AAV particles produced without peptone supplementation.
  • a plurality' of recombinant AAV particles produced using a cell culture system and/or a method disclosed herein has an AAV titer within a range of 1E12 vector genomes (vg)/L to 1E17 vg/L.
  • an AAV titer is about 1E12 vg/L to about 1E17 vg/L, about 5E12 vg/L to about 1E17 vg/L.
  • the present Example demonstrates, among other things, that inclusion of peptones can enhance AAV production and achieve higher AAV titer yield from HEK293 cells.
  • HEK293 suspension cells were grown in Fl 7 FreeStyle Medium (Thermo Fisher Scientific) supplemented with 4 mM L-Glutamine (Gibco). For what we have considered to be "small” scales (2.5 mL or 30 mL), suspension cells were cultured in 24 deep well plates (NEST Scientific) or in a single use 125- mL Erlenmeyer flask (Thermo Fisher Scientific) agitated at 275 rpm or 110 rpm in a shaking incubator (Kuhner Shaker Inc).
  • Cell preparation for transfection Before transfection, cells were collected bycentrifugation at 300xg for 5 min and the cell pellets were diluted to optimal cell density in Freestyle F17 medium including protein hydrolysates, Casein El, Gelatin N2, or C-Cell S146B at various concentrations. Cell viability- was checked at each step with Vi-CELL Blu (Beckman Coulter) and cells with 90% or higher viability were proceeded for AAV production.
  • AAV8 production was significantly increased when any of Casein Peptone El, Gelatin Peptone N2, and/or C-cell Peptone S146B was included as described herein.
  • AAV8 production was assessed to be about 2-4 times higher in the presence of an appropriate amount of such peptone, relative to its absence.
  • AAV8 production was not increased, and in fact was statistically significantly decreased in the presence of Tryptone Nl.
  • the present disclosure demonstrates that peptone impact on heterologous polypeptide production from cultured mammalian cells may show a concentration effect, specifically including a biphasic impact.
  • the present disclosure teaches the feasibility and desirability of determining an appropriate concentration (e.g..
  • polypeptide or type thereof for production of a particular polypeptide or type thereof - such as, for example, antibody vs enzyme vs hormone vs viral capsid, or even type of viral capsid, or alternatively or additionally, size of polypeptide and/or charge or other characteristic feature, etc., and/or for production out of a particular cell or cell type
  • an “intermediate” concentration i.e., between a first concentration at which enhanced polypeptide production is observed relative to no peptone and a second, higher concentration, at which an inhibitory effect is observed, at least relative to the first concentration and/or, in some embodiments, relative to no peptone.
  • FIG. 4 demonstrates that positive impact of each of Casein Peptone Eland/or C-cell Peptone S146B from HEK293 cells is confirmed across AAV serotypes, with impacts ranging from about 2 fold to about 12 fold.
  • Gelatin N2 did not show as significant a positive impact on production, even of AAV8, as was observed in other assessments (e.g., as presented in FIGs. 1-3); without wishing to be bound by any particular theory, it is proposed that at least some of the observed variability may reflect the production scale (where those skilled in the art will appreciate that smaller- scale production as exemplified herein can sometimes contribute to variability) as much as any characteristic of Gelatin N2.
  • FIG. 4 further surprisingly demonstrates a positive effect of peptones (e.g., specifically of Casein Peptone El and/or C-cell Peptone S146B) on production of AAV serot pes known to traditionally be low-yield, for example AAV2, AAV3, and AAV4.
  • peptone supplementation, and particularly supplementation with one or both of Casein Peptone El and/or C-cell Peptone S146B may be particularly useful and/or effective for low yield AAVs, thus addressing a long-felt need in the industry.
  • cells are contacted with peptones at least 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or more prior to transfection; alternatively or additionally, in some embodiments cells are in contact with peptones during part or all of the transfection process.
  • provided technologies comprise contacting mammalian cells (e.g., HEK293 cells, including variants thereof) with a peptone of interest (which, in some embodiments, may be or comprise, e.g., Casein Peptone El and/or C-cell Peptone S146B) at least 10-30 mins prior to transfection and/or maintaining such cells in contact with such peptone of interest during (e.g., through) transfection.
  • a peptone (Casein Peptone El and/or C-cell Peptone S146B) at such a concentration is contacted with mammalian cells (e.g., HEK293 cells) prior to and/or during transfection, for example at least 10 mins prior to transfection.
  • mammalian cells e.g., HEK293 cells
  • the present Example demonstrates, among other things, that inclusion of peptones can enhance AAV production and achieve higher titer from HEK293 cells cultured at large scales, e.g., in a bioreactor.
  • the exemplary bioreactor size used in this example is a 2L bioreactor and one with ordinary' skill would appreciate that these findings can be readily extrapolated to larger bioreactors such as 10L to 5000L bioreactors to enhance AAV production.
  • Materials and methods Cell culture
  • Seed train cell Culture HEK293 suspension cells were grow n in F17 FreeStyle Medium (Thermo Fisher Scientific) supplemented with 4 mM L-Glutamine (Gibco) and 0.1% poloxamer 188. Cultures were scaled up in Erlenmeyer flasks (Thermo Fisher Scientific) to expand enough cells for bioreactor inoculations. During the seed train scaling up, cells were agitated at 135 rpm in a shaking incubator (Kuhner).
  • Bioreactor inoculation/operation The day before transfection, cells were inoculated to the bioreactor by diluting them with fresh F17 FreeStyle Media (Thermo Fisher Scientific) supplemented with 4 mM L-Glutamine (Gibco) and 0.1% poloxamer 188. Cells were cultured at bioreactors until the target transfection cell densities were achieved. During bioreactor operations, nutrient levels (glucose and glutamine) were closely monitored and supplemented as depleted.
  • Peptone preparation Two peptones used in this study were purchased from Organotechnie: Casein Peptone El (cat#19546), and C-Cell S146B (cat#E0003). Peptones were dissolved in Freesty le F17 medium at 20% (W/V) and 0.2uM-filtered.
  • FectoVIR or VirusGEN was then added to DNA/DMEM and the mixture was incubated for 20 min at room temperature and added to each bioreactor vessel After 72h production, cells were lysed by adding 10% Triton XI 00 and Benzonase (Sigma-Millipore) to the culture and incubating for 1.5 hrs at the shaking incubator. For titration, all or partial crude lysate was removed.
  • peptone supplementation and particularly supplementation with one or both of Casein Peptone El and/or C-cell Peptone S146B, may be particularly useful and/or effective to obtain high yield of AAVs in large- scale cultures (e.g., in bioreactors used to manufacture a therapeutic).
  • FIG. 6 surprisingly demonstrates a positive effect of peptones (e.g., specifically of Casein Peptone El) on AAV8 production from cells transfected with two exemplary transfection reagents.
  • peptones e.g., specifically of Casein Peptone El
  • AAV8 production as assessed by vector genome (vg)/L titers or percentage of full capsid to empty 7 capsid, was enhanced when HEK293 cells were transfected with two exemplary transfection reagents in the presence of peptone (Casein Peptone El) supplementation.
  • the first transfection reagent (Transfection reagent 1), adding Casein Peptone Elto the cell culture medium increased AAV8 titer from about 3.5X10(14) vg/L to about 5.5x10(14) vg/L.
  • the second transfection reagent (Transfection reagent 2), adding Casein Peptone El to the cell culture medium increased AAV8 titer from about 4.5x10(14) vg/L to about 6.7x10(14) vg/L.
  • peptone supplementation (Casein Peptone El) also increased the percentage of full AAV8 capsids that were produced irrespective of which transfection reagent was used to transfect the HEK293 cells.
  • adding Casein Peptone Elto the cell culture medium increased the percentage of full capsid to empty capsid by about 5%.
  • peptone supplementation and particularly supplementation with one or both of Casein Peptone El and/or C-cell Peptone S146B may be particularly useful and/or effective in producing AAV particles from different production processes (e.g., production processes using any transfection reagent to introduce nucleic acid molecules encoding a payload and/or polypeptides required for viral production).
  • Embodiment 1 A cell culture system comprising: a population of mammalian cells; one or more transfection reagents; one or more nucleic acids sufficient to engineer the mammalian cells to express a heterologous polypeptide; and a peptone at a concentration in a range of about 0.05% to about 2%.
  • Embodiment 2 The cell culture system of embodiment 1, wherein the peptone comprises one or more of: tryptone N1 (TNI), Tryptone Plus. Casein Peptone El, Gelatin Peptone N2, C-Cell Pl 12, C-Cell G115. C-Cell S146B. C-Cell S204.
  • tryptone N1 Tryptone Plus
  • Casein Peptone El Gelatin Peptone N2, C-Cell Pl 12, C-Cell G115.
  • C-Cell S146B C-Cell S204.
  • Embodiment 3 The cell culture system of embodiment 2, wherein the peptone is or comprises Casein El.
  • Embodiment 4 The cell culture system of embodiment 2, wherein the peptone is or comprises Gelatin N2.
  • Embodiment 5 The cell culture system of embodiment 2, wherein the peptone is or comprises C-Cell S146B.
  • Embodiment 6 The cell culture system of embodiment 2, wherein the peptone is not TN 1.
  • Embodiment 7 The cell culture system of any one of the preceding embodiments, wherein the peptone is provided or otherwise present at a concentration of: (i) about 0.25%. about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5% about, 0.55%, about 0.6%, about 0.65%, about 0.7%. about 0.75%, about 0.8%, about 0.85%.
  • Embodiment 8 The cell culture system of any one of the preceding embodiments, wherein the peptone is provided or otherwise present at a concentration of about 0.05% to about 1.9%, about 0.05% to about 1.8%, about 0.05% to about 1.7%, about 0.05% to about 1.6%, about 0.05% to about 1.5%.
  • Embodiment 9 The cell culture system of any one of the preceding embodiments, wherein the population of mammalian cells is contacted with the peptone and the one or more transfection reagents at substantially the same time.
  • Embodiment 10 The cell culture system of any one of embodiments 1-9, wherein the population of mammalian cells is contacted with the peptone prior to contacting with the one or more transfection reagents.
  • Embodiment 11 The cell culture system of embodiment 10, wherein the population of mammalian cells is contacted with the peptone at least 5 minutes prior to contacting with the one or more transfection reagents.
  • Embodiment 12 The cell culture system of any one of the preceding embodiments, wherein the population of mammalian cells comprises: HEK cells, CHO cells, HeLa cells, PER.C6 cells, HKB11 cells, CAP cells, BHK cells, mouse myeloma cells, green African monkey kidney cells, or A549 cells.
  • Embodiment 13 The cell culture system of any one of the preceding embodiments, wherein the population of mammalian cells comprises HEK cells.
  • Embodiment 14 The cell culture system of embodiment 13. wherein the HEK cells comprise HEK293 cells.
  • Embodiment 16 The cell culture system of embodiment 15, wherein the heterologous polypeptide is or comprises a viral capsid polypeptide.
  • Embodiment 17 The cell culture system of embodiment 16. wherein the viral capsid polypeptide is part of a viral capsid.
  • Embodiment 18 The cell culture system of embodiment 17. wherein the viral capsid is enveloped.
  • Embodiment 19 The cell culture system of 18, wherein the enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • Embodiment 20 The cell culture system of any one of embodiments 15-19, wherein the viral capsid polypeptide is an AAV capsid polypeptide.
  • Embodiment 21 The cell culture system of 20, wherein the AAV capsid polypeptide is chosen from an AAV1; AAV2; AAV3; AAV4; AAV5; AAV6; AAV7; AAV8; AAV9; AAV10; AAV11; AAV12; or an AAV13 capsid polypeptide, or a variant or derivative of any of the foregoing.
  • Embodiment 22 The cell culture system of embodiment 20 or 21. wherein the AAV capsid polypeptide is or comprises an AAV8 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 23 The cell culture system of embodiment 20 or 21, wherein the AAV capsid polypeptide is or comprises an AAV1 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 24 The cell culture system of embodiment 20 or 21, wherein the AAV capsid polypeptide is or comprises an AAV2 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 25 The cell culture system of embodiment 20 or 21. wherein the AAV capsid is or comprises an AAV3 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 26 The cell culture system of embodiment 20 or 21. wherein the AAV capsid polypeptide is or comprises an AAV4 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 27 The cell culture system of embodiment 20 or 21, wherein the
  • AAV capsid polypeptide is or comprises an AAV5 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 28 The cell culture system of embodiment 20 or 21. wherein the AAV capsid polypeptide is or comprises an AAV6 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 29 The cell culture system of embodiment 20 or 21, wherein the AAV capsid polypeptide is or comprises an AAV9 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 30 The cell culture system of any one of embodiments 15-19, wherein the viral capsid polypeptide is or comprises a retrovirus capsid polypeptide.
  • Embodiment 31 The cell culture system of any one of embodiments 15-19, wherein the viral capsid polypeptide is or comprises a lentivirus capsid polypeptide.
  • Embodiment 32 The cell culture system of any one of embodiments 15-19. wherein the viral capsid polypeptide is or comprises a HSV capsid polypeptide.
  • Embodiment 33 The cell culture system of any one of embodiments 1-15, wherein the heterologous polypeptide is an antibody polypeptide.
  • Embodiment 34 The cell culture system of any one of 1-15. wherein the heterologous polypeptide is a cytokine polypeptide.
  • Embodiment 35 The cell culture system of any one of 1-15, wherein the heterologous polypeptide is a chemokine polypeptide.
  • Embodiment 36 The cell culture system of any one of 1-15, wherein the heterologous polypeptide is a hormone polypeptide.
  • Embodiment 37 The cell culture system of any one of 1-15, wherein the heterologous polypeptide is an enzyme polypeptide.
  • Embodiment 44 A method of manufacturing a pharmaceutical composition comprising, formulating an expressed heterologous polypeptide preparation described herein with one or more pharmaceutically acceptable excipients.
  • Embodiment 45 A method comprising delivering a heterologous polypeptide: produced from the cell culture system of any one of embodiments 1-37 or the method of any one of embodiments 39-43, a cell preparation of embodiment 38, or a pharmaceutical composition of embodiment 44, to a cell, tissue or subject.
  • Embodiment 46 The method of embodiment 45, wherein delivering comprises administering.
  • Embodiment 47 The method of embodiment 45 or 46, wherein the method is a treatment method.
  • Embodiment 48 The method of embodiment 45 or 46, wherein the method is a prevention method.
  • Embodiment 49 The method of any one of embodiments 45-49, wherein the subject is a mammal, e.g., a human.
  • Embodiment 53 The method of embodiment 50, wherein the peptone is or comprises C-Cell S146B.
  • Embodiment 55 The method of any one of embodiments 39-54, wherein the peptone is provided or otherwise present at a concentration of: (i) about 0.25%, about 0.3%, about 0.35%. about 0.4%, about 0.45%, about 0.5% about, 0.55%, about 0.6%.
  • Embodiment 56 The method of any one of embodiments 39-54, wherein the peptone is provided or otherwise present at a concentration of about 0.05% to about 1.9%, about 0.05% to about 1.8%, about 0.05% to about 1.7%, about 0.05% to about 1.6%, about 0.05% to about 1.5%, about 0.05% to about 1.4%.
  • Embodiment 57 The method of any one of embodiments 39-56, wherein the population of mammalian cells is contacted with the peptone and the one or more transfection reagents at substantially the same time.
  • Embodiment 58 The method of any one of embodiments 39-56, wherein the population of mammalian cells is contacted with the peptone prior to contacting with the one or more transfection reagents.
  • Embodiment 59 The method of embodiment 58, wherein the population of mammalian cells is contacted with the peptone at least 5 minutes prior to contacting with the one or more transfection reagents.
  • Embodiment 60 The method of any one of embodiments 39-59, wherein the population of mammalian cells comprises: HEK cells, CHO cells, HeLa cells, PER.C6 cells. HKB11 cells, CAP cells, BHK cells, mouse myeloma cells, green African monkey kidney cells, or A549 cells.
  • Embodiment 61 The method of embodiment 60, wherein the population of mammalian cells comprises HEK cells.
  • Embodiment 62 The method of embodiment 61, wherein the HEK cells comprise HEK293 cells.
  • Embodiment 63 The method of any one of embodiments 39-62, wherein the heterologous polypeptide is or comprises a viral capsid polypeptide, an antibody polypeptide, a cytokine polypeptide, a chemokine polypeptide, an enzy me polypeptide, or a hormone polypeptide.
  • Embodiment 64 The method of embodiment 63, wherein the heterologous polypeptide is or comprises a viral capsid polypeptide.
  • Embodiment 65 The method of embodiment 64, wherein the viral capsid polypeptide is part of a viral capsid.
  • Embodiment 66 The method of embodiment 65, wherein the viral capsid is enveloped.
  • Embodiment 67 The method of embodiment 66, wherein the enveloped viral capsid comprises a nucleic acid construct encoding a payload.
  • Embodiment 68 The method of any one of embodiments 63-67, wherein the viral capsid polypeptide is an AAV capsid polypeptide.
  • Embodiment 69 The method of embodiment 68, wherein the AAV capsid polypeptide is chosen from an AAV1; AAV2; AAV3; AAV4; AAV5; AAV6; AAV7; AAV8; AAV9; AAV 10; AAV11; AAV 12; or an AAV 13 capsid polypeptide, or a variant or derivative of any of the foregoing.
  • Embodiment 70 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV8 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 71 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV 1 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 72 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV2 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 73 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV3 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 74 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV4 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 75 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV5 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 76 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV6 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 77 The method of embodiment 68 or 69, wherein the AAV capsid polypeptide is or comprises an AAV9 capsid polypeptide or a variant or derivative thereof.
  • Embodiment 78 The method of any one of embodiments 63-67, wherein the viral capsid polypeptide is an adenovirus capsid polypeptide.
  • Embodiment 79 The method of any one of embodiments 63-67, wherein the viral capsid polypeptide is a retrovirus capsid polypeptide.
  • Embodiment 80 The method of any one of embodiments 63-67, wherein the viral capsid polypeptide is a lentivirus capsid polypeptide.
  • Embodiment 81 The method of any one of embodiments 63-67, wherein the viral capsid polypeptide is a HSV capsid polypeptide.
  • Embodiment 82 The method of any one of embodiments 39-63, wherein the heterologous polypeptide is an antibody polypeptide.
  • Embodiment 83 The method of any one of embodiments 39-63, wherein the heterologous polypeptide is a cytokine polypeptide.
  • Embodiment 84 The method of any one of embodiments 39-63, wherein the heterologous polypeptide is a chemokine polypeptide.
  • Embodiment 85 The method of any one of embodiments 39-63, wherein the heterologous polypeptide is a hormone polypeptide.
  • Embodiment 86 The method of any one of embodiments 39-63, wherein the heterologous polypeptide is an enzyme polypeptide.
  • Embodiment 87 The cell culture system of any of embodiments 1-37, wherein a cell culture system described herein is characterized in that a heterologous polypeptide (e.g., AAV particle) is produced at a higher titer and/or a higher quality compared to a heterologous polypeptide (e.g., AAV particle) produced with an otherwise similar cell culture system without one or more peptones.
  • a heterologous polypeptide e.g., AAV particle
  • Embodiment 88 The cell preparation of embodiment 38, wherein the cell preparation is characterized in that a heterologous polypeptide (e.g., AAV particle) is produced at a higher titer and/or a higher quality' compared to a heterologous polypeptide (e.g., AAV particle) produced with an otherwise similar cell preparation without one or more peptones.
  • a heterologous polypeptide e.g., AAV particle
  • Embodiment 89 The method of any one of embodiments 39-43, wherein the method is characterized in that a heterologous polypeptide (e.g., AAV particle) is produced at a higher titer and/or a higher quality compared to a heterologous polypeptide (e.g., AAV particle) produced with an otherwise similar method without one or more peptones.
  • a heterologous polypeptide e.g., AAV particle
  • Embodiment 90 The cell culture system of embodiment 87, the cell preparation of embodiment 88 or the method of embodiment 89, wherein a higher titer comprises a titer that is about 1.5-fold to about 100-fold higher.
  • Embodiment 91 The cell culture system of embodiment 87, the cell preparation of embodiment 88 or the method of embodiment 89, wherein a higher titer comprises a titer within a range of 1E12 vector genomes (vg)/L to 1 E 17 vg/L.
  • Embodiment 92 The cell culture system of embodiment 87. the cell preparation of embodiment 88 or the method of embodiment 89, wherein a higher quality comprises an increase in an amount (e.g., a percentage) of full capsids as compared to an amount (e.g., a percentage) of empty capsids, e.g., as determined by an assay described herein.
  • an amount e.g., a percentage
  • empty capsids e.g., as determined by an assay described herein.

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Abstract

La présente divulgation concerne des technologies utiles dans la production de polypeptides hétérologues, comprenant plus précisément des polypeptides de capside virale (et plus précisément des capsides virales telles que des capsides de virus adéno-associé) à partir de cellules de mammifère en culture. Entre autres, la présente divulgation concerne des technologies se rapportant à l'utilisation de peptones dans de telles applications de production de polypeptides.
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Citations (2)

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US6136787A (en) * 1990-04-04 2000-10-24 Vertex Pharmaceuticals Incorporated Interleukin 1β protease and interleukin 1β protease inhibitors
US20210363498A1 (en) * 2013-08-30 2021-11-25 Amgen Inc. High titer recombinant aav vector production in adherent and suspension cells

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Publication number Priority date Publication date Assignee Title
US6136787A (en) * 1990-04-04 2000-10-24 Vertex Pharmaceuticals Incorporated Interleukin 1β protease and interleukin 1β protease inhibitors
US20210363498A1 (en) * 2013-08-30 2021-11-25 Amgen Inc. High titer recombinant aav vector production in adherent and suspension cells

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Title
P.L. PHAM; S. PERRET; B. CASS; E. CARPENTIER; G. ST‐LAURENT; L. BISSON; A. KAMEN; Y. DUROCHER: "Transient gene expression in HEK293 cells: Peptone addition posttransfection improves recombinant protein synthesis", BIOTECHNOLOGY AND BIOENGINEERING, vol. 90, no. 3, 31 March 2005 (2005-03-31), Hoboken, USA, pages 332 - 344, XP071152811, ISSN: 0006-3592, DOI: 10.1002/bit.20428 *
PHUONG LAN PHAM , SYLVIE PERRET, HUYEN CHAU DOAN, BRIAN CASS, GILLES ST-LAURENT, AMINE KAMEN, YVES DUROCHER: "Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: Peptone additives improve cell growth and transfection efficiency.", BIOTECHNOLOGY AND BIOENGINEERING, vol. 84, no. 3, 5 November 2003 (2003-11-05), Hoboken, USA, pages 332 - 342, XP002296366, ISSN: 0006-3592, DOI: 10.1002/bit.10774 *

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