WO2022067028A1 - Engineered perkinsus marinus cells - Google Patents

Engineered perkinsus marinus cells Download PDF

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WO2022067028A1
WO2022067028A1 PCT/US2021/051933 US2021051933W WO2022067028A1 WO 2022067028 A1 WO2022067028 A1 WO 2022067028A1 US 2021051933 W US2021051933 W US 2021051933W WO 2022067028 A1 WO2022067028 A1 WO 2022067028A1
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cells
cell
perkinsus
marinus
engineered
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PCT/US2021/051933
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WO2022067028A9 (en
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José Antonio FERNÁNDEZ ROBLEDO
Raghavendra YADAVALLI
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Bigelow Laboratory For Ocean Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/14011—Filoviridae
    • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
    • C12N2760/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • Perkinsozoa is considered one of the earliest diverging groups of the lineage leading to dinoflagellates, and it includes the species Perkinsus marinus (1-2).
  • Perkinsus marinus is a marine protozoan parasite of oysters with a broad distribution in the Gulf and East Coasts.
  • Perkinsus marinus cells have been successfully cultured in host-free cell medium, and previous studies using cultured Perkinsus marinus cells have demonstrated low heterologous expression of protozoan proteins (3).
  • Perkinsus marinus cells such that the cells exhibit improved production of a protein(s) of interest (e.g., a viral protein).
  • a protein(s) of interest e.g., a viral protein
  • the disclosure relates to engineered Perkinsus cells.
  • an engineered Perkinsus cell is derived from a Perkinsus andrewsi cell, a Perkinsus beihaiensis cell, a Perkinsus chesapeaki cell, a Perkinsus honshuensis cell, a Perkinsus marinus cell, a Perkinsus mediterraneus cell, a Perkinsus olseni cell, or a Perkinsus qugwadii cell.
  • an engineered Perkinsus cell is an engineered Perkinsus marinus cell.
  • an engineered Perkinsus marinus cell expresses a polypeptide comprising a viral protein.
  • the polypeptide comprising the viral protein is expressed on the surface of the cell.
  • the polypeptide comprises a tag.
  • the polypeptide comprises an amino acid sequence of a signal peptide that directs the polypeptide comprising the viral protein to the cell surface.
  • the signal peptide is an MOE signal peptide.
  • the MOE signal peptide comprises an amino acid sequence of MRFIVGLYSCLAVLVLGQSSCPTGSAQC (SEQ ID NO: 1).
  • the cell comprises a heterologous polynucleotide comprising a nucleic acid sequence encoding the polypeptide comprising the viral protein.
  • the heterologous polynucleotide is a plasmid.
  • the heterologous polynucleotide is integrated into the genome of the cell.
  • the heterologous polynucleotide comprises a nucleic acid sequence encoding two or more polypeptides comprising viral proteins. In some embodiments, at least two of the viral proteins encoded by the heterologous polynucleotide comprise the same amino acid sequence. In some embodiments, at least two of the viral proteins encoded by the heterologous polynucleotide comprise different amino acid sequences.
  • the cell comprises two or more heterologous polynucleotides comprising a nucleic acid sequence encoding a polypeptide comprising a viral protein.
  • the nucleic acid sequences of at least two of the heterologous polynucleotides are the same.
  • the nucleic acid sequences of at least two of the heterologous polynucleotides are different.
  • the disclosure relates to populations of Perkinsus marinus cells.
  • at least 8% of the cells in the population express a protein of interest consequent to transfection of the population with a plurality of heterologous polynucleotides encoding the protein of interest.
  • the cells are trophozoites.
  • the number of cells in the population is from about 1 x 10 6 to about 50 x 10 6 cells. In some embodiments, the number of cells is about 25 x 10 6 cells.
  • the protein of interest comprises the amino acid sequence of a viral protein.
  • the disclosure relates to methods of engineering Perkinsus marinus cells to produce a protein of interest.
  • the method comprises contacting a population of Perkinsus marinus trophozoites with a transfection reagent and a plurality of heterologous polynucleotides encoding the protein of interest, wherein: the population of Perkinsus marinus trophozoites comprises from about 1 x 10 6 to about 50 x 10 6 cells; the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg; or a combination thereof.
  • the population of Perkinsus marinus trophozoites comprises from about 1 x 10 6 to about 50 x 10 6 cells and the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg.
  • the population of Perkinsus marinus trophozoites has about 25 x 10 6 cells.
  • the plurality of heterologous polynucleotides is in an amount of about 20 pg.
  • the population of Perkinsus marinus trophozoites are in a log growth phase when contacted with the transfection reagent and the plurality of heterologous polynucleotides. In some embodiments, the population of Perkinsus marinus trophozoites have an absorbance of 1.66 at ODeoo when contacted with the transfection reagent and the plurality of heterologous polynucleotides.
  • the protein of interest comprises the amino acid sequence of a viral protein.
  • the disclosure relates to methods of producing a polypeptide comprising a viral protein.
  • the method comprises culturing an engineered Perkinsus marinus cell described herein under conditions in which it expresses a polypeptide comprising the viral protein.
  • the method further comprises isolating the polypeptide comprising the viral protein.
  • FIG. 1 shows EGFP expression in engineered P. marinus trophozoites. High numbers of engineered cells were seen when using 20 pg of plasmid DNA and 25 x 10 6 P. marinus trophozoites for transfection.
  • FIGs. 2A-2B relate to a strategy for knock-in of GFP in P. marinus using the CRISPR/Cas9 system.
  • FIG. 2A depicts the strategy followed and reagents used.
  • FIG. 2B shows replication of the mutant PmM0E[M0E]:GFP after knocking in GFP in P. marinus MOE wild type. The knock-in was observed less than 24 hours after the delivery of the CRISPR/Cas9 components.
  • FIGs. 3A-3C relate to heterologous expression of Plasmodium falciparum genes in Perkinsus marinus cells.
  • FIG. 3A shows expression of P. falciparum CSP (circumsporozoite protein) tagged with GFP and IFA using anti-PfCSP to confirm the production of CSP protein.
  • FIG. 3B shows expression of P. falciparum RPL36 (ribosomal protein L36) tagged with GFP.
  • FIG. 3C shows his-tag affinity purification of rPfRPL36-GFP.
  • FIG. 4 depicts heterologous protozoan proteins expressed in Perkinsus marinus cells in the studies described herein.
  • FIGs. 5A-5C relate to expression of SARS-CoV-2 spike protein in Perkinsus marinus cells.
  • FIG. 5A shows a schematic depicting SARS-CoV-2 morphology.
  • FIG. 5B shows a schematic depicting the SARS-CoV-2 genome organization.
  • FIG. 5C shows a plasmid containing partial spike (S) gene tagged with GFP.
  • FIG. 5D shows an image 24 hours after transfection showing numerous cells expressing the GFP tagged spike (S) gene.
  • FIGs. 6A-6B relate to expression of VHSV G protein proteins in Perkinsus marinus cells.
  • FIG. 6A depicts the typical rhabdoviral genome organization.
  • FIG. 6B shows the G protein gene cloned into vector p[MOE]:GFP to give p[MOE]:G-GFP.
  • FIG. 7 shows a Perkinsus marinus cell expressing GFP-tagged ebola glycoprotein (GP1,2) gene 24 hours after transfection.
  • GP1,2 GFP-tagged ebola glycoprotein
  • FIGs. 8A-8D show plasmid amount and cell number studies.
  • FIG. 8A shows fifty million parasites transfected with 5 mg, 10 mg, 20 mg, and 40 mg of pPmMOE[MOEl]:GFP, respectively. Bar graphs show the % GFP-positive cells (y-axis) that were detected by flow cytometry at 24, 72, and 120 hours post-transfection time points (x-axis).
  • FIG. 8B shows GFP expression in parasites transfected with 20 mg of pPmMOE[MOEl]:GFP 24 and 120 hours post-transfections.
  • FIG. 8C shows % GFP expression in parasites transfected with 20 mg of pPmMOE[MOEl]:GFP.
  • the bar graphs show the % GFP-positive cells (y-axis) which were detected by flow cytometry at 24, 72, 120 hours, and 3 months post-transfection time points (x-axis).
  • FIG. 8D shows a scattered plot from FCM where no GFP expression in untransfected controls was observed, and 98% GFP-positive cells in 25.0 x 10 6 cells which were transfected with 20 mg of pPmMOE[MOEl]:GFP as indicated in the box.
  • FIGs. 9A-9E show a comparison of transfection protocols. 25 x 10 6 cells were transfected with 20 mg of pPmMOE[MOEl]:GFP plasmid using different protocols.
  • FIG. 9A shows a flow cytometry scattered plot of un-transfected (wild-type) cells, where GFP expression was not detected.
  • FIG. 9B shows a scattered plot of flow cytometry where GFP- positive cells were identified in transfection performed using the Lonza method.
  • FIG. 9C shows a scatterplot representation of GFP-positive cells in transfection performed using 3R buffer and BTX cuvette.
  • FIG. 9D is a scatterplot showing GFP-positive cells when transfected with 3R buffer utilizing the Lonza cuvette.
  • FIG. 9E is a bar graph showing the % of GFP-positive cells that were transfected with the Lonza system, 3R buffer in combination with BTX cuvette, 3R buffer using Lonza cuvette, and Lonza buffer with BT
  • FIGs. 10A-10D show SpCas9-RNP and sgRNA-mediated GFP knock-in in P. marinus trophozoites.
  • FIG. 10A is a schematic representation of dDNA with 396 bp homology on the 50 and 30 of the GFP coding sequence.
  • FIG. 10B is a schematic representation of the guide RNA target sites on PmMOEl coding sequences; sgRNA-1 targets the top strand indicated by the arrow direction; sgRNA-2 targets the bottom strand indicated by the arrow direction.
  • FIG. 10C shows a confocal microscopy panel showing successful GFP expression in cells transfected with sgRNA-l/SpCas9 and sgRNA-2/SpCas9.
  • FIG. 10D shows a scattered plot from FCM where no GFP expression in mock (dDNA+sgRNA alone) control was observed, and 0.2% GFP-positive cells were knocked in using sgRNA-1, and 0.35% in the case of sgRNA-2 as indicated in the box.
  • FIGs. 11A-11D show the sorting of P. marinus GFP-positive cells for endogenous PmMOEl C-terminus GFP tagging analysis.
  • FIG. 11A is a scattered plot showing 81% GFP- positive cells indicated with a box in the experiment where cells transfected with sgRNA-1 - Cas9.
  • FIG. 11B show a scattered plot showing 87% GFP-positive cells indicated with a box in the experiment where cells transfected with sgRNA-l-Cas9.
  • FIG. 11C shows the PCR intended to amplify the knock-in (expected sized 3,300 bp) using Fwd 1 and Rev 1 primers resulting in the amplification of the wildtype 2,600 bp amplicon (left panel).
  • FIG. 11D shows the sequencing results of the nPCR product from the sgRNA-2 targeted GFP knock-in experiment.
  • FIG. 12 shows the observed GFP expression among cell populations of 1.62, 3.12, 6.25, 12.5, 25.0, and 50.0 million.
  • FIGs. 13A-13B show PCR and nested PCR based genotyping of PMAR_Pmar027036 for the GFP knock in analysis.
  • the genomic DNA was isolated from two different clones labeled as 4 and 5, which were hand-picked 3 months after the cell sorting from the experiments involved in the utilization of SgRNA-1 and SgRNA-2, respectively.
  • FIG. 13A shows the PCR product showing the successful amplification of 2,600 bp of DNA sequencing containing flanking regions, M0E1 CDS encoded by PMAR_Pmar027036 and the GFP.
  • PCR product was diluted 100 times and used in the nested PCR to identify the knock in of the GFP, which successfully amplified the 748 bp of DNA fragment. The fragment was sequenced to confirm the successful knock in of GFP.
  • FIG. 13B shows a repeat of the gel analysis of the nested PCR products from (FIG. 13 A), showing the DNA size of 748 bp.
  • Described herein are engineered Perkinsus marinus cells, and populations thereof, that express proteins of interest.
  • engineered Perkinsus marinus cells are described that express viral genes (z.e., encoding viral proteins). Such engineered cells have not been described or contemplated previously.
  • Compositions e.g., vaccines
  • improved methods of engineering Perkinsus marinus cells to produce proteins of interest e.g., a viral protein
  • proteins of interest e.g., a viral protein
  • an engineered Perkinsus cell e.g., Perkinsus marinus cells
  • an engineered Perkinsus cell is derived from a Perkinsus andrewsi cell, a Perkinsus beihaiensis cell, a Perkinsus chesapeaki cell, a Perkinsus honshuensis cell, a Perkinsus marinus cell, a Perkinsus mediterraneus cell, a Perkinsus olseni cell, or a Perkinsus qugwadii cell.
  • an engineered Perkinsus cell is an engineered Perkinsus marinus cell.
  • heterologous protein refers to a protein that is not produced by a wild-type Perkinsus cell corresponding to the species from which the engineered Perkinsus cell is derived.
  • a heterologous protein of interest may be: (i) a protein from any source other than a Perkinsus cell from which the engineered Perkinsus cell is derived; or (ii) a modified protein from a Perkinsus cell from which the engineered Perkinsus cell is derived (/'. ⁇ ?., such that the modified protein is not produced by the corresponding wildtype Perkinsus cell).
  • an engineered Perkinsus cell expresses more than one heterologous protein of interest.
  • a Perkinsus cell expresses at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 heterologous proteins of interest.
  • an engineered Perkinsus cell expresses 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2- 10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10.
  • an engineered Perkinsus cell expresses 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous proteins of interest.
  • an engineered Perkinsus cell expresses a heterologous protein from a protozoan organism.
  • the protozoan organism is a protozoan parasite.
  • the protozoan organism is Plasmodium berghei (Pb), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg), or Cryptosporidium parvum (Cp).
  • the protein of interest comprises or consists of an HAP2 protein (e.g., PbHAP2, or a fragment thereof), a MSP8 protein (e.g., PbMSP8, or a fragment thereof), an Rpl36 protein (e.g., PfRpl36, or a fragment thereof), a CSP protein (e.g., PfCSP, or a fragment thereof), an ACP protein (e.g., TgACP or a fragment thereof), an FNR protein (e.g., TgFNR or a fragment thereof), or an AdoT protein (e.g., CpAdoT or a fragment thereof).
  • HAP2 protein e.g., PbHAP2, or a fragment thereof
  • MSP8 protein e.g., PbMSP8, or a fragment thereof
  • Rpl36 protein e.g., PfRpl36, or a fragment thereof
  • a CSP protein e.g., PfCSP, or
  • an engineered Perkinsus cell expresses a heterologous protein from a virus.
  • the virus is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, an Ebola virus, or a viral hemorrhagic septicemia virus (VHSV). Proteins encoded by the genomes of SARS-CoV-2, Ebola, and VHSV have been identified previously. See e.g., FIGs. 5A-5B and FIG. 6A.
  • the virus is SARS-CoV-2 and the protein of interest comprises or consists of spike (S) protein (or a portion thereof).
  • the virus is Ebola and the protein of interest comprises or consists of GP1,2 (or a portion thereof). In some embodiments, the virus is VHSV and the protein of interest comprises or consists of glycoprotein (G) (or a portion thereof).
  • a protein of interest described herein is expressed on the surface of the engineered Perkinsus cell.
  • a protein of interest described herein comprises a tag.
  • a protein of interest comprises a viral protein and a tag.
  • a tag may be an affinity tag (e.g., a chitin binding protein (CBP) tag, a maltose binding protein (MBP) tag, a streptavidin tag, a glutathione-S-transferase (GST) tag, or a poly His tag), a solubilization tag (e.g., a thioredoxin (TRX) tag, a poly(NANP), an MBP tag, or a GST tag), an epitope tag (e.g., a FLAG-tag, an ALFA-tag, a V5-tag, a Myc-tag, an HA-tag, a Spot-tag, a T7-tag, or an NE-tag.), and/or a fluorescence tag (e.g., TagBFP, mTagBFP2, Azu
  • CBP chitin
  • a protein of interest described herein comprises a signal peptide.
  • Signal peptides facilitate translocation of proteins to a specific location within the cell, e.g., to the cellular membrane.
  • a protein of interest comprises a signal peptide that directs the protein of interest to the cell surface.
  • a signal peptide is an MOE signal peptide.
  • a an MOE signal peptide comprises an amino acid sequence of MRFIVGLYSCLAVLVLGQSSCPTGSAQC (SEQ ID NO: 1) or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 1.
  • BLAST® Basic Local Alignment Search Tool
  • an engineered Perkinsus cell described herein comprises a heterologous polynucleotide having a nucleic acid sequence that encodes a heterologous protein of interest (e.g., comprising or consisting of a viral protein).
  • heterologous polynucleotide refers to a polynucleotide that is not found in the Perkinsus cell from which the engineered cell is derived.
  • an engineered Perkinsus cell described herein comprises more than one heterologous polynucleotide.
  • an engineered Perkinsus cell described herein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 heterologous polynucleotides.
  • an engineered Perkinsus cell described herein comprises 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9,
  • an engineered Perkinsus cell described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous polynucleotides.
  • the nucleic acid sequences of two or more of the heterologous polynucleotides are the same. In some embodiments, the nucleic acid sequences of two or more of the heterologous polynucleotides are different.
  • a heterologous polynucleotide encodes more than one protein of interest. For example, in some embodiments a heterologous polynucleotide encodes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proteins of interest. In some embodiments, a heterologous polynucleotide encodes 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8,
  • a heterologous polynucleotide encodes 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins of interest.
  • two or more of the proteins of interest encoded by a heterologous polynucleotide comprise the same amino acid sequence.
  • two or more of the proteins of interest encoded by a heterologous polynucleotide comprise different amino acid sequences.
  • a heterologous polynucleotide is an extrachromosomal DNA (z.e., found off the chromosomes of the cell, either inside or outside of the nucleus).
  • a heterologous polynucleotide is a plasmid.
  • a heterologous polynucleotide is integrated within a chromosome (z.e., within the genome of) the cells.
  • a heterologous polynucleotide may further comprise a promoter sequence that regulates transcription of the nucleic acid sequence that encodes for the heterologous protein of interest.
  • the promoter is a bacterial promoter or a viral promoter (e.g., CMV).
  • the disclosure relates to populations of Perkinsus cells (e.g., populations of Perkinsus marinus cells).
  • a population of Perkinsus cells comprises an engineered Perkinsus cell described herein (e.g., an engineered Perkinsus marinus cell).
  • the cells in the population are Perkinsus trophozoites (e.g., Perkinsus marinus trophozoites).
  • a population of Perkinsus cells comprises a plurality of engineered Perkinsus cells described herein.
  • each of the engineered cells in the plurality express the same protein(s) of interest (e.g., comprising or consisting of the same viral protein).
  • a plurality of engineered Perkinsus cells comprises two or more distinct engineered cells described herein.
  • At least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% of the cells in the population of Perkinsus cells express a protein of interest (z.e., are engineered Perkinsus cells, as described herein) consequent to transfection of the population with a plurality of heterologous polynucleotides encoding a protein(s) of interest.
  • a cell expresses a protein of interest “consequent to transfection” if the cell uptakes a heterologous polynucleotide and expresses a protein of interest in an amount that is sufficient enough to detect (e.g., by visual observation, immuno staining, immunofluorescence, cell sorting, etc.).
  • At least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% of the cells in the population of Perkinsus cells express a protein of interest (z.e., are engineered Perkinsus cells, as described herein) consequent to transfection of the population with a plurality of heterologous polynucleotides encoding a protein(s) of interest within a certain time following transfection; for example, within 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours following transfection; or within 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 20-21, 20-22,
  • the number of cells transfected with the heterologous polynucleotide is used to determine the percentage of cells expressing the protein(s) of interest.
  • populations of Perkinsus cells that express the protein(s) of interest can be obtained and purified using known methods subsequent to transfection.
  • the number of cells in the population is about 1 x 10 6 cells, about 5 x 10 6 cells, about 10 x 10 6 cells, about 15 x 10 6 cells, about 20 x 10 6 cells, about 25 x 10 6 cells, about 30 x 10 6 cells, about 35 x 10 6 cells, about 40 x 10 6 cells, about 45 x 10 6 cells, or about 50 x 10 6 cells.
  • the number of cells is from about 1 x 10 6 to about 50 x 10 6 , about 5 x 10 6 to about 50 x 10 6 , about 10 x 10 6 to about 50 x 10 6 , about 15 x 10 6 to about 50 x 10 6 , about 20 x 10 6 to about 50 x 10 6 , about 25 x 10 6 to about 50 x 10 6 , about 1 x 10 6 to about 25 x 10 6 , about 5 x 10 6 to about 25 x 10 6 , about 10 x 10 6 to about 25 x 10 6 , about 15 x 10 6 to about 25 x 10 6 , about 20 x 10 6 to about 25 x 10 6 cells.
  • the number of cells is about 25 x 10 6 cells.
  • Compositions Comprising Engineered Perkinsus Cells or a Protein of Interest Obtained Therefrom
  • the disclosure relates to compositions comprising an engineered Perkinsus cell described herein, e.g., engineered Perkinsus marinus cells (or a population of engineered Perkinsus cells described herein) or a protein(s) of interest obtained therefrom.
  • a composition is a vaccine comprising an engineered Perkinsus cell described herein. In some embodiments, a composition is a vaccine comprising a protein of interest that is obtained from an engineered Perkinsus cell described herein.
  • the vaccine is formulated for oral delivery (/'. ⁇ ?., the vaccine is an oral vaccine).
  • a method of engineering a Perkinsus cell for production of a protein(s) of interest comprises contacting a population of Perkinsus cells (e.g., Perkinsus marinus cells or trophozoites) with a transfection reagent and a plurality of heterologous polynucleotides encoding the protein(s) of interest.
  • Suitable transfection reagents are known to those having ordinary skill in the art.
  • the population of Perkinsus cells comprises about 1 x 10 6 cells, about 5 x 10 6 cells, about 10 x 10 6 cells, about 15 x 10 6 cells, about 20 x 10 6 cells, about 25 x 10 6 cells, about 30 x 10 6 cells, about 35 x 10 6 cells, about 40 x 10 6 cells, about 45 x 10 6 cells, or about 50 x 10 6 cells.
  • the number of cells is from about 1 x 10 6 to about 50 x 10 6 , about 5 x 10 6 to about 50 x 10 6 , about 10 x 10 6 to about 50 x 10 6 , about 15 x 10 6 to about 50 x 10 6 , about 20 x 10 6 to about 50 x 10 6 , about 25 x 10 6 to about 50 x 10 6 , about 1 x 10 6 to about 25 x 10 6 , about 5 x 10 6 to about 25 x 10 6 , about 10 x 10 6 to about 25 x 10 6 , about 15 x 10 6 to about 25 x 10 6 , about 20 x 10 6 to about 25 x 10 6 cells.
  • the population of Perkinsus cells comprises about 25 x 10 6 cells.
  • the plurality of heterologous polynucleotides that are contacted with the population of Perkinsus cells is in an amount of about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25
  • the plurality of heterologous polynucleotides is in an amount of about 15 pg to about 25 pg, about 16 pg to about 25 pg, about 17 pg to about 28 pg, about 19 pg to about 25 pg, about 20 pg to about 25 pg, about 15 pg to about 20 pg, about 16 pg to about 20 pg, about 17 pg to about 20 pg, about 18 pg to about 20 pg, about 18 pg to about 22 pg, or about 19 pg to about 21 pg. In some embodiments, the plurality of heterologous polynucleotides is in an amount of about 20 pg.
  • the population of Perkinsus cells (e.g., Perkinsus marinus cells or trophozoites) comprises from about 1 x 10 6 to about 50 x 10 6 cells and the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg.
  • the population of Perkinsus cells are in a log growth phase when contacted with the transfection reagent and the plurality of heterologous polynucleotides. In some embodiments, the population of Perkinsus cells have an absorbance of 1.66 at ODeoo when contacted with the transfection reagent and the plurality of heterologous polynucleotides.
  • the disclosure relates to methods of producing a protein of interest (e.g., comprising or consisting of a viral protein).
  • a method of producing a protein of interest comprises culturing an engineered Perkinsus cell described herein under conditions in which it expresses the protein of interest.
  • the method further comprises isolating (or purifying) the protein of interest.
  • Perkinsus marinus cells were engineered to produce a heterologous protein by: (i) transfection methodologies; and (ii) targeted genome editing.
  • the CRISPR/Cas9 system was used for targeted genome editing.
  • the CRISPR/Cas9 system was tested by delivering a guide RNA (gRNA), ribonuclease Cas9, and donor DNA (dDNA) targeting PmMOE for knock-in GFP and for knock-out of GFP from a previously described P. marinus variant expressing PmMOE-GFP (FIG. 2A).
  • the mutant phenotype was successfully reproduced by knocking-in GFP at the 3’ end of PmMOE in the wild type strain (FIG. 2B) and knocking-out the GFP from the previously transformed PmMOE-GFP variant.
  • the gRNA, dDNA, and ribonuclease Cas9 were delivered into the trophozoites using electroporation.
  • the efficiency of CRISPR/Cas9 transformation was 0.5%.
  • Cells with the phenotype expressing PmMOE-GFP were observed 18 hours post-transfection. Both PCR and sequence analysis indicated that the GFP was integrated at the designated position and with the intended length or mutated at the designated position (null mutants).
  • Example 2 Stable expression of heterologous proteins from protozoan parasites in Perkinsus marinus cells.
  • Prokaryotic expression systems e.g., E. coli expression systems
  • E. coli expression systems are the most commonly used systems for industrial production of recombinant proteins.
  • the overall success rate for obtaining soluble, recombinant proteins in prokaryotic systems remains low.
  • expression of heterologous proteins in yeast may result in N- and O-linked glycosylation patterns, which might be different from those in the native parasite protein, resulting in inactive products.
  • the use of mammalian cells to produce recombinant proteins from parasites has been hindered by the labor-intensive and expensive methodology required for establishing stable recombinant cells.
  • the full-length PfCSP coding sequence was codon-optimized and cloned into p[MOE]:GFP. 25.0 x 10 6 P. marinus cells were electroporated with the plasmids cloned with the PfRPL36 and PfCSP. Expression was observed 18 hours post-transfection.
  • the IFA using a human monoclonal IgG ab specific to PfCSP confirmed PfCSP protein synthesis.
  • the GFP expression was followed for 3 months, which confirmed the stable expression of full-length PfRPL36 and PfCSP in the P. marinus (FIGs.
  • Toxoplasma gondii apicoplast specific genes acyl carrier protein 1 and ferredoxin NAD+- reductase and the Cryptosporidium parvum adenosine transporter and tryptophan synthase B were also produced successfully using P. marinus cells (FIG. 4).
  • Example 3 Expression of viral proteins in Perkinsus marinus cells.
  • Stable heterologous expression of two viral genes in P. marinus were tested: (i) SARS-Cov-2 spike protein (223 aa); and (ii) Ebola GP1,2 (676 aa).
  • the SARS-CoV-2 virion is encoded in a 30 Kb genome with 14 ORFs.
  • the first two ORFs code for polyprotein (ppla/ab) required for virus replication, followed by structural proteins for spike, membrane, and nucleoprotein.
  • the longest ORF is located at the 5' terminus, encoding 15 nonstructural proteins collectively involved in virus replication and possibly in immune evasion.
  • accessory genes (3a, 3b, p6, 7a, 7b, 8b, 9b, and orfl4) are found with adjacent ORFs.
  • Accessory proteins are not required for virus replication or other known functions (5) (FIGs. 5A-5B).
  • P. marinus trophozoites were transfected with a bacterial plasmid containing a partial SARS-CoV-2 spike gene fused to GFP and under control of the CMV promoter (FIG. 5C).
  • CMV viral
  • bacterial promoters expression was observed 18 hours post-transfection using confocal microscopy (FIG. 5D).
  • IFA immunofluorescence assay
  • CDS full-length coding sequence
  • VHSV viral hemorrhagic septicemia virus
  • N nucleoprotein
  • P phosphoprotein
  • M matrix protein
  • G glycoprotein
  • L large polymerase
  • the G-protein is important as it is inserted into the host cell membrane during assembly of the newly generated virion, at which point it becomes visible to host immune system (FIG. 6B).
  • VHSV is closely related to infectious haematopoietic necrosis virus (IHNV), another significant pathogen of salmonid culture.
  • Example 4 CRISPR/Cas9 ribonucleoprotein-based genome editing methodology in the marine protozoan parasite Perkinsus marinus
  • Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there has not been clinical evidence of this parasite infecting humans, studies of HLA-DR4 0 transgenic mice have strongly suggested the parasite may act as a natural adjuvant in oral vaccines.
  • P. marinus has been developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. The transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8 has been previously reported.
  • Described herein is an electroporation-based protocol used to establish a stable heterologous expression method. Using 20 pg of pPmM0E[M0El]:GFP and 25.0 x 10 6 P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the protocol described herein, successful knock- in of GFP at the C-terminus of the PmMOEl was observed using ribonucleoprotein (RNP)- based CRISPR/Cas9 gene editing methodology. GFP was expressed 18 hours posttransfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.
  • RNP ribonucleoprotein
  • Perkinsus marinus (original name Dermocystidium marinum). first described in 1950 as infecting the eastern oyster (Crassostrea virginica), has been a constant threat to the oyster industry (Mackin et al., 1950; Andrews, 1996; Perkins, 1996). In North America, P. marinus and Perkinsus chesapeaki can coexist in the same bivalve host (McEaughlin and Faisal, 1998; Coss et al., 2001a, b; Pecher et al., 2008; Reece et al., 2008; Arzul et al., 2012).
  • the parasite is taken up by hemocytes and uses them as a vehicle for migration into other host tissues (Eau et al., 2018a; Schott et al., 2019; Yadavalli et al., 2020).
  • Previous studies based on intracellular structures and phylogeny have suggested P. marinus may be a close relative to the apicomplexan, a lineage leading to intracellular parasitism having shared genomic and physiological affinities (Matsuzaki et al., 2008; Joseph et al., 2010; Bachvaroff et al., 2011; Fernandez Robledo et al., 2011; Van Voorhis et al., 2016).
  • the methods described herein are focused on developing molecular tools that establish P. marinus as a heterologous expression system to express genes of pathogens of medical and veterinary relevance.
  • the plasmid MOE[MOEl ]:GFP (formerly known as pMOE:GFP) was built by expanding 1 kb each of 5’ and 3’ flanking regions for PmMOEl coding sequence tagged with GFP, which developed an electroporation-based transfection protocol to deliver the plasmid, and successfully showed a single integration event into the genome via non-homologous recombination (Fernandez Robledo et al., 2008).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 CRISPR-associated protein 9
  • CRISPR/Cas9 technology utilizes machinery such as Cas9 protein, an RNA-guided endonuclease protein, as well as a guide RNA (gRNA) for the nuclease to generate a doublestrand break, which is repaired by nonhomologous end joining (NHEJ) and random mutations incorporated to disrupt the target gene (Mali et al., 2013; Bortesi and Fischer, 2015).
  • gRNA guide RNA
  • NHEJ nonhomologous end joining
  • a donor DNA (dDNA) molecule with homologous templates on either side of the knock-in sequence is required in addition to Cas9 and gRNA.
  • the incorporation of the gene of interest into the genome happens via a homologous- dependent repair mechanism.
  • electroporation-based transfection methodology was developed to improve heterologous gene expression in P. marinus. Furthermore, using the improved protocol described herein, Cas9-gRNA ribonucleoprotein coupled with dDNA was successfully delivered into the P. marinus wild-type trophozoites and tagged the PmMOEl gene with GFP at the C-terminus to achieve mutants phenotypically similar to the previously reported P. marinus mutant strain (PRA-393)(Fernandez Robledo et al., 2008).
  • the trophozoites transfected with 5 and 10 pg of pPmM0E[M0El]:GFP were detected as 0.002 and 0.03%, respectively.
  • Parasites transfected with 5.0 pg of pPmM0E[M0El]:GFP yielded 0.05 and 0.2% GFP-positive cells at 72 and 120 hour time points, respectively (FIG. 8A).
  • parasites transfected with 10.0 pg of pPmM0E[M0El]:GFP yielded 1 and 7.7% GFP-positive cells at 72 and 120 hour time points, respectively (FIG. 8A).
  • parasites transfected with 40 pg at 72 hours post-transfection.
  • a dDNA plasmid containing 396 bp of PmMOEl coding sequence lacking a start codon on the 5’ of the GFP coding sequence was generated which also contained 396 bp of 3’ UTR of PmMOEl at the 3’ of the GFP- coding sequence.
  • the dDNA with GFP and templates were amplified using PCR from previously reported plasmid pPmMOE[MOEl]:GFP (schematic representation in FIG. 10A; Fernandez Robledo et al., 2008).
  • the sgRNA targeting at position 314 on the top strand (sgRNA-1) and another sgRNA targeting position 395 on the bottom strand (sgRNA-2) of the PmMOEl coding sequence were designed using the Benchling software (FIG. 10B). Twenty-five million P. marinus trophozoites were transfected with 20 pg of SpCas9 and sgRNA (1:1) along with 20 pg of dDNA. The parasites transfected with sgRNA- l/SpCas9 and sgRNA-2/SpCas9 and dDNA exhibited GFP expression 24 hour post-transfection (FIG.
  • GFP-positive cells from the sgRNA- 1 and sgRNA- 2 transfections were sorted and cultured for 3 months.
  • Flow cytometry analysis detected approximately 81% of GFP-positive cells transfected with sgRNA- 1 (FIG. 11 A) and 87% of GFP-positive cells transfected with sgRNA-2 (FIG. 11B), respectively.
  • Attempts of amplification of the knock- in resulted in around 2,600 bp amplicon, which would include the 5’ flanking, 5’ UTR, PmMOEl, but not the GFP knock-in (FIG. 11C, 2,600 bp arrow and FIG.
  • Perkinsus marinus a marine protozoan parasite which causes devastating infections to eastern oysters, is under development as a model organism for the protozoan parasite of mollusks (Yadavalli et al., 2020).
  • the success of such attempts varied (Wijayalath et al., 2014; Cold et al., 2017), and prior studies have reported an inconsistency in gene expression.
  • the original transfection method uses 5 pg of plasmid and 50.0 x 10 6 cells; to start, the plasmid amount was increased by 2-fold to 40 pg.
  • the plasmid amount increased in higher GFP-positive cells, especially cells transfected with 20 and 40 pg of the plasmid. In all the cases, fluorescent cells were observed as early as 24 hours post-transfection. Cells needed 3 days to recover and for the GFP expression to be quantifiable.
  • the cells transfected with 40 pg yielded twofold higher GFP-positive cells than cells transfected with 20 pg of plasmid after 72 hours.
  • the CRISPR/Cas9 methodology is broadly adopted by numerous parasitology labs around the world (Mali et al., 2013; Ghorbal et al., 2014; Peng et al., 2014; Shen et al., 2014; Sollelis et al., 2015; Janssen et al., 2018; Lin et al., 2019). Utilizing the improved conditions described herein, further steps were taken to develop the CRISPR/Cas9-based gene editing methodology for P. marinus. For the proof of concept, the PmMOEl gene that has a defined phenotype when tagged with GFP was targeted (Fernandez Robledo et al., 2008).
  • Fluorescent trophozoites were detected within 18 hours of delivering the CRISPR/Cas9 system components. Lack of GFP expression in the transfection of dDNA alone (lacking CRISPR/Cas9 components) ruled out the possibilities of non-homologous recombination in frame with any expressed gene; however, with this fluorescence screening, plasmid fragmentation and integration at the transposable element sites could not be excluded.
  • the GFP expression pattern in the transfectants was similar to that of P. marinus PRA393 (FIG. 10C).
  • GFP-positive cells sorted from sgRNA-l/Cas9 and sgRNA-2/Cas9 experiments were PCR amplified to check for the knock-in of the GFP.
  • the PCR in the sorted cells did not result in the 3,300 bp amplicon.
  • the nested PCR produced the expected size amplicons whose direct sequences confirmed the successful knock-in of GFP at the C -terminus of PmMOEl.
  • previous studies have suggested the trend is to build a plasmid vector that incorporates both the expression of Cas9 and the sgRNA or even generate a mutant conditionally expressing Cas9.
  • CRISPR/Cas9 components were chosen for delivery, including the SpCas9 nuclease, directly by electroporation.
  • the data reported herein are from a single trial targeting PmMOEl using 25.0 x 10 6 log-phase trophozoites, 20 pg of dDNA, 10 pg of sgRNAs, and SaCas9 nuclease (chosen based on Beneke et al. (2017) and Soares Medeiros et al. (2017)) and resulted in a successful knock-in.
  • Genome editing tools like CRISPR/Cas9 in parasite biology are used for gene disruption, fluorescent tagging, and single nucleotide mutation incorporation to study genes involved in the parasite growth, invasion, and drug resistance (Wagner et al., 2014; Di Chandica et al., 2017).
  • CRISPR/Cas9 is widely used in high- throughput and genome screening studies to identify essential genes involved in parasite invasion and antiparasitic drug candidates (Di Ci et al., 2017; Di Mandarin and Carruthers, 2018).
  • CRISPR/Cas9-based knock-out studies in Cryptosporidium parvum are used to understand the mechanism of the parasite’s resistance to antifolate drugs and nutrient acquisition pathways (Vinayak et al., 2015; Pawlowic et al., 2017, 2019).
  • the P. marinus genome encodes for 23,454 genes embedded in 17,000 supercontigs.
  • tetra-polyploidy pose a significant bottleneck for the assembly (El-Sayed et al., 2007; Bogema et al., 2020).
  • Proteome studies identified that P. marinus possess 4,073 non- redundant hypothetical proteins, of which 36 and 27% are involved in metabolic and cellular processes, respectively (Marcia et al., 2017).
  • the rhoptry proteins such as serine-threonine kinases, protein phosphatases, proteosomes, and a virulent candidate merozoite surface protein 3, which are known to play a crucial role in parasite invasion and cell-cell communication during the invasion in P.
  • P. marinus possess extracellular proteins such as high molecular weight cell wall protein 1 (Montes et al., 2002); glycosylation, mucin, and sugar-binding domain protein Pmar_XP_002783417.1 encoded by Pmar_PMAR006943; sensory signal transduction-related histidine kinase encoded by Pmar_PMAR009211; and a family of cysteine-rich modular proteins whose function in the parasite life cycle are yet to be investigated (Montes et al., 2002).
  • extracellular proteins such as high molecular weight cell wall protein 1 (Montes et al., 2002); glycosylation, mucin, and sugar-binding domain protein Pmar_XP_002783417.1 encoded by Pmar_PMAR006943; sensory signal transduction-related histidine kinase encoded by Pmar_PMAR009211; and a family of cysteine-rich modular proteins whose function in the parasite life cycle are yet to be investigated (Montes
  • apoptotic genes such as apoptosis inhibitory molecule (Fas), apoptosis-inducing factor (Tadesse et al., 2017; Lau et al., 2018b), peroxiredoxin, and superoxide dismutase have been shown to favor parasite survival by reducing the host cell (Schott and Vasta, 2003; Schott et al., 2003; Box et al., 2020). The function of these apoptotic genes responsible for the disease in the oysters is limited.
  • the transfection vector pPmMOE[MOEl]:GFP (former pPmMOE-GFP) (Fernandez Robledo et al., 2008) was propagated in Escherichia coli JM109. Plasmid minipreps were prepared using a commercial kit (E.Z.N.A.® Plasmid mini Kit I, Omega-Tek, Norcross, GA, United States), and DNA concentration and purity were estimated with a NanodropTM 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The isolated plasmid DNA was air dried using speedVac for all the experiments. P.
  • marinus cells were prepared following the Cell Line Optimization Nucleofector Kit before electroporation using the NucleofectorTM 2b (Lonza, Walkersville, MD, United States). For all the experiments, the pre-set program D-023 and Lonza’s solution V (Fernandez Robledo et al., 2008) were used. Briefly, dried plasmid was resuspended in 100 pl of Solution V containing supplement 1. pPmMOE[MOEl]:GFP was tested at 5, 10, 20, and 40 pg with 50 million P. marinus cells. Once the optimal plasmid amount was established (20 pg), it was tested with variable P.
  • the 3R-transfection buer composed of 200 mM Na2HPO4, 70 mM NathPCF, 15 mM KC1, and 150 mM HEPES, was prepared and pHed to 7.3. Dried 20 pg of circular pPmMOE[MOEl]:GFP plasmid was resuspended in 60 pl of milliQ water. Once dissolved, 35 pl of 3R transfection buer and 10 pl of 1.5 mM CaCh were added (Protocols. io). Twenty-five million P. marinus trophozoites were transfected.
  • PAM-target site selection was identified using the PmMOEl sequence (Pmar_PMAR027036) and the software (Benchling, Inc.). The output sequences were searched using BLASTx (NCBI-Blast, 2021) against the P. marinus nr database (RefSeq assembly: GCF_000006405.1), which predicted Pmar_PMAR025337 as another possible target.
  • sgRNA Single-guide RNA
  • sgRNA- 1 Single-guide RNA
  • sgRNA-2 Single-guide RNA targeting positive strand at position 339 of PmMOEl CDS
  • sgRNA-2 Single-guide RNA
  • sgRNA-2 Single-guide RNA targeting positive strand at position 339 of PmMOEl CDS
  • sgRNA-2 5’-CCC TGT AAA TGT GGT GGT GG-3’
  • sgRNA targeting negative strand at position 382 of PmMOE CDS sgRNA-2) 5’-CAT GTC GGC TTC GTC GTA GT CGG-3’
  • SEQ ID NO: 3 Single-guide RNA targeting positive strand at position 339 of PmMOEl CDS
  • sgRNA-2 Single-guide RNA targeting negative strand at position 382 of PmMOE CDS
  • sgRNA-2 5’-CAT GTC GGC TTC GTC GTA GT CGG-3’
  • the dDNA was amplified from pPmMOE[MOEl]:GFP (Fernandez Robledo et al., 2008) using primers forward 5’-CGC TTC ATT GTT GGT CTG TAC-3’ (SEQ ID NO: 4) and reverse 5’-CAG TAC GAA ATT ACG CGA GAT G-3’ (SEQ ID NO: 5).
  • the amplicon was cloned into the pGEM®-T vector by T-A cloning (pGEM-T Vector Systems, Promega Corporation, Madison, WI), propagated in Escherichia coli JM109 (L1001, Promega), and sent for sequencing. Plasmid minipreps were prepared using a commercial kit (E.Z.N.A.® Plasmid Mini Kit I, Omega Bio-Tek, Norcross, GA, United States), and DNA concentration and purity were estimated with a Nanodrop 1000 spectrophotometer.
  • Perkinsus marinus cells were prepared following the Cell Line Optimization Nucleofector Kit protocol before electroporation. Using the NucleofectorTM 2b, 10 pg of Streptococcus pyrogenes Cas9 (SpCas9) nuclease TrueCutTM Cas9 protein v2 (Thermo Fisher Scientific, Vilnius, Lithuania) and 10 pg of sgRNAs (Synthego, Silicon Valley, CA, United States) were mixed in 100 pl of Lonza’s solution V and incubated at room temperature for 15 minutes for hybridization of sgRNA and Cas9 protein (Beneke et al., 2017).
  • SpCas9 Streptococcus pyrogenes Cas9
  • sgRNAs Synthego, Silicon Valley, CA, United States
  • the primer pair Fwd 1 5’-CTC GTA ATG AGC CCA ACC AT-3’ (SEQ ID NO: 6) and Rev 1 5’-GGA GGA CTT GAG GCT CTG TG 3’ (SEQ ID NO: 7) (Fernandez Robledo et al., 2008) were designed using the available supercontig (Ensembl, 2021) results in 2,600 bp of PmMOEl (wildtype) and yielded 3,300 bp after successful GFP knock-in.
  • primers were designed spanning 136 bp of the 5’ flanking region, PmMOEl CDS, and 201 bp of the GFP sequence (Fwd 2 5’-TGT TGT AAG GCG AGA CGC TA-3’ (SEQ ID NO: 8) and Rev 2 5’-GTA GGT CAG GGT GGT CAC GA-3’ (SEQ ID NO: 9), respectively.
  • 50 ng of the gDNA and primers mentioned herein were used to amplify by polymerase chain reaction. The amplicons were purified from the 1% agarose gel using the ZymocleanTM Gel DNA Recovery kit (Tustin, CA, United States).
  • Parasites were fixed with 3% paraformaldehyde (Thermo Fisher Scientific, preserved 37% reagent) for 15 minutes at room temperature. Parasites were washed three times at 1,000 x g for 5 minutes using 1 x phosphate-buered saline (l x PBS). Following the washes, parasites were treated with 0.1% Triton X-100 for 15 minutes and washed three times with 1 x PBS. The cells were stained with 25 pg/ml concentration of 4’,6-diamidino- 2- phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, United States).
  • DAPI diamidino- 2- phenylindole
  • the flow cytometry experiments were performed on the live parasite, using ZE5 Cell Analyzer; data was collected using Everest software version 2.0 and analyzed. A minimum of 100,000 events was collected for parasites based on forward and side scatterplot, and a singlet gate was applied to collect a minimum of 30,000 cells.
  • BD Sciences Influx Cell Sorter (BD Sciences, NJ, United States) was used for cell sorting, and cells were sorted based on eGFP-positive gates.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

Described herein are engineered Perkinsus cells, and populations thereof, that express a protein of interest, such as a viral protein. Also described herein are methods of engineering a Perkinsus cell to produce a protein of interest.

Description

ENGINEERED PERKINSUS MARINUS CELLS
RELATED APPLICATION
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/083,418 filed September 25, 2020, the entirety of which is incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 23, 2021, is named B156970004WO00-SEQ-CRP and is 2799 bytes in size.
BACKGROUND
Perkinsozoa is considered one of the earliest diverging groups of the lineage leading to dinoflagellates, and it includes the species Perkinsus marinus (1-2). Perkinsus marinus is a marine protozoan parasite of oysters with a broad distribution in the Gulf and East Coasts. Perkinsus marinus cells have been successfully cultured in host-free cell medium, and previous studies using cultured Perkinsus marinus cells have demonstrated low heterologous expression of protozoan proteins (3).
SUMMARY
Although previous studies showed heterologous expression of protozoan proteins in the protozoa Perkinsus marinus, heterologous expression of non-protozoan proteins in Perkinsus marinus has remained largely unexplored; indeed, heterologous expression of viral proteins in Perkinsus marinus has not been previously considered or tested. The inventors of the instant disclosure have appreciated that poor engineering methods (e.g., transfection methods) and complexities related to RNA trans-splicing inherent in Perkinsus marinus may have contributed to the lack of development of Perkinsus marinus as a heterologous expression platform; in particular, for the expression of non-protozoan proteins, such as viral proteins. Disclosed herein are improved methods for engineering Perkinsus marinus cells such that the cells exhibit improved production of a protein(s) of interest (e.g., a viral protein). These studies demonstrate for the first time that Perkinsus marinus cells can be engineered to heterologously express viral proteins.
In some aspects, the disclosure relates to engineered Perkinsus cells.
In some embodiments an engineered Perkinsus cell is derived from a Perkinsus andrewsi cell, a Perkinsus beihaiensis cell, a Perkinsus chesapeaki cell, a Perkinsus honshuensis cell, a Perkinsus marinus cell, a Perkinsus mediterraneus cell, a Perkinsus olseni cell, or a Perkinsus qugwadii cell.
In some embodiments, an engineered Perkinsus cell is an engineered Perkinsus marinus cell.
In some embodiments, an engineered Perkinsus marinus cell expresses a polypeptide comprising a viral protein. In some embodiments, the polypeptide comprising the viral protein is expressed on the surface of the cell.
In some embodiments, the polypeptide comprises a tag.
In some embodiments, the polypeptide comprises an amino acid sequence of a signal peptide that directs the polypeptide comprising the viral protein to the cell surface. In some embodiments, the signal peptide is an MOE signal peptide. In some embodiments, the MOE signal peptide comprises an amino acid sequence of MRFIVGLYSCLAVLVLGQSSCPTGSAQC (SEQ ID NO: 1).
In some embodiments, the cell comprises a heterologous polynucleotide comprising a nucleic acid sequence encoding the polypeptide comprising the viral protein. In some embodiments, the heterologous polynucleotide is a plasmid. In some embodiments, the heterologous polynucleotide is integrated into the genome of the cell.
In some embodiments, the heterologous polynucleotide comprises a nucleic acid sequence encoding two or more polypeptides comprising viral proteins. In some embodiments, at least two of the viral proteins encoded by the heterologous polynucleotide comprise the same amino acid sequence. In some embodiments, at least two of the viral proteins encoded by the heterologous polynucleotide comprise different amino acid sequences.
In some embodiments, the cell comprises two or more heterologous polynucleotides comprising a nucleic acid sequence encoding a polypeptide comprising a viral protein. In some embodiments, the nucleic acid sequences of at least two of the heterologous polynucleotides are the same. In some embodiments, the nucleic acid sequences of at least two of the heterologous polynucleotides are different. In some aspect, the disclosure relates to populations of Perkinsus marinus cells. In some embodiments, at least 8% of the cells in the population express a protein of interest consequent to transfection of the population with a plurality of heterologous polynucleotides encoding the protein of interest. In some embodiments, the cells are trophozoites.
In some embodiments, the number of cells in the population is from about 1 x 106 to about 50 x 106 cells. In some embodiments, the number of cells is about 25 x 106 cells.
In some embodiments, the protein of interest comprises the amino acid sequence of a viral protein.
In some aspects, the disclosure relates to methods of engineering Perkinsus marinus cells to produce a protein of interest. In some embodiments, the method comprises contacting a population of Perkinsus marinus trophozoites with a transfection reagent and a plurality of heterologous polynucleotides encoding the protein of interest, wherein: the population of Perkinsus marinus trophozoites comprises from about 1 x 106 to about 50 x 106 cells; the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg; or a combination thereof.
In some embodiments, the population of Perkinsus marinus trophozoites comprises from about 1 x 106to about 50 x 106 cells and the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg.
In some embodiments, the population of Perkinsus marinus trophozoites has about 25 x 106 cells.
In some embodiments, the plurality of heterologous polynucleotides is in an amount of about 20 pg.
In some embodiments, the population of Perkinsus marinus trophozoites are in a log growth phase when contacted with the transfection reagent and the plurality of heterologous polynucleotides. In some embodiments, the population of Perkinsus marinus trophozoites have an absorbance of 1.66 at ODeoo when contacted with the transfection reagent and the plurality of heterologous polynucleotides.
In some embodiments, the protein of interest comprises the amino acid sequence of a viral protein.
In some aspects, the disclosure relates to methods of producing a polypeptide comprising a viral protein. In some embodiments, the method comprises culturing an engineered Perkinsus marinus cell described herein under conditions in which it expresses a polypeptide comprising the viral protein. In some embodiments, the method further comprises isolating the polypeptide comprising the viral protein.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.
FIG. 1 shows EGFP expression in engineered P. marinus trophozoites. High numbers of engineered cells were seen when using 20 pg of plasmid DNA and 25 x 106 P. marinus trophozoites for transfection.
FIGs. 2A-2B relate to a strategy for knock-in of GFP in P. marinus using the CRISPR/Cas9 system. FIG. 2A depicts the strategy followed and reagents used. FIG. 2B shows replication of the mutant PmM0E[M0E]:GFP after knocking in GFP in P. marinus MOE wild type. The knock-in was observed less than 24 hours after the delivery of the CRISPR/Cas9 components.
FIGs. 3A-3C relate to heterologous expression of Plasmodium falciparum genes in Perkinsus marinus cells. FIG. 3A shows expression of P. falciparum CSP (circumsporozoite protein) tagged with GFP and IFA using anti-PfCSP to confirm the production of CSP protein. FIG. 3B shows expression of P. falciparum RPL36 (ribosomal protein L36) tagged with GFP. FIG. 3C shows his-tag affinity purification of rPfRPL36-GFP.
FIG. 4 depicts heterologous protozoan proteins expressed in Perkinsus marinus cells in the studies described herein.
FIGs. 5A-5C relate to expression of SARS-CoV-2 spike protein in Perkinsus marinus cells. FIG. 5A shows a schematic depicting SARS-CoV-2 morphology. FIG. 5B shows a schematic depicting the SARS-CoV-2 genome organization. FIG. 5C shows a plasmid containing partial spike (S) gene tagged with GFP. FIG. 5D shows an image 24 hours after transfection showing numerous cells expressing the GFP tagged spike (S) gene.
FIGs. 6A-6B relate to expression of VHSV G protein proteins in Perkinsus marinus cells. FIG. 6A depicts the typical rhabdoviral genome organization. FIG. 6B shows the G protein gene cloned into vector p[MOE]:GFP to give p[MOE]:G-GFP. FIG. 7 shows a Perkinsus marinus cell expressing GFP-tagged ebola glycoprotein (GP1,2) gene 24 hours after transfection.
FIGs. 8A-8D show plasmid amount and cell number studies. FIG. 8A shows fifty million parasites transfected with 5 mg, 10 mg, 20 mg, and 40 mg of pPmMOE[MOEl]:GFP, respectively. Bar graphs show the % GFP-positive cells (y-axis) that were detected by flow cytometry at 24, 72, and 120 hours post-transfection time points (x-axis). FIG. 8B shows GFP expression in parasites transfected with 20 mg of pPmMOE[MOEl]:GFP 24 and 120 hours post-transfections. FIG. 8C shows % GFP expression in parasites transfected with 20 mg of pPmMOE[MOEl]:GFP. The bar graphs show the % GFP-positive cells (y-axis) which were detected by flow cytometry at 24, 72, 120 hours, and 3 months post-transfection time points (x-axis). FIG. 8D shows a scattered plot from FCM where no GFP expression in untransfected controls was observed, and 98% GFP-positive cells in 25.0 x 106 cells which were transfected with 20 mg of pPmMOE[MOEl]:GFP as indicated in the box.
FIGs. 9A-9E show a comparison of transfection protocols. 25 x 106 cells were transfected with 20 mg of pPmMOE[MOEl]:GFP plasmid using different protocols. FIG. 9A shows a flow cytometry scattered plot of un-transfected (wild-type) cells, where GFP expression was not detected. FIG. 9B shows a scattered plot of flow cytometry where GFP- positive cells were identified in transfection performed using the Lonza method. FIG. 9C shows a scatterplot representation of GFP-positive cells in transfection performed using 3R buffer and BTX cuvette. FIG. 9D is a scatterplot showing GFP-positive cells when transfected with 3R buffer utilizing the Lonza cuvette. FIG. 9E is a bar graph showing the % of GFP-positive cells that were transfected with the Lonza system, 3R buffer in combination with BTX cuvette, 3R buffer using Lonza cuvette, and Lonza buffer with BTX cuvette.
FIGs. 10A-10D show SpCas9-RNP and sgRNA-mediated GFP knock-in in P. marinus trophozoites. FIG. 10A is a schematic representation of dDNA with 396 bp homology on the 50 and 30 of the GFP coding sequence. FIG. 10B is a schematic representation of the guide RNA target sites on PmMOEl coding sequences; sgRNA-1 targets the top strand indicated by the arrow direction; sgRNA-2 targets the bottom strand indicated by the arrow direction. FIG. 10C shows a confocal microscopy panel showing successful GFP expression in cells transfected with sgRNA-l/SpCas9 and sgRNA-2/SpCas9. The observed localization patterns were similar to those of the PRA-393 MOE-GFP mutant strain. FIG. 10D shows a scattered plot from FCM where no GFP expression in mock (dDNA+sgRNA alone) control was observed, and 0.2% GFP-positive cells were knocked in using sgRNA-1, and 0.35% in the case of sgRNA-2 as indicated in the box.
FIGs. 11A-11D show the sorting of P. marinus GFP-positive cells for endogenous PmMOEl C-terminus GFP tagging analysis. FIG. 11A is a scattered plot showing 81% GFP- positive cells indicated with a box in the experiment where cells transfected with sgRNA-1 - Cas9. FIG. 11B show a scattered plot showing 87% GFP-positive cells indicated with a box in the experiment where cells transfected with sgRNA-l-Cas9. FIG. 11C shows the PCR intended to amplify the knock-in (expected sized 3,300 bp) using Fwd 1 and Rev 1 primers resulting in the amplification of the wildtype 2,600 bp amplicon (left panel). This PCR product was used as a template in the nested PCR (nPCR) to confirm the GFP knock-in using Fwd 2 and Rev 2 primers, which yielded the expected 748 bp amplicon (right panel), 1 kb Plus DNA Ladder (New England Biolabs, Ipswich, MA, United States). FIG. 11D shows the sequencing results of the nPCR product from the sgRNA-2 targeted GFP knock-in experiment.
FIG. 12 shows the observed GFP expression among cell populations of 1.62, 3.12, 6.25, 12.5, 25.0, and 50.0 million.
FIGs. 13A-13B show PCR and nested PCR based genotyping of PMAR_Pmar027036 for the GFP knock in analysis. The genomic DNA was isolated from two different clones labeled as 4 and 5, which were hand-picked 3 months after the cell sorting from the experiments involved in the utilization of SgRNA-1 and SgRNA-2, respectively. FIG. 13A shows the PCR product showing the successful amplification of 2,600 bp of DNA sequencing containing flanking regions, M0E1 CDS encoded by PMAR_Pmar027036 and the GFP. PCR product was diluted 100 times and used in the nested PCR to identify the knock in of the GFP, which successfully amplified the 748 bp of DNA fragment. The fragment was sequenced to confirm the successful knock in of GFP. FIG. 13B shows a repeat of the gel analysis of the nested PCR products from (FIG. 13 A), showing the DNA size of 748 bp.
DETAILED DESCRIPTION
Described herein are engineered Perkinsus marinus cells, and populations thereof, that express proteins of interest. In particular, engineered Perkinsus marinus cells are described that express viral genes (z.e., encoding viral proteins). Such engineered cells have not been described or contemplated previously. Compositions (e.g., vaccines) comprising Perkinsus marinus cells, and populations thereof, are also described herein. Additionally, improved methods of engineering Perkinsus marinus cells to produce proteins of interest (e.g., a viral protein) with high production yields are described.
Engineered Perkinsus Cells
In some aspects, the disclosure relates to engineered Perkinsus cells (e.g., Perkinsus marinus cells) that express a heterologous protein of interest. In some embodiments an engineered Perkinsus cell is derived from a Perkinsus andrewsi cell, a Perkinsus beihaiensis cell, a Perkinsus chesapeaki cell, a Perkinsus honshuensis cell, a Perkinsus marinus cell, a Perkinsus mediterraneus cell, a Perkinsus olseni cell, or a Perkinsus qugwadii cell. In some embodiments, an engineered Perkinsus cell is an engineered Perkinsus marinus cell.
As used herein, the term “heterologous protein” refers to a protein that is not produced by a wild-type Perkinsus cell corresponding to the species from which the engineered Perkinsus cell is derived. A heterologous protein of interest may be: (i) a protein from any source other than a Perkinsus cell from which the engineered Perkinsus cell is derived; or (ii) a modified protein from a Perkinsus cell from which the engineered Perkinsus cell is derived (/'.<?., such that the modified protein is not produced by the corresponding wildtype Perkinsus cell).
In some embodiments, an engineered Perkinsus cell expresses more than one heterologous protein of interest. For example, in some embodiments a Perkinsus cell expresses at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 heterologous proteins of interest. In some embodiments, an engineered Perkinsus cell expresses 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2- 10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10. 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 heterologous proteins of interest. In some embodiments, an engineered Perkinsus cell expresses 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous proteins of interest.
In some embodiments, an engineered Perkinsus cell expresses a heterologous protein from a protozoan organism. In some embodiments, the protozoan organism is a protozoan parasite. For example, in some embodiments, the protozoan organism is Plasmodium berghei (Pb), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg), or Cryptosporidium parvum (Cp). In some embodiments, the protein of interest comprises or consists of an HAP2 protein (e.g., PbHAP2, or a fragment thereof), a MSP8 protein (e.g., PbMSP8, or a fragment thereof), an Rpl36 protein (e.g., PfRpl36, or a fragment thereof), a CSP protein (e.g., PfCSP, or a fragment thereof), an ACP protein (e.g., TgACP or a fragment thereof), an FNR protein (e.g., TgFNR or a fragment thereof), or an AdoT protein (e.g., CpAdoT or a fragment thereof).
In some embodiments, an engineered Perkinsus cell expresses a heterologous protein from a virus. In some embodiments, the virus is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, an Ebola virus, or a viral hemorrhagic septicemia virus (VHSV). Proteins encoded by the genomes of SARS-CoV-2, Ebola, and VHSV have been identified previously. See e.g., FIGs. 5A-5B and FIG. 6A. In some embodiments, the virus is SARS-CoV-2 and the protein of interest comprises or consists of spike (S) protein (or a portion thereof). In some embodiments, the virus is Ebola and the protein of interest comprises or consists of GP1,2 (or a portion thereof). In some embodiments, the virus is VHSV and the protein of interest comprises or consists of glycoprotein (G) (or a portion thereof).
In some embodiments, a protein of interest described herein is expressed on the surface of the engineered Perkinsus cell.
In some embodiments, a protein of interest described herein comprises a tag. For example, in some embodiments, a protein of interest comprises a viral protein and a tag. A tag may be an affinity tag (e.g., a chitin binding protein (CBP) tag, a maltose binding protein (MBP) tag, a streptavidin tag, a glutathione-S-transferase (GST) tag, or a poly His tag), a solubilization tag (e.g., a thioredoxin (TRX) tag, a poly(NANP), an MBP tag, or a GST tag), an epitope tag (e.g., a FLAG-tag, an ALFA-tag, a V5-tag, a Myc-tag, an HA-tag, a Spot-tag, a T7-tag, or an NE-tag.), and/or a fluorescence tag (e.g., TagBFP, mTagBFP2, Azurite, EBFP2, EBFP2, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, DIKOK, mK02, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP- T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, or NirFP). In some embodiments, a tag is removable by chemical or enzymatic means (e.g., by TEV protease, Thrombin, Factor Xa or Enteropeptidase).
In some embodiments, a protein of interest described herein comprises a signal peptide. Signal peptides facilitate translocation of proteins to a specific location within the cell, e.g., to the cellular membrane. As such, in some embodiments, a protein of interest comprises a signal peptide that directs the protein of interest to the cell surface. In some embodiments, a signal peptide is an MOE signal peptide. In some embodiments, a an MOE signal peptide comprises an amino acid sequence of MRFIVGLYSCLAVLVLGQSSCPTGSAQC (SEQ ID NO: 1) or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 1. Methods of determining the extent of identity between two sequences (e.g., two amino acid sequences or two polynucleic acids) are known to those having ordinary skill in the art. One exemplary method is the use of Basic Local Alignment Search Tool (BLAST®) software with default parameters (blast.ncbi.nlm.nih.gov/Blast.cgi).
In some embodiments, an engineered Perkinsus cell described herein comprises a heterologous polynucleotide having a nucleic acid sequence that encodes a heterologous protein of interest (e.g., comprising or consisting of a viral protein). As used herein the term “heterologous polynucleotide” refers to a polynucleotide that is not found in the Perkinsus cell from which the engineered cell is derived. In some embodiments, an engineered Perkinsus cell described herein comprises more than one heterologous polynucleotide. For example, in some embodiments, an engineered Perkinsus cell described herein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 heterologous polynucleotides. In some embodiments, an engineered Perkinsus cell described herein comprises 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9,
2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10. 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 heterologous polynucleotides. In some embodiments, an engineered Perkinsus cell described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous polynucleotides. In some embodiments, the nucleic acid sequences of two or more of the heterologous polynucleotides are the same. In some embodiments, the nucleic acid sequences of two or more of the heterologous polynucleotides are different.
In some embodiments, a heterologous polynucleotide encodes more than one protein of interest. For example, in some embodiments a heterologous polynucleotide encodes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proteins of interest. In some embodiments, a heterologous polynucleotide encodes 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8,
3-9, 3-10. 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 proteins of interest. In some embodiments, a heterologous polynucleotide encodes 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins of interest. In some embodiments, two or more of the proteins of interest encoded by a heterologous polynucleotide comprise the same amino acid sequence. In some embodiment, two or more of the proteins of interest encoded by a heterologous polynucleotide comprise different amino acid sequences.
In some embodiments, a heterologous polynucleotide is an extrachromosomal DNA (z.e., found off the chromosomes of the cell, either inside or outside of the nucleus). For example, in some embodiments, a heterologous polynucleotide is a plasmid.
In some embodiments, a heterologous polynucleotide is integrated within a chromosome (z.e., within the genome of) the cells.
A heterologous polynucleotide may further comprise a promoter sequence that regulates transcription of the nucleic acid sequence that encodes for the heterologous protein of interest. In some embodiments, the promoter is a bacterial promoter or a viral promoter (e.g., CMV).
Populations of Perkinsus Cells
In some aspects, the disclosure relates to populations of Perkinsus cells (e.g., populations of Perkinsus marinus cells). In some embodiments, a population of Perkinsus cells comprises an engineered Perkinsus cell described herein (e.g., an engineered Perkinsus marinus cell). In some embodiments, the cells in the population are Perkinsus trophozoites (e.g., Perkinsus marinus trophozoites).
In some embodiments, a population of Perkinsus cells comprises a plurality of engineered Perkinsus cells described herein. In some embodiments, each of the engineered cells in the plurality express the same protein(s) of interest (e.g., comprising or consisting of the same viral protein). In some embodiments, a plurality of engineered Perkinsus cells comprises two or more distinct engineered cells described herein.
In some embodiments, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% of the cells in the population of Perkinsus cells express a protein of interest (z.e., are engineered Perkinsus cells, as described herein) consequent to transfection of the population with a plurality of heterologous polynucleotides encoding a protein(s) of interest. A cell expresses a protein of interest “consequent to transfection” if the cell uptakes a heterologous polynucleotide and expresses a protein of interest in an amount that is sufficient enough to detect (e.g., by visual observation, immuno staining, immunofluorescence, cell sorting, etc.).
In some embodiments, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% of the cells in the population of Perkinsus cells express a protein of interest (z.e., are engineered Perkinsus cells, as described herein) consequent to transfection of the population with a plurality of heterologous polynucleotides encoding a protein(s) of interest within a certain time following transfection; for example, within 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours following transfection; or within 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 20-21, 20-22, 20-23, 20-24, 20-36, 20-48, 24-36, 24-48, or 36-48 hours following transfection.
The number of cells transfected with the heterologous polynucleotide (z.e., “transfection of the population”) is used to determine the percentage of cells expressing the protein(s) of interest. One having ordinary skill in the art will appreciate that populations of Perkinsus cells that express the protein(s) of interest can be obtained and purified using known methods subsequent to transfection.
In some embodiments, the number of cells in the population (z.e., that are subject to the transfection with a plurality of heterologous polynucleotides) is about 1 x 106 cells, about 5 x 106 cells, about 10 x 106 cells, about 15 x 106 cells, about 20 x 106 cells, about 25 x 106 cells, about 30 x 106 cells, about 35 x 106 cells, about 40 x 106 cells, about 45 x 106 cells, or about 50 x 106 cells. In some embodiments, the number of cells is from about 1 x 106 to about 50 x 106, about 5 x 106 to about 50 x 106, about 10 x 106 to about 50 x 106, about 15 x 106 to about 50 x 106, about 20 x 106 to about 50 x 106, about 25 x 106 to about 50 x 106, about 1 x 106 to about 25 x 106, about 5 x 106 to about 25 x 106, about 10 x 106 to about 25 x 106, about 15 x 106 to about 25 x 106, about 20 x 106 to about 25 x 106 cells. In some embodiments, the number of cells is about 25 x 106 cells. Compositions Comprising Engineered Perkinsus Cells or a Protein of Interest Obtained Therefrom In some aspects, the disclosure relates to compositions comprising an engineered Perkinsus cell described herein, e.g., engineered Perkinsus marinus cells (or a population of engineered Perkinsus cells described herein) or a protein(s) of interest obtained therefrom.
In some embodiments, a composition is a vaccine comprising an engineered Perkinsus cell described herein. In some embodiments, a composition is a vaccine comprising a protein of interest that is obtained from an engineered Perkinsus cell described herein.
In some embodiments, the vaccine is formulated for oral delivery (/'.<?., the vaccine is an oral vaccine).
Methods of Engineering Perkinsus Cells
In some aspects, the disclosure relates to improved methods of engineering Perkinsus cells (e.g., engineered Perkinsus marinus cells) to produce a protein of interest (e.g., comprising or consisting of a viral protein). In some embodiments, a method of engineering a Perkinsus cell for production of a protein(s) of interest comprises contacting a population of Perkinsus cells (e.g., Perkinsus marinus cells or trophozoites) with a transfection reagent and a plurality of heterologous polynucleotides encoding the protein(s) of interest. Suitable transfection reagents are known to those having ordinary skill in the art.
In some embodiments of the methods described herein, the population of Perkinsus cells (e.g., Perkinsus marinus cells or trophozoites) comprises about 1 x 106 cells, about 5 x 106 cells, about 10 x 106 cells, about 15 x 106 cells, about 20 x 106 cells, about 25 x 106 cells, about 30 x 106 cells, about 35 x 106 cells, about 40 x 106 cells, about 45 x 106 cells, or about 50 x 106 cells. In some embodiments, the number of cells is from about 1 x 106 to about 50 x 106, about 5 x 106 to about 50 x 106, about 10 x 106 to about 50 x 106, about 15 x 106 to about 50 x 106, about 20 x 106 to about 50 x 106, about 25 x 106 to about 50 x 106, about 1 x 106 to about 25 x 106, about 5 x 106 to about 25 x 106, about 10 x 106 to about 25 x 106, about 15 x 106 to about 25 x 106, about 20 x 106 to about 25 x 106 cells. In some embodiments, the population of Perkinsus cells (e.g., Perkinsus marinus cells or trophozoites) comprises about 25 x 106 cells.
In some embodiments of the methods described herein, the plurality of heterologous polynucleotides that are contacted with the population of Perkinsus cells is in an amount of about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 |ag. In some embodiments, the plurality of heterologous polynucleotides is in an amount of about 15 pg to about 25 pg, about 16 pg to about 25 pg, about 17 pg to about 28 pg, about 19 pg to about 25 pg, about 20 pg to about 25 pg, about 15 pg to about 20 pg, about 16 pg to about 20 pg, about 17 pg to about 20 pg, about 18 pg to about 20 pg, about 18 pg to about 22 pg, or about 19 pg to about 21 pg. In some embodiments, the plurality of heterologous polynucleotides is in an amount of about 20 pg.
In some embodiments, the population of Perkinsus cells (e.g., Perkinsus marinus cells or trophozoites) comprises from about 1 x 106 to about 50 x 106 cells and the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg.
In some embodiments of the methods described herein, the population of Perkinsus cells are in a log growth phase when contacted with the transfection reagent and the plurality of heterologous polynucleotides. In some embodiments, the population of Perkinsus cells have an absorbance of 1.66 at ODeoo when contacted with the transfection reagent and the plurality of heterologous polynucleotides.
Methods of Producing a Protein of Interest
In some aspects, the disclosure relates to methods of producing a protein of interest (e.g., comprising or consisting of a viral protein). In some embodiments, a method of producing a protein of interest comprises culturing an engineered Perkinsus cell described herein under conditions in which it expresses the protein of interest.
In some embodiments, the method further comprises isolating (or purifying) the protein of interest.
EXAMPLES Example 1: Engineering Perkinsus marinus cells to produce a heterolo ous protein.
Perkinsus marinus cells were engineered to produce a heterologous protein by: (i) transfection methodologies; and (ii) targeted genome editing.
Improved methods for engineering P. marinus cells via transfection were developed. Various parameters were analyzed, including plasmid amounts (2, 5, 10, 20 pg; 1:1 supercoiled), cell numbers (5, 10, 25, 50 x 106 cells), and transfection reagents. Transfection reactions were carried out with cells in log growth phase (ODeoo = 1.66), and the numbers of viable engineered cells were analyzed at various time points following transfection. The highest numbers of engineered cells (> 8% of the starting population) were seen when using 20 pg of plasmid DNA and 25 x 106 P. marinus trophozoites (FIG. 1). The transfection reagent Nucleofector (Lonza) also provided superior results.
For targeted genome editing, the CRISPR/Cas9 system was used. The CRISPR/Cas9 system was tested by delivering a guide RNA (gRNA), ribonuclease Cas9, and donor DNA (dDNA) targeting PmMOE for knock-in GFP and for knock-out of GFP from a previously described P. marinus variant expressing PmMOE-GFP (FIG. 2A). The mutant phenotype was successfully reproduced by knocking-in GFP at the 3’ end of PmMOE in the wild type strain (FIG. 2B) and knocking-out the GFP from the previously transformed PmMOE-GFP variant. The gRNA, dDNA, and ribonuclease Cas9 were delivered into the trophozoites using electroporation. The efficiency of CRISPR/Cas9 transformation was 0.5%. Cells with the phenotype expressing PmMOE-GFP were observed 18 hours post-transfection. Both PCR and sequence analysis indicated that the GFP was integrated at the designated position and with the intended length or mutated at the designated position (null mutants).
Example 2: Stable expression of heterologous proteins from protozoan parasites in Perkinsus marinus cells.
Prokaryotic expression systems (e.g., E. coli expression systems) are the most commonly used systems for industrial production of recombinant proteins. However, for proteins from protozoan parasites, the overall success rate for obtaining soluble, recombinant proteins in prokaryotic systems remains low. In addition, expression of heterologous proteins in yeast may result in N- and O-linked glycosylation patterns, which might be different from those in the native parasite protein, resulting in inactive products. And, the use of mammalian cells to produce recombinant proteins from parasites has been hindered by the labor-intensive and expensive methodology required for establishing stable recombinant cells.
It was previously hypothesized that protein from protozoan parasites, like the malaria parasites in the genus Plasmodium and Perkinsus, are more likely to be produced using Perkinsus as a heterologous system than other common transgene expression systems like bacteria or yeast (4). It was previously shown that, indeed, P. marinus can express mouse malaria candidate genes (Plasmodium berghei HAP2 and MSP8) (3). Additional experiments were performed herein to test the heterologous expression of two Plasmodium falciparum genes: (i) a highly conserved P. falciparum gene encoding for 60S ribosomal protein L36 (652 bp, 112 aa); and (ii) a P. falciparum CSP encoded by the gene (1,194 bp, 397 aa). The full-length PfCSP coding sequence was codon-optimized and cloned into p[MOE]:GFP. 25.0 x 106 P. marinus cells were electroporated with the plasmids cloned with the PfRPL36 and PfCSP. Expression was observed 18 hours post-transfection. In the case of PfCSP, the IFA using a human monoclonal IgG ab specific to PfCSP confirmed PfCSP protein synthesis. The GFP expression was followed for 3 months, which confirmed the stable expression of full-length PfRPL36 and PfCSP in the P. marinus (FIGs. 3A-3B). Following the stable expression, the N-terminal 6 x His tag was used to purify the PfRPE36 protein using Ni2+- NTA affinity column. The purification was also successful, and a yield of 213 pg/ml was obtained. SDS-PAGE gel confirmed the purification (FIG. 3C).
The Toxoplasma gondii apicoplast specific genes acyl carrier protein 1 and ferredoxin NAD+- reductase and the Cryptosporidium parvum adenosine transporter and tryptophan synthase B were also produced successfully using P. marinus cells (FIG. 4).
Example 3: Expression of viral proteins in Perkinsus marinus cells.
Although previous studies showed heterologous expression of protozoan proteins in the protozoa Perkinsus marinus, heterologous expression of viral proteins in Perkinsus marinus has not been previously considered or tested. Successful production of viral proteins in other expression systems has been unpredictable. For example, production of a high yields of viral protein product may be impacted by toxicity to the cell expressing the viral protein and by codon usage frequencies in the cell expressing the viral protein. In P. marinus specifically, expression of viral genes may be impacted by trans-splicing mechanisms that have been found to occur robustly therein.
Stable heterologous expression of two viral genes in P. marinus were tested: (i) SARS-Cov-2 spike protein (223 aa); and (ii) Ebola GP1,2 (676 aa).
The SARS-CoV-2 virion is encoded in a 30 Kb genome with 14 ORFs. The first two ORFs code for polyprotein (ppla/ab) required for virus replication, followed by structural proteins for spike, membrane, and nucleoprotein. The longest ORF is located at the 5' terminus, encoding 15 nonstructural proteins collectively involved in virus replication and possibly in immune evasion. At the 3' terminus, accessory genes (3a, 3b, p6, 7a, 7b, 8b, 9b, and orfl4) are found with adjacent ORFs. Accessory proteins are not required for virus replication or other known functions (5) (FIGs. 5A-5B).
P. marinus trophozoites were transfected with a bacterial plasmid containing a partial SARS-CoV-2 spike gene fused to GFP and under control of the CMV promoter (FIG. 5C). Preliminary studies demonstrated that P. marinus can express genes under viral or bacterial promoters. Under both viral (CMV) and bacterial promoters, expression was observed 18 hours post-transfection using confocal microscopy (FIG. 5D). An immunofluorescence assay (IFA) using a polyclonal ab specific to SARS-CoV-2 spike gene confirmed SARS-CoV-2 spike gene protein synthesis (FIG. 5D). In this case, a pcDNA3-SARS-CoV-2-SRBD-sGFP was used, which uses the viral CMV promoter for heterologous expression of the SARS- CoV-2 spike protein fragment.
For Ebola GP1,2, the full-length coding sequence (CDS) was codon-optimized for P. marinus, and it was cloned into plasmid p[MOE]:GFP. Heterologous expression was observed (FIG. 7).
Proteins from VHSV are additional exemplary viral candidate proteins for expression in P. marinus. The viral hemorrhagic septicemia virus (VHSV) is a negative-sense, enveloped, single- stranded RNA rhabdovirus. It is an important pathogen of over 50 different species of fish in the northern hemisphere. This virus has a fairly simple genome; VHSV is approximately l lkb. The typical rhabdoviral genome encodes five basic structural proteins, including; the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the large polymerase (L) protein (FIG. 6A). The G-protein is important as it is inserted into the host cell membrane during assembly of the newly generated virion, at which point it becomes visible to host immune system (FIG. 6B). VHSV is closely related to infectious haematopoietic necrosis virus (IHNV), another significant pathogen of salmonid culture.
Example 4: CRISPR/Cas9 ribonucleoprotein-based genome editing methodology in the marine protozoan parasite Perkinsus marinus
Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there has not been clinical evidence of this parasite infecting humans, studies of HLA-DR40 transgenic mice have strongly suggested the parasite may act as a natural adjuvant in oral vaccines. P. marinus has been developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. The transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8 has been previously reported. Described herein is an electroporation-based protocol used to establish a stable heterologous expression method. Using 20 pg of pPmM0E[M0El]:GFP and 25.0 x 106 P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the protocol described herein, successful knock- in of GFP at the C-terminus of the PmMOEl was observed using ribonucleoprotein (RNP)- based CRISPR/Cas9 gene editing methodology. GFP was expressed 18 hours posttransfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.
Introduction
Perkinsus marinus (original name Dermocystidium marinum). first described in 1950 as infecting the eastern oyster (Crassostrea virginica), has been a constant threat to the oyster industry (Mackin et al., 1950; Andrews, 1996; Perkins, 1996). In North America, P. marinus and Perkinsus chesapeaki can coexist in the same bivalve host (McEaughlin and Faisal, 1998; Coss et al., 2001a, b; Pecher et al., 2008; Reece et al., 2008; Arzul et al., 2012). In the oysters, the parasite is taken up by hemocytes and uses them as a vehicle for migration into other host tissues (Eau et al., 2018a; Schott et al., 2019; Yadavalli et al., 2020). Previous studies based on intracellular structures and phylogeny have suggested P. marinus may be a close relative to the apicomplexan, a lineage leading to intracellular parasitism having shared genomic and physiological affinities (Matsuzaki et al., 2008; Joseph et al., 2010; Bachvaroff et al., 2011; Fernandez Robledo et al., 2011; Van Voorhis et al., 2016).
Human exposure to Perkinsus spp. may occur when infected oysters or clams are consumed, due to the high prevalence of the parasite in oysters (Marquis et al., 2015, 2020). Nevertheless, the effect of consuming P. marinus-vcA c d oysters has not been investigated in humans. In previous studies using humanized mice which expressed HLA-DR40 genes and lacked expression of mouse MHC-class II genes (DR4.EA0), DR4.EA0 mice did not develop detectable pathology or systemic inflammation (Wijayalath et al., 2014). Notably, naive (unfed) DR4.EA0 mice had antibodies (IgM and IgG) that reacted against P. marinus, whereas parasite- specific T-cell responses were undetectable. Upon oral feeding with P. marinus, parasite-specific IgM and IgG antibodies were boosted with parasite- specific cellular (INFy) responses detected in the spleen, suggesting P. marinus as a natural adjuvant (Wijayalath et al., 2014).
The methods described herein are focused on developing molecular tools that establish P. marinus as a heterologous expression system to express genes of pathogens of medical and veterinary relevance. Previously, the plasmid MOE[MOEl ]:GFP (formerly known as pMOE:GFP) was built by expanding 1 kb each of 5’ and 3’ flanking regions for PmMOEl coding sequence tagged with GFP, which developed an electroporation-based transfection protocol to deliver the plasmid, and successfully showed a single integration event into the genome via non-homologous recombination (Fernandez Robledo et al., 2008). Previous studies using developed electroporation-based transfection protocol and MOE[MOEl ]:GFP plasmids have reported possibilities of plasmid fragmentation and transposable element-dependent genome integration (Faktorova et al., 2020). Considering the phylogenetic relationship of P. marinus with apicomplexans, P. marinus was transfected with plasmids carrying Plasmodium berghei HAP2 and MSP8 and observed transient expression of both genes (Cold et al., 2016). However, 37.8% efficiency was not replicated when the transfection methodology was developed (Fernandez Robledo et al., 2008).
Other than transfection using pMOE[MOEl ]:GFP-dcrivcd plasmids, there are no systems for functional studies of P. marinus genes like gene knock-out. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system is a powerful tool for editing genomes (Jinek et al., 2012; Lander, 2016). CRISPR/Cas9 technology utilizes machinery such as Cas9 protein, an RNA-guided endonuclease protein, as well as a guide RNA (gRNA) for the nuclease to generate a doublestrand break, which is repaired by nonhomologous end joining (NHEJ) and random mutations incorporated to disrupt the target gene (Mali et al., 2013; Bortesi and Fischer, 2015). However, to knock-in a gene of interest, a donor DNA (dDNA) molecule with homologous templates on either side of the knock-in sequence is required in addition to Cas9 and gRNA. The incorporation of the gene of interest into the genome happens via a homologous- dependent repair mechanism. Previous studies have utilized plasmid-based endogenous expression of Cas9 and gRNA (Peng et al., 2014; Soares Medeiros et al., 2017). However, studies have reported the toxicity and instability due to the transgenic expression of Cas9 (Peng et al., 2014). Alternatively, a Cas9-gRNA ribonucleoprotein complex-based genome editing method was established in kinetoplastids (Beneke et al., 2017; Soares Medeiros et al., 2017; Verruto et al., 2018).
As described herein, electroporation-based transfection methodology was developed to improve heterologous gene expression in P. marinus. Furthermore, using the improved protocol described herein, Cas9-gRNA ribonucleoprotein coupled with dDNA was successfully delivered into the P. marinus wild-type trophozoites and tagged the PmMOEl gene with GFP at the C-terminus to achieve mutants phenotypically similar to the previously reported P. marinus mutant strain (PRA-393)(Fernandez Robledo et al., 2008).
Plasmid Amount and Cell Number Experiments
To increase the heterologous gene expression efficiency, the previously developed Lonza-based electroporation method was used (Fernandez Robledo et al., 2008). In the first round, a rate of 50.0 x 106 P. marinus trophozoites per transfection was maintained. The pPmM0E[M0El]:GFP plasmid amount was varied with a twofold increase (5.0, 10.0, 20.0, and 40.0 pg). In all cases, green fluorescent cells were observed under a UV-microscope as early as 24 hours post-transfection. The flow cytometer was used to detect the number of GFP-positive cells. The trophozoites transfected with 5 and 10 pg of pPmM0E[M0El]:GFP were detected as 0.002 and 0.03%, respectively. Parasites transfected with 5.0 pg of pPmM0E[M0El]:GFP yielded 0.05 and 0.2% GFP-positive cells at 72 and 120 hour time points, respectively (FIG. 8A). Furthermore, parasites transfected with 10.0 pg of pPmM0E[M0El]:GFP yielded 1 and 7.7% GFP-positive cells at 72 and 120 hour time points, respectively (FIG. 8A). Interestingly, at 72 hours post-transfection, parasites transfected with 40 pg (FIG. 8A) of pPmM0E[M0El]:GFP yielded 2 x higher GFP-positive cells compared with parasites that received 20 pg (FIG. 8A). Surprisingly, at 120 hours posttransfection, 9% and 11% of GFP-positive cells were detected in the cases of parasites transfected with 20 and 40 pg of the plasmid, respectively (FIG. 8A). Observing the plateau of GFP-positive cells when parasites were transfected with 20 and 40 pg, 20 pg of plasmid was chosen for cell number studies.
In the second round, the amount of pPmM0E[M0El]:GFP plasmid was kept constant at 20 pg/transfection, and P. marinus trophozoites cell numbers were varied by a twofold increase between 1.56 x 106 and 50.0 x 106 cells/transfection. Using the confocal microscope, it was observed that the 25.0 million parasites transfected with 20 pg of the plasmid yielded the highest levels of GFP-expressing cells qualitatively at 24 and 120 hours post-transfection (FIG. 8B). The flow cytometer and detected 2% of GFP-positive cells (FIG. 8C) as early as 24 hours and 68% in 72 hours (FIG. 8C) and achieved 98% of GFP-positive cells 120 hours post-transfection (FIG. 8C). The trophozoites were monitored for 3 months, where a constant 95% GFP-positive cells were detected (FIG. 8C and FIG. 8D).
Comparison of Transfection Reagents and Materials
To establish an affordable and reliable transfection methodology, various protocols were tested, such as using 3R buer with Lonza and commercial cuvettes (BTX Disposable Cuvettes Plus), and Lonza buer-based transfection utilizing Lonza cuvette and commercial cuvette. In all the cases, 25 million cells were transfected with 20 pg of plasmid, and GFP- positive cells were detected 120 hours post-transfection using flow cytometry (FIGs. 9A-9D). As expected, 98% of GFP-positive cells were identified using the Lonza system (FIGs. 9B and 9E). Notably, flow cytometry evaluated 90% of GFP-positive cells using 3R-buer and BTX cuvette (FIGs. 9C and 9E). By using a 3R buer in combination with a Lonza cuvette, 48% of GFP-positive cells were detected (FIGs. 9D and 9E). Finally, transfection utilizing Lonza-buer and BTX cuvette yielded a meager 2% GFP-positive cells (FIG. 9E).
SPCAS9-RNP and SGRNA mediated GFP knock-in in Perkinsus marinus trophozoites
To establish an HDR-based gene-editing method, a dDNA plasmid containing 396 bp of PmMOEl coding sequence lacking a start codon on the 5’ of the GFP coding sequence was generated which also contained 396 bp of 3’ UTR of PmMOEl at the 3’ of the GFP- coding sequence. The dDNA with GFP and templates were amplified using PCR from previously reported plasmid pPmMOE[MOEl]:GFP (schematic representation in FIG. 10A; Fernandez Robledo et al., 2008). The sgRNA targeting at position 314 on the top strand (sgRNA-1) and another sgRNA targeting position 395 on the bottom strand (sgRNA-2) of the PmMOEl coding sequence were designed using the Benchling software (FIG. 10B). Twenty-five million P. marinus trophozoites were transfected with 20 pg of SpCas9 and sgRNA (1:1) along with 20 pg of dDNA. The parasites transfected with sgRNA- l/SpCas9 and sgRNA-2/SpCas9 and dDNA exhibited GFP expression 24 hour post-transfection (FIG. 10C, +sgRNA- l+dDNA+SpCas9 and +dDNA+sgRNA-2+SpCas9), showing a similar pattern of GFP mutant strain PRA-393 (FIG. 10C, PRA-393 panel). The parasites transfected without SpCas9, and only with dDNA and sgRNA used (i.e., +dDNA+sgRNA) as mock transfection, did not show GFP expression (FIG. 10C, mock transfection panel). Two months post-transfection, using the flow cytometer, 0.2% GFP-positive cells were detected in the experiment transfected with sgRNA- l+dDNA+SpCas9. Furthermore, parasites transfected with sgRNA-2+dDNA+SpCas9 complex yielded 0.26%. GFP-positive cells were not detected in mock transfections (FIG. 10D).
Sorting Perkinsus marinus GFP-Positive Cells for Genotyping Validation
Ten thousand GFP-positive cells from the sgRNA- 1 and sgRNA- 2 transfections were sorted and cultured for 3 months. Flow cytometry analysis detected approximately 81% of GFP-positive cells transfected with sgRNA- 1 (FIG. 11 A) and 87% of GFP-positive cells transfected with sgRNA-2 (FIG. 11B), respectively. Attempts of amplification of the knock- in (expected size 3,300 bp) resulted in around 2,600 bp amplicon, which would include the 5’ flanking, 5’ UTR, PmMOEl, but not the GFP knock-in (FIG. 11C, 2,600 bp arrow and FIG. 13A), suggesting that the knock-in of GFP was less represented compared with the wild type. The PCR product was diluted, and a nested PCR with specific primers was run targeting the putative knock-in; sequencing of the nested PCR product (748 bp) confirmed the accurate integration by HDR (FIG. 11C, 748 bp arrow and FIG. 13B). The chromatogram validated the successful knock-in of GFP at the C-terminus PmMOEl (FIG. 11D).
Discussion
Perkinsus marinus, a marine protozoan parasite which causes devastating infections to eastern oysters, is under development as a model organism for the protozoan parasite of mollusks (Yadavalli et al., 2020). The availability of axenic culture and transfection methodology, the parasite’s ability to naturally trigger an immune response in mice, and its phylogenetic affinities drove its use to express apicomplexan genes. However, the success of such attempts varied (Wijayalath et al., 2014; Cold et al., 2017), and prior studies have reported an inconsistency in gene expression.
The original transfection method uses 5 pg of plasmid and 50.0 x 106 cells; to start, the plasmid amount was increased by 2-fold to 40 pg. The plasmid amount increased in higher GFP-positive cells, especially cells transfected with 20 and 40 pg of the plasmid. In all the cases, fluorescent cells were observed as early as 24 hours post-transfection. Cells needed 3 days to recover and for the GFP expression to be quantifiable. The cells transfected with 40 pg yielded twofold higher GFP-positive cells than cells transfected with 20 pg of plasmid after 72 hours. However, at 120 hours, the number of GFP-expressing cells plateaued to 10% in both cases, indicating that the number of cells also affects transfection efficiency. It was determined that 25.0 x 106 cells transfected with 20 pg of plasmid resulted in 98% GFP-positive cells 120 hours post-transfection. The Nucleofector™ 2b uses cuvettes, and the transfection occurs in a 100 pl reaction, and it appears that the delivery of the electrical pulse is optimal when 25.0 x 106 cells are used.
Interestingly, cell numbers above and below 25.0 x 106 cells resulted in quite a low transfection efficiency (FIG. 12). P. marinus cells may also be transfected with the transfection buer (3R buer), which provides an efficiency above 40% and provides savings when the research budgets are tight. Experiments utilizing the combination of transfection buer and BTX cuvettes were successful, although with a low number of transfectants. More than 90% of GFP-expressing cells were observed, even at 3 months post-transfection in all the cases, suggesting a stable GFP expression.
The CRISPR/Cas9 methodology is broadly adopted by numerous parasitology labs around the world (Mali et al., 2013; Ghorbal et al., 2014; Peng et al., 2014; Shen et al., 2014; Sollelis et al., 2015; Janssen et al., 2018; Lin et al., 2019). Utilizing the improved conditions described herein, further steps were taken to develop the CRISPR/Cas9-based gene editing methodology for P. marinus. For the proof of concept, the PmMOEl gene that has a defined phenotype when tagged with GFP was targeted (Fernandez Robledo et al., 2008). Fluorescent trophozoites were detected within 18 hours of delivering the CRISPR/Cas9 system components. Lack of GFP expression in the transfection of dDNA alone (lacking CRISPR/Cas9 components) ruled out the possibilities of non-homologous recombination in frame with any expressed gene; however, with this fluorescence screening, plasmid fragmentation and integration at the transposable element sites could not be excluded. The GFP expression pattern in the transfectants was similar to that of P. marinus PRA393 (FIG. 10C). GFP-positive cells sorted from sgRNA-l/Cas9 and sgRNA-2/Cas9 experiments were PCR amplified to check for the knock-in of the GFP. Interestingly, the PCR in the sorted cells did not result in the 3,300 bp amplicon. However, the nested PCR produced the expected size amplicons whose direct sequences confirmed the successful knock-in of GFP at the C -terminus of PmMOEl. In the protozoan parasites with a large trajectory of genetic manipulation, previous studies have suggested the trend is to build a plasmid vector that incorporates both the expression of Cas9 and the sgRNA or even generate a mutant conditionally expressing Cas9. CRISPR/Cas9 components were chosen for delivery, including the SpCas9 nuclease, directly by electroporation. The data reported herein are from a single trial targeting PmMOEl using 25.0 x 106 log-phase trophozoites, 20 pg of dDNA, 10 pg of sgRNAs, and SaCas9 nuclease (chosen based on Beneke et al. (2017) and Soares Medeiros et al. (2017)) and resulted in a successful knock-in.
Genome editing tools like CRISPR/Cas9 in parasite biology are used for gene disruption, fluorescent tagging, and single nucleotide mutation incorporation to study genes involved in the parasite growth, invasion, and drug resistance (Wagner et al., 2014; Di Cristina et al., 2017). For example, in a Plasmodium falciparum study, the development of an artemisinin-resistant parasite by single-nucleotide substitution, identification of the multidrug resistance mutation 1 (PfMDRl) in response to the drug ACT-451840, and incorporation of a point mutation in the PfATP4 gene to generate the drug-resistant strain were all possible by utilizing the CRISPR/Cas9 system (Ghorbal et al., 2014; Ng et al., 2016; Crawford et al., 2017). In Toxoplasma gondii, CRISPR/Cas9 is widely used in high- throughput and genome screening studies to identify essential genes involved in parasite invasion and antiparasitic drug candidates (Di Cristina et al., 2017; Di Cristina and Carruthers, 2018). CRISPR/Cas9-based knock-out studies in Cryptosporidium parvum are used to understand the mechanism of the parasite’s resistance to antifolate drugs and nutrient acquisition pathways (Vinayak et al., 2015; Pawlowic et al., 2017, 2019).
The P. marinus genome encodes for 23,454 genes embedded in 17,000 supercontigs. However, tetra-polyploidy pose a significant bottleneck for the assembly (El-Sayed et al., 2007; Bogema et al., 2020). Proteome studies identified that P. marinus possess 4,073 non- redundant hypothetical proteins, of which 36 and 27% are involved in metabolic and cellular processes, respectively (Marcia et al., 2017). Additionally, the rhoptry proteins such as serine-threonine kinases, protein phosphatases, proteosomes, and a virulent candidate merozoite surface protein 3, which are known to play a crucial role in parasite invasion and cell-cell communication during the invasion in P. falciparum were also identified in P. marinus. Prior studies have reported that P. marinus possess extracellular proteins such as high molecular weight cell wall protein 1 (Montes et al., 2002); glycosylation, mucin, and sugar-binding domain protein Pmar_XP_002783417.1 encoded by Pmar_PMAR006943; sensory signal transduction-related histidine kinase encoded by Pmar_PMAR009211; and a family of cysteine-rich modular proteins whose function in the parasite life cycle are yet to be investigated (Montes et al., 2002). Furthermore, apoptotic genes such as apoptosis inhibitory molecule (Fas), apoptosis-inducing factor (Tadesse et al., 2017; Lau et al., 2018b), peroxiredoxin, and superoxide dismutase have been shown to favor parasite survival by reducing the host cell (Schott and Vasta, 2003; Schott et al., 2003; Box et al., 2020). The function of these apoptotic genes responsible for the disease in the oysters is limited.
Materials and Methods
Perkinsus Marinus Cell Culture
Experiments were carried out with cultures of the wild-type P. marinus CB5D4 (ATCC#PRA-240) (Shridhar et al., 2013) maintained in DME:Ham’s F12 (1:2) supplemented with 5% fetal bovine serum (FBS), in a 25 cm2 (5-8 ml) polystyrene canted neck cell culture flasks with vent caps (Corning®, Corning, New York, United States) at 24-28°C in a microbiology incubator as reported elsewhere (Gauthier and Vasta, 1995). Trophozoites in the log phase (OD595 = 0.4-0.5) were aliquoted in Eppendorf tubes to contain 1.56 x 106, 3.13 x 106, 6.25 x 106, 12.50 x 106, 25.0 x 106, and 50.0 x 106.
Perkinsus marinus Transfection
The transfection vector pPmMOE[MOEl]:GFP (former pPmMOE-GFP) (Fernandez Robledo et al., 2008) was propagated in Escherichia coli JM109. Plasmid minipreps were prepared using a commercial kit (E.Z.N.A.® Plasmid mini Kit I, Omega-Tek, Norcross, GA, United States), and DNA concentration and purity were estimated with a Nanodrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The isolated plasmid DNA was air dried using speedVac for all the experiments. P. marinus cells were prepared following the Cell Line Optimization Nucleofector Kit before electroporation using the Nucleofector™ 2b (Lonza, Walkersville, MD, United States). For all the experiments, the pre-set program D-023 and Lonza’s solution V (Fernandez Robledo et al., 2008) were used. Briefly, dried plasmid was resuspended in 100 pl of Solution V containing supplement 1. pPmMOE[MOEl]:GFP was tested at 5, 10, 20, and 40 pg with 50 million P. marinus cells. Once the optimal plasmid amount was established (20 pg), it was tested with variable P. marinus cell number (1.56 x 106, 3.13 x 106, 6.25 x 106, 12.5 x 106, 25.0 x 106, and 50.0 x 106). Immediately after electroporation, the individual electroporation cuvettes’ contents were transferred to a 24-well plate, each well containing 1 ml of DME:Ham’s F12 (1:2) supplemented with 5% FBS (Gauthier and Vasta, 1995). The cuvettes were gently washed with 500 pl of fresh culture medium and pooled with those wells corresponding to each original sample. The transfection buer (3R buer)-based transfection protocol (Faktorova et al., 2020) was also tested. The 3R-transfection buer, composed of 200 mM Na2HPO4, 70 mM NathPCF, 15 mM KC1, and 150 mM HEPES, was prepared and pHed to 7.3. Dried 20 pg of circular pPmMOE[MOEl]:GFP plasmid was resuspended in 60 pl of milliQ water. Once dissolved, 35 pl of 3R transfection buer and 10 pl of 1.5 mM CaCh were added (Protocols. io). Twenty-five million P. marinus trophozoites were transfected.
Protospacer Adjacent Motif-Target Site Selection and Donor DNA Construction
Selection and Donor DNA Construction PAM-target site selection was identified using the PmMOEl sequence (Pmar_PMAR027036) and the software (Benchling, Inc.). The output sequences were searched using BLASTx (NCBI-Blast, 2021) against the P. marinus nr database (RefSeq assembly: GCF_000006405.1), which predicted Pmar_PMAR025337 as another possible target. Based on the string searches, the PAM sites were rated according to their “uniqueness.” Single-guide RNA (sgRNA) targeting positive strand at position 339 of PmMOEl CDS (sgRNA- 1) 5’-CCC TGT AAA TGT GGT GGT GG-3’ (SEQ ID NO: 2) and sgRNA targeting negative strand at position 382 of PmMOE CDS (sgRNA-2) 5’-CAT GTC GGC TTC GTC GTA GT CGG-3’ (SEQ ID NO: 3) with unique PAM sequence “CGG” were synthesized (Synthego, Silicon Valley, CA, United States). Although the sgRNA-2 sequence identified three target sites on PmMOEl CDS, the sequence reported herein is the only fragment that exhibited 100% complementarity. The dDNA was amplified from pPmMOE[MOEl]:GFP (Fernandez Robledo et al., 2008) using primers forward 5’-CGC TTC ATT GTT GGT CTG TAC-3’ (SEQ ID NO: 4) and reverse 5’-CAG TAC GAA ATT ACG CGA GAT G-3’ (SEQ ID NO: 5). The amplicon was cloned into the pGEM®-T vector by T-A cloning (pGEM-T Vector Systems, Promega Corporation, Madison, WI), propagated in Escherichia coli JM109 (L1001, Promega), and sent for sequencing. Plasmid minipreps were prepared using a commercial kit (E.Z.N.A.® Plasmid Mini Kit I, Omega Bio-Tek, Norcross, GA, United States), and DNA concentration and purity were estimated with a Nanodrop 1000 spectrophotometer.
RNP Complex and Donor DNA Delivery into Perkinsus Marinus
Perkinsus marinus cells were prepared following the Cell Line Optimization Nucleofector Kit protocol before electroporation. Using the Nucleofector™ 2b, 10 pg of Streptococcus pyrogenes Cas9 (SpCas9) nuclease TrueCut™ Cas9 protein v2 (Thermo Fisher Scientific, Vilnius, Lithuania) and 10 pg of sgRNAs (Synthego, Silicon Valley, CA, United States) were mixed in 100 pl of Lonza’s solution V and incubated at room temperature for 15 minutes for hybridization of sgRNA and Cas9 protein (Beneke et al., 2017). Twenty micrograms of dried dDNA plasmid were resuspended with a SpCas9-gRNA complex and electroporated into 25.0 x 106 P. marinus trophozoites, using pre-set D-023 program (Fernandez Robledo et al., 2008). Immediately after electroporation, the individual electroporation cuvettes’ contents were transferred to a 12-well plate, each well containing 1 ml of DME:Ham’s F12 (1:2) supplemented with 5% FBS, to allow cells to recover (Gauthier and Vasta, 1995). Upon identifying the fluorescent cells, the cells were spun down at 1,000 x g for 5 minutes at room temperature and resuspended into fresh media. Cells were screened for green fluorescence at 24, 72, and 120 hours, and 2, 4, and 6 weeks post-transfection using confocal microscopy and flow cytometry.
DNA Isolation and Genotyping for GFP Knock-In by DNA Sequencing
Upon observing green fluorescent P. marinus trophozoites, cells were allowed to recover for 1 week. The genomic DNA from P. inarinus w}'\d-typc (PRA-240), GFP-mutant (PRA-393), parasites transfected with dDNA alone, lacking CRISPR elements (sgRNA and Cas9), and parasites transfected with sgRNA- 1 and sgRNA-2 with Cas9 were isolated using E.Z.N.A. ® tissue DNA kit (Norcross, GA, United States) according to the manufacturer protocol. The purity and concentration of isolated DNA were analyzed using Nanodrop™ 1000 spectrophotometer. For DNA genotyping, the primer pair Fwd 1 5’-CTC GTA ATG AGC CCA ACC AT-3’ (SEQ ID NO: 6) and Rev 1 5’-GGA GGA CTT GAG GCT CTG TG 3’ (SEQ ID NO: 7) (Fernandez Robledo et al., 2008) were designed using the available supercontig (Ensembl, 2021) results in 2,600 bp of PmMOEl (wildtype) and yielded 3,300 bp after successful GFP knock-in. To identify the GFP knock-in site at the 3’ PmMOE CDS, primers were designed spanning 136 bp of the 5’ flanking region, PmMOEl CDS, and 201 bp of the GFP sequence (Fwd 2 5’-TGT TGT AAG GCG AGA CGC TA-3’ (SEQ ID NO: 8) and Rev 2 5’-GTA GGT CAG GGT GGT CAC GA-3’ (SEQ ID NO: 9), respectively. Briefly, 50 ng of the gDNA and primers mentioned herein were used to amplify by polymerase chain reaction. The amplicons were purified from the 1% agarose gel using the Zymoclean™ Gel DNA Recovery kit (Tustin, CA, United States).
Confocal Microscopy
Parasites were fixed with 3% paraformaldehyde (Thermo Fisher Scientific, preserved 37% reagent) for 15 minutes at room temperature. Parasites were washed three times at 1,000 x g for 5 minutes using 1 x phosphate-buered saline (l x PBS). Following the washes, parasites were treated with 0.1% Triton X-100 for 15 minutes and washed three times with 1 x PBS. The cells were stained with 25 pg/ml concentration of 4’,6-diamidino- 2- phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, United States). Excess DAPI was washed with 1 x PBS, and parasites were resuspended in the fresh 1 x PBS and placed in NunC® Lab-Tek® II (Millipore Sigma, Darmstadt, Germany) for live-cell imaging. Parasites were imaged at a total magnification of 630 x on Carl Zeiss LSM-700 multiphoton scanning laser microscope.
Flow Cytometry Sorting and Analysis
The flow cytometry experiments were performed on the live parasite, using ZE5 Cell Analyzer; data was collected using Everest software version 2.0 and analyzed. A minimum of 100,000 events was collected for parasites based on forward and side scatterplot, and a singlet gate was applied to collect a minimum of 30,000 cells. BD Sciences Influx Cell Sorter (BD Sciences, NJ, United States) was used for cell sorting, and cells were sorted based on eGFP-positive gates.
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Survey for protozoan parasites in eastern oysters (Crassostrea virginica) from the Gulf of Maine using PCR-Based Assays. Parasitol. Int. 64, 299-302. doi: 10.1016/j.parint.2015.04.001 Matsuzaki, M., Kuroiwa, H., Kuroiwa, T., Kita, K., and Nozaki, H. (2008). A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus. Mol. Biol. Evol. 25, 1167-1179. doi: 10.1093/molbev/msn064 McLaughlin, S. M., and Faisal, M. (1998). In vitro propagation of two Perkinsus species from the Softshell clam Mya arenaria. Parasite 5, 341-348. Montes, J. F., Durfort, M., Llado, A., and Garcia- Valero, J. (2002). Characterization and immunolocalization of a main proteinaceous component of the cell wall of the protozoan parasite Perkinsus atlanticus. Parasitology 124(Pt 5), 477-484. doi: 10.1017/s0031182002001415 NCBLBlast (2021). NCBI-Blast. Available online at: https://blast.ncbi. nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSea rch&LINK_LOC=blasthome Ng, C. L., Siciliano, G., Lee, M. C., de Almeida, M. J., Corey, V. C., Bopp, S. E., et al. (2016). CRISPR-Cas9-modified Pfmdrl protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine-containing compounds but potentiates artemisinin-based combination therapy partner drugs. Mol. Microbiol. 101, 381-393. Pawlowic, M. C., Somepalli, M., Sateriale, A., Herbert, G. T., Gibson, A. R., Cuny, G. D., et al. (2019). Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell. Proc. Natl. Acad. Sci. U.S.A. 116, 21160-21165. doi: 10.1073/pnas.1908239116 Pawlowic, M. C., Vinayak, S., Sateriale, A., Brooks, C. F., and Striepen, B. (2017). Generating and maintaining transgenic Cryptosporidium parvum parasites. Curr. Protoc. Microbiol. 46, 20B 2 1-20B 2 32. Pecher, W. T., Alavi, M. R., Schott, E. J., Fernandez Robledo, J. A., Roth, L., Berg, S. T., et al. (2008). Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays. J. Parasitol. 94, 410-422. doi: 10.1645/ge- 1282.1 Peng, D., Kurup, S. P., Yao, P. Y., Minning, T. A., and Tarleton, R. L. (2014). CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. mBio 6:e02097-14. Perkins, F. O. (1996). The structure of Perkinsus marinus (Mackin, Owen and Collier, 1950) Levine, 1978 with comments on taxonomy and phylogeny of Perkinsus spp. J. Shellfish Res. 15, 67-87. Reece, K. S., Dungan, C. F., and Burreson, E. M. (2008). Molecular epizootiology of Perkinsus marinus and P. chesapeaki infections among wild oysters and clams in Chesapeake Bay, USA. Dis. Aquat. Organ. 82, 237-248. doi: 10.3354/dao01997 Schott, E. J., Di Leila, S., Bachvaroff, T. R., Amzel, L. M., and Vasta, G. R. (2019). Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase. BMC Evol. Biol. 19:146. doi: 10.1186/sl2862-019-1465-5 Schott, E. J., Pecher, W. T., Okafor, F., and Vasta, G. R. (2003). The protistan parasite Perkinsus marinus is resistant to selected reactive oxygen species. Exp. Parasitol. 105, 232-240. doi: 10.1016/j.exppara.2003.12.012 Schott, E. J., and Vasta, G. R. (2003). The PmSODl gene of the protistan parasite Perkinsus marinus complements the Sod2Delta mutant of Saccharomyces cerevisiae, and directs an iron superoxide dismutase to mitochondria. Mol. Biochem. Parasitol. 126, 81-92. doi: 10.1016/s0166-6851(02)00271-2 Shen, B., Brown, K. M., Lee, T. D., and Sibley, L. D. (2014). Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. mBio 5:el l l4. Shridhar, S., Hassan, K., Sullivan, D. J., Vasta, G. R., and Fernandez Robledo, J. A. (2013). 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Trends Parasitol. 36, 1013-1014. OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
EQUIVALENTS
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

What is claimed is: CLAIMS
1. An engineered Perkinsus marinus cell that expresses a polypeptide comprising a viral protein.
2. The engineered Perkinsus marinus cell of claim 1, wherein the viral protein is expressed on the surface of the cell.
3. The engineered Perkinsus marinus cell of claim 2, wherein the polypeptide further comprises a tag.
4. The engineered Perkinsus marinus cell of any one of claims 1-3, wherein the polypeptide comprises an amino acid sequence of a signal peptide that directs the polypeptide comprising the viral protein to the cell surface.
5. The engineered Perkinsus marinus cell of claim 4, wherein the signal peptide is an MOE signal peptide.
6. The engineered cell of claim 5, wherein the MOE signal peptide comprises an amino acid sequence of MRFIVGLYSCLAVLVLGQSSCPTGSAQC (SEQ ID NO: 1).
7. The engineered Perkinsus marinus cell of any one of claims 1-6, wherein the cell comprises a heterologous polynucleotide comprising a nucleic acid sequence encoding the polypeptide comprising the viral protein.
8. The engineered Perkinsus marinus cell of claim 7, wherein the heterologous polynucleotide is a plasmid.
9. The engineered Perkinsus marinus cell of claim 7, wherein the heterologous polynucleotide is integrated into the genome of the cell.
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10. The engineered Perkinsus marinus cell of any one of claims 7-9, wherein the heterologous polynucleotide comprises a nucleic acid sequence encoding two or more polypeptides comprising viral proteins.
11. The engineered Perkinsus marinus cell of claim 10, wherein at least two of the viral proteins encoded by the heterologous polynucleotide comprise the same amino acid sequence.
12. The engineered Perkinsus marinus cell of claim 10, wherein at least two of the viral proteins encoded by the heterologous polynucleotide comprise different amino acid sequences.
13. The engineered Perkinsus marinus cell of any one of claims 1-5, wherein the cell comprises two or more heterologous polynucleotides comprising a nucleic acid sequence encoding a polypeptide comprising a viral protein.
14. The engineered Perkinsus marinus cell of claim 13, wherein the nucleic acid sequences of at least two of the heterologous polynucleotides are the same.
15. The engineered Perkinsus marinus of claim 13, wherein the nucleic acid sequences of at least two of the heterologous polynucleotides are different.
16. A population of Perkinsus marinus cells, wherein at least 8% of the cells in the population express a protein of interest consequent to transfection of the population with a plurality of heterologous polynucleotides encoding the protein of interest.
17. The population of Perkinsus marinus cells of claim 16, wherein the cells are trophozoites.
18. The population of Perkinsus marinus cells of claim 16 or claim 17, wherein the number of cells in the population is from about 1 x 106 to about 50 x 106 cells.
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19. The population of Perkinsus marinus cells of any one of claims 16-18, wherein the number of cells is about 25 x 106 cells.
20. The population of Perkinsus marinus cells of any one of claims 16-19, wherein the protein of interest comprises the amino acid sequence of a viral protein.
21. A method of engineering a Perkinsus marinus cell to produce a protein of interest, the method comprising contacting a population of Perkinsus marinus trophozoites with a transfection reagent and a plurality of heterologous polynucleotides encoding the protein of interest, wherein: the population of Perkinsus marinus trophozoites comprises from about 1 x 106to about 50 x 106 cells; the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg; or a combination thereof.
22. The method of claim 21, wherein the population of Perkinsus marinus trophozoites comprises from about 1 x 106to about 50 x 106 cells and the plurality of heterologous polynucleotides is in an amount from about 15 pg to about 25 pg.
23. The method of claim 21 or claim 22, wherein the population of Perkinsus marinus trophozoites has about 25 x 106 cells.
24. The method of any one of claims 21-23, wherein the plurality of heterologous polynucleotides is in an amount of about 20 pg.
25. The method of any one of claims 21-24, wherein the population of Perkinsus marinus trophozoites are in a log growth phase when contacted with the transfection reagent and the plurality of heterologous polynucleotides.
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26. The method of any one of claims 21-25, wherein the population of Perkinsus marinus trophozoites have an absorbance of 1.66 at ODeoo when contacted with the transfection reagent and the plurality of heterologous polynucleotides.
27. The method of any one of claims 21-26, wherein the protein of interest comprises the amino acid sequence of a viral protein.
28. A method of producing a polypeptide comprising a viral protein, the method comprising culturing an engineered Perkinsus marinus cell according to any one of claims 1- 15 under conditions in which it expresses the polypeptide comprising the viral protein.
29. The method of claim 28, further comprising isolating the polypeptide comprising the viral protein.
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