Classifications

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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4727—Mucins, e.g. human intestinal mucin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L12/00—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor
  • A61L12/08—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor using chemical substances
  • A61L12/14—Organic compounds not covered by groups A61L12/10 or A61L12/12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2101/00—Chemical composition of materials used in disinfecting, sterilising or deodorising
  • A61L2101/32—Organic compounds
  • A61L2101/46—Macromolecular compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4725—Proteoglycans, e.g. aggreccan
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  • C07K2319/00—Fusion polypeptide
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  • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

• the disclosure provided improved glycoproteins, and compositions and methods related to the same. BACKGROUND OF THE DISCLOSURE
• Protein therapeutic agents represent a large and rapidly growing portion of the pharmaceutical market.
• Current biologics enable the treatment of a wide variety of human diseases, including cancer, autoimmune disorders, arthritis and infectious diseases.
• the commercial success of biologics has been a major impetus for the development of improved manufacturing technologies that reliably produce the biological agents on a large scale.
• Mammalian cells are preferred over prokaryotic organisms for production of protein therapeutics because eukaryote-specific post-translational modifications are often required for protein functionality and appropriate pharmacokinetics.
• monoclonal antibodies a major class of protein therapeutics, must be post-translationally modified with sugar structures called glycans in a post-translational modification process called glycosylation. Without glycosylation, therapeutic antibodies typically have poor stability and pharmacokinetics in vivo.
• CHO cells for bio-manufacturing are their capacity to generate glycans that are not native to humans. These glycans can produce deleterious immune responses and have been implicated in therapeutic resistance, which remains a significant concern for physicians and patients.
• the risk of patient immune responses from CHO-derived products has motivated a deeper consideration of the use of human cell lines for manufacturing recombinant protein therapies.
• Suspension adapted human embryonic kidney 293 cells (293-F) have become the most popular host cell line for the production of biological therapeutics with human glycosylation patterns.
• the 293-F cell system has several desirable features for recombinant protein production, including a fast proliferation rate, a high level of protein production, and ease of transient transfection.
• FDA United States Food and Drug Administration
• 293-F cells can exhibit a higher propensity to form large aggregates in suspension, limiting their yield and reliability for bio-manufacturing.
• the disclosure provides modified mammalian cells that express modified
• the cells comprise recombinant polypeptides expressed from recombinant polynucleotides introduced into the cells.
• the polypeptides comprise a transmembrane anchor and a segment external to the cells.
• the segment external to the cells includes repeated amino acid sequences.
• the repeated amino acid sequences are selected from:
• KEPAPTTP SEQ ID NO:1
• DAATPAP SEQ ID NO:2
• DAATPAPP SEQ ID NO:3
• PPASTSAPG SEQ ID NO:4
• PDTRPAPGATAPPAHGVTSA SEQ ID NO:5
• the repeated amino acid sequence may be repeated contiguously 10- 120 times. In certain embodiments, the repeated amino acid sequence is repeated contiguously 21, 40, 42, 59 or 80 times.
• the cells are modified mammalian cells.
• the cells are modified human cells, which may be human embryonic kidney cells, which in certain embodiments include human 293-F cells.
• the modified human cells that express modified mucins are adapted to growth in a suspension culture.
• the modified cells in the suspension culture exhibit less aggregation relative to a suitable control value.
• a non-limiting example of a suitable control value is a value obtained from a suspended cell culture comprising cells that do not express the recombinant polypeptide comprising the repeated amino acid sequences.
• the modified cells may be present in a suspension cell culture that is present in a suspended cell bioreactor.
• the modified cells express a second, distinct polypeptide. This is achieved by further modifying the cells such that they comprise an introduced polynucleotide encoding a distinct polypeptide that is different from the polypeptide comprising the repeated amino acid sequences.
• the polypeptides expressed by the modified cells exhibit O-glycans on the segment external to the cells.
• This segment can comprise one or a combination of Core 2 O-glycan, GlcNAc b1-6(Gal b1-3)GalNAc and/or the Core 2 derivatives of GlcNAc b1- 6(Gal b1-3)GalNAc at an abundance of at least 5% relative to all Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, and Core 8 O-glycans.
• the polypeptides expressed by the cells as described above include a transmembrane anchor that comprises a cytoplasmic recycling motif.
• Isolated polynucleotides that encode the polypeptides of this disclosure are included, as are expression vectors comprising such polynucleotides.
• the expression vectors comprising such polynucleotides.
• polynucleotides are incorporated into the modified cells such that they are integrated into a chromosome of the cells, which may be achieved by random integration.
• the disclosure includes a method of making cells that express the described polypeptides. This approach comprises introducing an isolated/recombinant polynucleotide that encodes a described polypeptide into the cells such that the polypeptide is expressed.
• a method for producing a desired polypeptide or another agent which may be distinct from the polypeptide comprising the repeated sequences.
• the method comprises expressing the desired polypeptide or producing another agent in modified mammalian cells that express a modified mucin polypeptide described herein.
• the method may further comprise separating the desired polypeptide or the other agent from the cells.
• Part I Part I, Part II, Part III, and Part IV
• Part I Part I, Part II, Part III, and Part IV
• FIG. 1 Combinatorial Genetic Encoded Library for Sequence-Specific Mucins.
• (a) Schematic diagram of the combinatorial sequence-specific mucins.
• (b) Schematic shows the swappable bio-bricks and flanking restriction sites for complete mucin construction.
• (c) Work flow for the design and fabrication of cDNAs for the mucin tandem-repeat backbones.
• the Wild-type Muc1 sequence is SEQ ID NO:8.
• the Muc1 single mutant (Muc1_S) is SEQ ID NO:5.
• the Muc1 double mutant (Muc1_D) is SEQ ID NO:6.
• the Muc1 triple mutant (Muc1_T) is SEQ ID NO:7.
• the Synthetic 1 (Syn1) is DAATPAP is SEQ ID NO:2.
• the Synthetic 2 (Syn2) is SEQ ID NO:3.
• the Synthetic 3 (Syn3) is SEQ ID NO:4.
• the Lubricin consensus sequence (Syn4) is SEQ ID NO:1.
• FIG. 2 Construction and Validation of Sequence-Specific Mucin Expression.
• (a) Components and features of codon-optimized Muc1 variants with GFP reporters.
• the amino acid sequence in (a) is SEQ ID NO:8.
• (b) Predicted Molecular Weight of the polypeptide backbone.
• (c) Biosynthesis of Tn antigen, Core 1, and Core 2 glycans, and specificity of relevant lectins for their detection.
• the MCF10A cells were stably transfected with native Muc1.
• the surface sialic acids were labeled with AFDye 568 through periodate labeling prior to lysate collection.
• the blot was stained in multiple colors with MUC1 TR (CD227 HPMV) Ab- FITC, and PNA-CF640 or biotinylated VVA (Secondary: NeutrAvidin-Dylight 650).
• MUC1 TR CD227 HPMV
• Ab- FITC Ab- FITC
• PNA-CF640 biotinylated VVA (Secondary: NeutrAvidin-Dylight 650).
• e Western blot analysis of native and codon-scrambled Muc1 in extracts of transiently trnsfected HEK293T cells.
• HEK293T cells expressing indicated constructs and probed with PNA lectin (left), anti-Muc1 antibody (center left), GFP(center right) and Hoescht nuclear stain (right) (scale bar 10 mm).
• PNA lectin blot analysis left
• intensity profiles right
• mucins of varying sizes in extracts of transiently transfected HEK293T cells.
• Figure 3 Engineering the Frequency of Glycosylation Sites in the Muc1 Polymer Backbone Tunes O-glycan Maturation.
• sMuc1 tandem repeats sMuc1 tandem repeats
• transmembrane anchors TM21 and engineered cytoplasmic motifs; native CT refers to a native cytoplasmic tail adapted from Muc1.
• TM21 transmembrane anchors
• native CT refers to a native cytoplasmic tail adapted from Muc1.
• PNA-lectin blot analysis of the indicated mucin isoforms before and after sialidase treatment blots are representative of three independent experiments.
• Figure 6 Western blot analysis of MCF10A cells edited with lentivirus with native repetitive (Native_Muc1) versus codon-scrambled Muc1 cDNAs (Muc1_42).
• Figure 7 Mucins with Tunable Sizes. The sequences shown in Figure 7 are
• PDTRPAPGSTAPPAHGVTSA SEQ ID NO:8.
• Figure 8 Western blot Image of affinity-purified recombinant secreted mucins from FreeStyleTM 293-F cell culture media probed with anti-6x His antibody and VVA lectin
• PDARPAPGATAPPAHGVTAA SEQ ID NO:7.
• FIG. 10 MALDI-TOF_MS spectra of mucin-type O-glycans as reported by Cellular O-Glycome Reporter/Amplification (CORA).
• CORA Cellular O-Glycome Reporter/Amplification
• Figure 11 Mucins Constructed with Designer Tandem Repeats.
• the sequences shown in Figure 11 are DAATPAP (SEQ ID NO:2) DAATPAPP (SEQ ID NO:3) and PPASTSAPG (SEQ ID NO:4).
• the vector includes a tetracycline responsive element (tetO), a minimal CMV promoter, the Muc1 signal sequence (Muc1 N-terminus), the tandem repeats of the biopolymer (0, 21, or 42 repeats of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8), the transmembrane domain of Muc1 (Muc1 TM), the bicistronic green fluorescent protein reporter (IRES GFP), a EF-1a promoter, the reverse tetracycline transactivator (rtTA), and a second bicistronic neomycin resistance cassette (IRES NeoR).
• tetO tetracycline responsive element
• Muc1 signal sequence Muc1 N-terminus
• the tandem repeats of the biopolymer (0, 21, or 42 repeats of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8)
• the transmembrane domain of Muc1 Muc1
• IVS GFP bic
• ITRs inverted terminal repeat sequences
• AmpR ampicillin resistance cassette
• ori origin of replication
• B Schematic representation of membrane bound biopolymers expressed by the cells and localized to the cells surface.
• C Schematic of the relative size of the extracellular domain of the engineered biopolymers designated Mucin-0, Mucin-135, and Mucin-270 for their respective length in nm. The predicted molecular weight of these proteins was 42 kDa, 81 kDa, and 120 kDa, respectively.
• Figure 13 Validation of Biopolymer Coatings. Expression and cell-surface localization of biopolymer coatings was validated for the new, engineered 293-F cell lines.
• A Representative confocal microscopy images of stable suspension adapted human embryonic kidney 293 (293-F) cell lines– wild type (w.t.), or stably expressing the Mucin-0, Mucin- 135, or Mucin-270 biopolymer.
• Images show the cell membrane (shown in blue, CF633 Wheat Germ Agglutinin, WGA), O-glycans covalently attached to the Mucin-135 and Mucin-270 biopolymers (shown in red, CF568 Peanut Agglutinin, PNA), and green- fluorescent protein (shown in green, GFP) which is co-expressed on the plasmid with the Mucin-0, Mucin-135 and Mucin-270 biopolymer.
• B Representative flow cytometry histograms showing the polydisperse population of biopolymer expressing cell lines compared to w.t. cells, y-axis is scaled to show the population distribution of GFP positive cells. >50,000 cells per histogram.
• G Agarose gel showing polymerase chain reaction (PCR) product of Mucin-270 gene from DNA extracted from non-transfected cells (Mock), w.t. cells transiently transfected (Transient), or cells with the Mucin-270 gene incorporated in the genome and cultured for 2 months (2 mo.) or 12 days (12 d) after gentamycin selection.
• Star indicates the predicted molecular weight of Mucin-270 PCR product. #1 and #2 are biological replicates. Mean and S.D. shown, ns– not significant.
• Figure 14 Biopolymer Coatings Reduced Cell Aggregation. Genetically-encoded biopolymer coatings of Mucin-135 and Mucin-270 size reduce cell aggregation in suspension cell culture. A, Representative phase contrast images for w.t. and biopolymer cell lines.
• FIG. 15 Mucin-270 Reduced Aggregation in High Calcium Culture Media.
• the Mucin-270 cell line out-performs commercial anti-clumping solution in highly aggregating conditions.
• A Image of Mucin-270 and w.t. cultures grown in media with 2 mM CaCl 2 (+ Ca 2+ ). Mucin-270 expression significantly decreases cell aggregation, even compared to commercially available anti-clumping reagent (+ anti-clump).
• B Quantification of the concentration of w.t.
• FIG. 17 Biopolymer Coated Cells can be Transfected. Transfection was determined for the biopolymer coated cell lines by transfection with a cytoplasmic red- fluorescent protein (RFP).
• RFP red- fluorescent protein
• B Representative flow cytometry histogram showing the distribution of expression among transfected cell populations. The peak to the left of the gray line, centered around zero, represented the non-transfected population for each cell line which is further validated by the overlapping histogram of non-transfected w.t. cells (w.t.-null).
• C Representative flow cytometry histogram showing the distribution of expression among transfected cell populations. The peak to
• Figure 19 Additional data to accompany Figure 14 acquired 24 hr prior. A,
• SynLubricin for secretion and purification SynLubricin also retains the two somatomedin B domains (SMB 1 and 2) and the two Hemopexin domains of the native protein.
• the PRG4A sequence in the alignment is amino acids 347 - 853 of SEQ ID NO:66.
• the SynLub sequence in the alignment is amino acids 347-818 of SEQ ID NO:68.
• E Vector map illustrating the tetracycline-inducible promoter, multiple cloning site (MCS) for cDNA of interest, bicistronic GFP reporter (IRES2 CopGFP), and second expression cassette for the rtTA-M2 tetracycline transactivator and neomycin-resistance gene.
• Figure 21 Sorting strategy to isolate stable polyclonal cell populations that produce high levels of SynLubricin.
• C Quantification of the relative intensity of signal from anti-PRG4 Western blots in B.
• FIG 22 Integrated SynLubricin cDNA is stable in the cellular genome. PCR amplification of SynLubricin coding region in genomic DNA extracts of wild-type and stably integrated 293-F cells cultured continuously for 2 months. As positive controls, PCR amplifications of SynLubricin plasmid and DNA extract from SynLubricin transiently transfected 293-F cells (Transient) are shown. The expected size of full-length SynLubricin is indicated by the star.
• Figure 23 Optimization of SynLubricin production.
• C) Time course for glucose consumption in sorted 293-F cells induced at day 0 with 1 mg /mL doxycycline with or without 3.5 mM VPA. Mean and S.D. shown, n 3.
• Figure 24 Purification of SynLubricin by anionic exchange chromatography.
• FIG. 25 Lubrication of cartilage explants shows functionality of SynLubricin. Friction coefficients of NaCl-extracted cartilage explants bathed in saline (PBS), bovine synovial fluid, or SynLubricin. Prior to lubrication analysis, the SynLubricin was purified with DEAE Sepharose, eluting either without washing or after a stringent 500 mM NaCl wash. Mean and S.D. are shown with independent measurements indicated. ***p ⁇ 0.001, ****p ⁇ 0.0001; NA: statistical testing is not applicable due to sample size.
• PBS saline
• NA statistical testing is not applicable due to sample size.
• Figure 28 Application of the codon-scrambling strategy for Muc1.
• the sequence shown on Figure 28 is PDTRPAPGSTAPPAHGVTSA (unmodified Muc1 repeat) (SEQ ID NO:8).
• FIG. 29 SynLubricin has low affinity for immobilized-metal-affinity- chromatography (IMAC) resin.
• IMAC immobilized-metal-affinity- chromatography
• Figure 30 Glycocalyx polymers induce membrane projections.
• A Schematic and table illustrating the genetically encoded biopolymers that were constructed and used throughout this work. The gene library encoded native and synthetic mucins comprised of a central polypeptide core, sugar side chains linked to serine (S) and threonine (T) residues, and a transmembrane anchor.
• B Quantification of membrane tube density in epithelial cells, showing mucin polymers induce dramatic tubularization compared to wild-type (Control) cells. Number of cells analyzed is shown on the x-axis for each condition. Box notches here and elsewhere indicate 95% confidence intervals.
• GUVs giant unilamellar vesicles
• Podxl membrane-anchored Podocalyxin
• HSA human serum albumin
• High HSA High HSA
• n 10-20. All GUVs were formulated with 10 mole % Ni-NTA-lipid for protein anchorage.
• center Quantification of the fraction of GUVs with or without tubes; n is the number of GUVs analyzed for each protein.
• right Representative confocal images of GUVs. *** p ⁇ 0.001 (post-hoc student’s two tailed t test).
• FIG. 31 Membrane morphology of tissue synoviocytes is regulated by the glycocalyx.
• A Experimental workflow for resected equine synovial tissues.
• B Second
• HAS3 hyaluronic acid synthase 3
• HyA hyaluronidase
• HyA hyaluronidase
• C Quantification showing tubule density was dependent on the presence of HA.
• D Images of freshly resected synovial tissue showing the nucleus (DAPI), surface-anchored HA (hyaluronic acid binding protein, HABP) of a representative synoviocyte, and the tissue collagen (second harmonic generation, SHG). Depth along the z-axis is coded according to the color bar. Note the HA-enriched membrane extensions protruding from the synovial tissue surface.
• FIG. 1 Lower right panel shows a cartoon representation of the observed tissue synoviocyte.
• E Membrane tubules are visible, by SEM, on synoviocytes in freshly excised equine synovial tissue. The synoviocyte head is pseudo-colored in orange protruding from the synovial tissue. HyA treatment to digest HA resulted in the rapid retraction of synoviocyte tubules (right). *** p ⁇ 0.001 (post-hoc student's two-tailed t test).
• Figure 32 Polymer brush model of the glycocalyx and generation of preferred membrane shapes
• A Polymer model of membrane bending illustrating proposed spontaneous membrane curvature induced by the cellular glycocalyx.
• Low density polymers are non-interacting and adopt a compact structure in the“mushroom” regime.
• R G the average distance between polymers
• H the height of the polymer brush
• Entropic pressures are the basis for membrane curvature generation by polymer mushrooms and brushes.
• B Muc1 construct with SUMO and GFP tags flanking the polymer domain for visualization of polymer extension with expansion microscopy (ExM).
• Figure 33 Preferred membrane shape depends on cell-surface biopolymer concentrations.
• A Strategy for sorting cells into populations with varying levels of cell surface mucin (Muc1-42TR-GFP DCT) using fluorescence-activated cell sorting (FACS).
• B Representative SEM images showing the transition of membrane morphological features of sorted cell populations with the indicated mucin surface density. Mucin densities were chosen to match the indicated points on the theoretical graphs (Fig.3D).
• C Average radius of bleb structures measured in the mushroom regime and tube structures measured in the brush regime.
• D Observed density of membrane blebs on sorted cell populations having the indicated average mucin surface density.
• Figure 34 Glycocalyx-mediated membrane instabilities and extracellular vesicle biogenesis.
• B Fluorescent intensity line trace from (A) (PNA image, red line). Values are normalized for their respective maximum intensities for phalloidin and PNA stains.
• C Fluorescent intensity line trace from (A) (PNA image, red line). Values are normalized for their respective maximum intensities for phalloidin and PNA stains.
• FIG. 35 Validation of genetically encoded mucins.
• A Cartoon representations of the genetically-encoded glycoproteins.
• Mucin-1 (Muc1) contains 42 repeats of
• PDTRPAPGSTAPPAHGVTSA SEQ ID NO:8
• Podocalyxin S/T-Rich
• the engineered glycoproteins lack the native cytoplasmic tail signaling domain ( ⁇ CT) while retaining the native transmembrane domain (TM) or exchanged with a synthetic 21-amino-acid transmembrane anchor (TM21).
• the rationally designed mucin contains 80 repeats of PPASTSAPG (SEQ ID NO:4) fused to a fluorescent marker (GFP) and the native stalk and TM without the native cytoplasmic tail signaling domain ( ⁇ CT).
• (B) Representative confocal microscopy images showing membrane tubularization induced by various engineered glycoproteins compared to wild-type (Control) cells. The cell surface is visualized with lectin WGA (wheat germ agglutinin). Mucin staining with lectin PNA (peanut agglutinin) confirms glycoprotein O- glycosylation and surface localization on MCF10A cells, n 3.
• FIG. 1 Representative confocal microscopy images of endocytosed 488 TNF after 0.5 h of treatment.
• F Quantification of tube density for the indicated mucin size. Number of cells analyzed is shown on the x-axis for each condition. Box notches indicate 95% confidence intervals. Statistical comparison is to 42TR. ns– not significant, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 (post-hoc student’s two-tailed t test).
• FIG. 36 Hyaluronic acid localizes on the cell surface and induces cell-surface projections.
• A Cartoon of hyaluronic acid (HA) extruded by the transmembrane protein hyaluronic acid synthase 3 (HAS3).
• HAS3 transmembrane protein hyaluronic acid synthase 3
• MECs human mammary epithelial cells
• ELISA quantification of HA secreted by MECs into their media, normalized to the number of cells in the sample and the HA secretion of Control cells, n 3.
• C Representative confocal microscopy images of human MECs, either wild-type (Control) or stably expressing HAS3. Cells are stained with Hoescht (nucleus) and Alexa Fluor 568 hyaluronic acid binding protein (HABP).
• D Representative SEM images showing highly elongated membrane tubules in HAS3-expressing human MECs (left) and a zoomed in region on the same cell (right). ** p ⁇ 0.01 (post-hoc student’s two-tailed t test).
• Figure 37 Mucins cause tubularization of model lipid membranes.
• FIG. 1 Representative confocal images of DOPC giant unilamellar vesicles (GUVs) labeled with Bodipy-PC with an increasing fraction of Ni-NTA lipids.
• Recombinant Alexa Fluor 568-labeled Podocalyxin (Podxl) associates with the GUV via a polyhistidine tag. Scale bar is 5 ⁇ m in each BODIPY-PC image.
• Figure 38 Supporting information for physical characterization of individual mucins and mucin ensembles.
• A Cartoon representation of the recombinant Muc142 tandem repeat (Muc1-42TR) polymer fused to a 10x-histidine tag.
• B Western blot validation of recombinant Muc1-42TR production (Media + Muc1-42TR 10xHis), Ni-NTA resin binding of the protein (Flowthrough), wash of non-specific proteins (Wash), and purified recombinant Muc1-42TR polymer (Elution). Samples are probed with anti-Muc1 and anti-His antibodies as well as PNA (peanut agglutinin) to bind O-linked glycans.
• PNA peanut agglutinin
• A Graph for the predicted brush thickness as a function of biopolymer surface density in the brush regime. Brush thickness scales approximately as a power law with biopolymer concentration.
• B Plot showing energetic contributions as functions of the biopolymer density. In the mushroom regime, polymers have only elastic energy, while in an extended brush, excluded volume and electrostatic interactions contribute to biopolymer free energy.
• C Plot depicting variation of spontaneous curvature generated with biopolymer density and molecular length.
• D Graph displaying trend of spontaneous curvature as a function of biopolymer density and Kuhn length.
• Kuhn length equal to twice the persistence length, is directly proportional to polymer bending stiffness, and is referred to as the length of a monomeric segment in the manuscript.
• Figure 40 Fluorescence-activated sorting and quantification of Muc1 surface densities.
• A Extended workflow for quantitative experiments at different Muc1 surface densities.
• B SDS-Page calibration of Alexa Fluor 647 labeled nanobody.
• C Calibration curve between the log value for integrated density of fluorescence signal from nanobody dilution series (shown in (B)) versus the log value of the number of molecule loaded. A linear regression fit and R 2 value are shown.
• D Residuals for the linear regression fit shown in (C).
• E Fluorescence-activated cell sorting (FACS) histogram showing the nanobody
• Figure 41 Tubular membrane shapes contain filamentous actin cores and resemble microvilli.
• B Fluorescent intensity line trace from (A) (bottom row, yellow line). Values are normalized for their respective maximum intensities.
• (C) Representative confocal microscopy images of epithelial cells expressing Muc1-42TR ⁇ CT showing actin staining with Alexa Fluor 568 phalloidin. Mucins are labeled with Alexa Fluor 647 PNA. The bottom row shows the region of interest from the composite image (yellow box), n 3. This data repeats and elaborates on (Fig.5A, B).
• the disclosure includes every amino acid sequence described herein, and every polynucleotide sequence that encodes the amino acid sequences, including but not limited to cDNA sequences, and mRNA sequences. Complementary sequences, and reverse
• the disclosure relates generally to improved glycoproteins, compositions comprising the proteins for use in diverse applications, and methods of making and using the
• glycoproteins are mucins and/or lubricins.
• the disclosure includes cells and cell cultures that express the proteins described herein.
• the disclosure includes cell cultures that are improved for producing any of a variety of proteins due to reduced clumping, aggregation, etc. of the cells.
• the disclosure relates to reducing clumping, including but not limited to extreme clumping.“Extreme clumping” as used herein refers to the association of multiple cells to hundreds of cells) into irregular masses or aggegates. Cell clumping/aggregation can be measured by direct observation of large cell aggregates observed by eye or microscopically as compared to single cells.
• cells can be stained with a nuclear stain (e.g., DAPI) and lysed to determine how many cells are comprised the cell aggregate.
• a nuclear stain e.g., DAPI
• An alternative method for quantitating the propensity of cells to clump involves the addition of calcium to precipitate cells, followed by turbidometric measurements.
• cells described in this disclosure exhibit less aggregation relative to a control. Any suitable control or reference value can be used.
• modified cells of this disclosure exhibit less aggregation compaired to cells without the same modifications (e.g., unmodified cells), wherein the modified cells and umodified cells are cultured in high calcium conditions, such as a higher than typical cell concentrations of CaCl 2 .
• a high calcium concentration is 2 mM CaCl 2 .
• less aggregation can comprise fewer cell aggregates relative to a control control value.
• a cell aggregate comprises a group of more than than three cells in contact with one another.
• cell cultures that have been engineered to express modified mucins and/or lubricins as described herein are also modified to recombinantly express at least one other protein.
• the disclosure provides for cells expressing a modified mucin and/or lubricin, the cells further expressing at least a second recombinant protein, wherein expression of the second recombinant protein is improved relative to a reference.
• An improvement in expression can comprise, for example, expression of more protein, secretion of more protein, recovery of more protein, and other parameters that will be apparent to those skilled in the art.
• the cells that express proteins of this disclosure are eukaryotic cells.
• the cells are eukaryotic cells, including but not limited to insect and mammalian cells.
• the mammalian cells are not Chinese hamster ovary (CHO) cells, although in certain instances CHO cells may be used.
• the cells are mammalian epithelial cells.
• the cells are human cells, and thus are better suited for producing, for example, human biologics, than non-human mammalian cells.
• the cells are human 293 cells.
• 293 cells are derived from 293 cells and stably express the SV40 large T antigen.
• the cells are human 293 cells adapted for growth in suspension cultures (293 suspsension cells).
• the cells are human 293-F cells, which are commercially available from a variety of vendors.
• the present disclosure includes modifying heterologous, or cells obtained from an individual, to express one or more of the glycoproteins described herein.
• human or non-human cells can be modified to, for example, correct a defect in a mucin or mucin-like protein, or the production thereof.
• cells modified according to this disclosure are totipotent, pluripotent, oligopotent stem, or multipotent stem cells.
• the cells are hematopoetic cells.
• the cells are chondrocytes.
• the cells are mesenchymal stem cells or marrow stromal cells.
• the cells are synovial cells.
• the cells are chondrogenic precursor cells.
• the cells endogenously produce cartilage-specific gene products, such as type II collagen and/or cartilage-specific chondroitin sulfate proteoglycan (CSPG).
• the cells are epithelial cells, or precursors thereof, or are goblet cells.
• the cells are immune cells, and include but are not necessarily limited to T cells, such as CD4+ and CD8+ T cells, and dendritic cells.
• Cells can be modified according to any established technique, including but not limited to use of viral expression vectors, or by chromosome editing, such as by any suitable CRISPR-based gene editing approach. Modified cells can be administered to an individual in need thereof.
• transgenic non-human animals that have been created to express one or more of the modified proteins of this disclosure can be produced and used to study a wide range of biological functions, disorders and conditions.
• cells modified according to this disclosure to improve protein production can be used to increase expression of any particular protein, without limitation.
• a modified mucin and/or lubricin protein described herein may be referred to as a“first” polypeptide
• a desired protein that is produced by the cells may be referred to as a“second” polypeptide.
• the second polypeptide thus may be distinct from the first polypeptide.
• the terms“first” and“second” are used for convenience, are not meant to indicate importance, or limit the disclosure to co-production of only two distinct polypeptides, but production of only two distinct polypeptides is also included within the scope of the disclosure.
• Representative second polypeptides include, for example, biologic agents that are or have a protein component, and thus include antibodies or fragments or derivatives thereof, protein or peptide vaccines, enzymes, structural proteins, and the like.
• the protein is any protein that can be suitable for use as a pharmaceutical/biologic agent, a nutraceutical, a dietary or other food supplement, a food additive, a filler, a binder, or for any other use or purpose.
• a modified cell as described herein is used to produce a viral particle.
• the viral particles are pathogenic.
• the viral particles are not pathogenic.
• the viral particles are attenuated viruses.
• the viral particles comprise a virus like particle (VLP).
• one or more than one viral protein can be produced.
• viral ribonucleoprotein (vRNP) complexes can be produced.
• viral particles produced using the compositions and methods described herein comprise viral proteins and a cell-derived envelope.
• second, or more than second polypeptides comprise lentiviruses and lentiviral particles, including but not limited to pseudoviral particles.
• the second of further polypeptides comprise one or more recombinant adeno-associated virus (rAAV) proteins.
• rAAV adeno-associated virus
• Such proteins include the well-known rep, cap and adeno-helper components.
• the rep component comprises four overlapping genes encoding Rep proteins required for the AAV life cycle (Rep78, Rep68, Rep52 and Rep40).
• the cap component comprises overlapping nucleotide sequences of capsid proteins VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.
• Adeno Helper function proteins may also be produced.
• the second polypeptide comprises an agent with binds to a target with specificity.
• the second polypeptide comprises an intact
• the second polypeptide comprises a T cell receptor (TCR), such as a TCR a and/or b chain.
• TCR T cell receptor
• the second polypeptide comprises a binding moiety of a bi-specific or tri-specific antibody.
• the second polypeptide comprises a chimeric antigen receptor (CAR), which can be expressed on immune cells such as T cells (CAR T cells).
• compositions and methods of this disclosure also include using the modified cells as described herein to prodice a secreted membrane vesicle.
• the type of vesicle is not particularly limited, and includes any membrane-bound vesicles secreted by cells, representative examples of which include exosomes, microvesicles, apoptotic bodies, and other extra-vesicular bodies, such as ectosomes.
• the disclosure includes expression, including increased expression, of a distinct protein, such as the aforementioned second polypeptide.
• the distinct protein e.g., the non-mucin or non mucin-like protein
• the distinct protein can be expressed from a separate gene, or mRNA, or can be encoded and expressed from the same mRNA, such as a bicistronic mRNA that may include any suitable feature, such as an internal ribosome entry sequence (an IRES).
• any glycoprotein described herein can be present in a fusion protein. Fusion proteins are produced recombinantly and contain in a single, contiguous polypeptide, segments of distinct proteins.
• a fusion protein described herein comprises a glycoprotein or segment thereof, and a second protein or segment that is not particularly limited.
• the second protein produced a detectable signal, and thus includes, for example, fluorescent proteins.
• cells including human cells, which express the described mucins and display them on their surface, are transfection competent.
• the described mucin coating does not impede transfection.
• anti-clumping agents e.g., agents designed to inhibit cell aggregation
• modified cells comprising the described mucins are able to produce and secrete high levels of recombinant protein, such as a representative red floursecent protein, as further described herein.
• a high levels of protein comprises 1-10 g/L of protein, inclusive, and including all ranges of numbers there between, and further including all expressions of ranges of weight and volumes, including, for example, milligrams and micrograms, and micrograms and microliters.
• compositions and methods of this disclosure involve recombinantly produced proteins that have repeated amino acid sequences, such as tandem repeat sequences.
• tandem repeat sequences are either modified relative to their naturally occurring sequences, or are the same as their naturally occurring sequences, but the number of repeats may have been altered, relative to the number of repeats in a naturally occurring protein. Combinations of distinct repeats may be included in the polypeptides described herein.
• the disclosure comprises introducing an expression vector described herein that encodes one or more proteins described herein, which may be a codon-optimized expression vector, into a suitable cell/cell culture, allowing expression of the protein(s), and recovering the protein(s) from the cells.
• cells in a cell culture are modified to express at least protein described herein using any suitable expression vector.
• the expression vector may be integrated into a chromosome of the cells, or may be maintained permanently or transiently as an epigenetic element.
• the expression vector may be configured to express the protein(s) in a constituent or inducible manner.
• a transposon based expression vector can be used, or a lentiviral expression system can be used.
• a lentiviral system can be excluded as a tool to express the proteins described herein.
• any protein described herein may, or may not include, a signal sequence.
• a signal sequence may be used.
• polynucleotide such as a cDNA encoding one or more of the proteins described herein, is randomly integrated into one or more chromosomes to produce the modified cells.
• a randomized transposition of a cDNA into the genome is used.
• codon-optimized expression vectors comprise a threshold number of altered codons, wherein the altered codons do not change the amino acid encoded by the particular codons.
• optimized codons may contain, for example, changes in wobble bases.
• at least one codon is altered, and from one codon to all of the codons that encode each amino acid in the particular protein may be altered.
• the codon optimized cDNAs reduce cDNA sequence repetitiveness to improve stability of the nucleotide sequence during DNA processing, including but not necessarily limited to slippage during replication, transcription, reverse transcription and other nucleotide processing operations on repetitive nucleotide sequences which often result in deletions or amplifications of cDNAs and mRNAs.
• codons with less than a predetermined threshold of frequency of usage in the pertinent cell type are replaced with codons that have a higher frequency of usage.
• codons that have less than or equal to 10% usage frequency in human cells can be replaced.
• the mucin/lubricin protein, or a protein for which improved production may be desired can be modified for recovery using any suitable approach, including but not limited to including one or more purification tags, including but not limited to a His-tag.
• a His-tag is a linear sequence of n histidine residues where n is typically 6-10. His-tags achieve purification by binding specifically to nickel or cobalt ions, which may be for example, attached to a substrate, such as any suitable beads.
• the His- tag, or any other suitable purification tag may be placed at the N-terminus of the protein, at the C-terminus of the protein, or interior to the protein.
• a FLAG-tag, or FLAG octapeptide, or FLAG epitope is may be included in proteins of this disclosure.
• Suitable FLAG sequences are known in the art.
• a Small ubiquitin-related modifier (SUMO) tag such as a His-SUMO tag can be included.
• protease cleavage sites can be included, such as for protein identification, separation, purification, etc. The proteins can be purified to any desired degree of purity.
• tandem repeats that are included in proteins of this disclosure comprise any one or any combination of the following amino acid segments:
• KEPAPTTP (SEQ ID NO:1) (lubricin-like repeat); KEPAPTP (SEQ ID NO:9) (modified lubricin-like repeat); KEPAPTTTP (SEQ ID NO:10) (modified lubricin-like repeat);
• DAATPAP (synthetic mucin repeat); DAATPAPP (SEQ ID NO:3)
• PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8) (Muc1 repeat);
• PDTRPAPGATAPPAHGVTSA SEQ ID NO:5 (Modified Muc1 repeat);
• PDTRPAPGATAPPAHGVTAA SEQ ID NO:6 (Modified Muc1 repeat);
• PDARPAPGATAPPAHGVTAA SEQ ID NO:7 (Modified Muc1 repeat).
• any amino acid sequence described herein can be a segment of a longer tandem repeat, and thus may have additional amino acid sequences on its N- or C-terminus.
• the amino acid sequence of a tandem repeat described herein comprises or consists of from 7-80 amino acids.
• a tandem repeat described herein exhibits an estimated length of approximately 135 nm, or 270 nm.
• a protein of this disclosure comprises at least one amino acid modification, and/or is expressed from a codon- optimized expression vector, and has an apparent molecular weights of approximately 470, 210, or 170 kDa.
• the repeats are perfect repeats, meaning the identical sequence is repeated in the protein, which differs from certain tandem repeats that occur naturally.
• the disclosure includes all cDNA and amino acid sequences disclosed in Parts I-IV of the Examples, and variants thereof as described herein. From time to time, such representative sequences are referred to for convenience as“biobricks.”
• the disclosure provides polypeptides, such as glycoproteins, and codon-optimized expression vectors encoding the glycoproteins, that are described herein as SynMuc1 and SynLubricin, as well as Muc1_42, Muc1_21, Muc1_10, Muc1_0, Muc1_21D, Muc1_21S, Muc1_21T, Syn1_40, Syn1_80, Syn2_40, Syn2_80, Syn3_40.
• mucin-135 and mucin-270 are the same mucin as as Muc1_21 and Muc1_42, respectively.
• Polypeptides comprising amino acid sequences that are at least 90% identical to the amino acid sequence of these sequences are included.
• the proteins comprise mutations, relative to an endogenous protein.
• An“endogenous” protein is a protein that is normally encoded by an unmodified gene.
• an endogenous gene or other polynucleotide comprises a DNA sequence that is unmodified, such as by recombinant, gene editing, or other approaches. Mutations, as further described below, can include amino acid insertions, deletions, and changes, and may also include additional repeated sequences, or fewer repeated sequences, relative to an endogenous sequence.
• tandem repeat amino acid sequences are introduced into a glycoprotein at its N-terminus, its C-terminus, or both the N-terminus and C-terminus.
• a recombinantly produced protein described herein comprises variants that have tandem repeats of any one or combination of the tandem repeat sequences described herein, wherein the variants comprise modifications of such sequences.
• Expression vectors encoding the variants are included.
• the modifications comprise amino acid segments that have between 90.0-99.9% amino acid identity, inclusive, and including all ranges of numbers there between to the first decimal point, with contiguous amino acid and polynucleotide sequences expressly described herein.
• tandem repeats comprised by recombinantly produced proteins of this disclosure have 90, 95, 97, 98, 99 or 99.5% amino acid sequence identity to the amino acid sequences described herein, across their full length(s).
• amino acid substitutions such as alanine substitutions
• Muc1-like variants can be used in any other protein described herein, with tandem repeats with altered frequencies of S/T glycosylation sites that can comprise a percentage of S/T sites in mucin backbones from, for example, at least 10%, and up to 33%, or more.
• an amino acid such as alanine can be substituted for S/T in one, two, or three, four, or five, or more glycosylation sites.
• a recombinant protein is a protein expressed from a polynucleotide that has been introduced to a cell that did not comprise a coding sequence for that protein prior to introducing the polynucleotide. The same applies to recombinant cDNA sequences.
• the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced).
• the nucleotides or amino acids at corresponding nucleotide or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position.
• tandem repeat variants described herein comprise a change of 1, 2, 3, 4, or 5 amino acids.
• an amino acid can be deleted, added, or changed.
• an amino acid that is changed is a serine, a threonine, or a combination of serine and threonine residues are changed. In embodiments, about 1-50% of serine and/or threonine residues are changed.
• a serine or threonine residue present in a native protein sequence is changed to an alanine, or to another amino acid.
• a protein of this disclosure comprises fewer, or no amino acids that are present in a native (non-modified and/or endogenous protein).
• a native protein comprises one or any combination of asparagine, aspartic acid, glycine, isoleucine, leucine, and/or serine, which can be engineered recombinantly out of representative proteins of this disclosure.
• amino acid changes introduced into proteins of this disclosure result in changed glycosylation patterns.
• the disclosure provides for production of recombinant proteins with controllable glycosylation patterns.
• the number of O-linked oligosaccharides present on a protein of this disclosure is modified.
• the glycosylation pattern is changed relative to a control, such as a protein in which a corresponding glycosylation site is not changed.
• one or more properties of the proteins, and or cells that express the proteins is changed.
• the stoichiometry of oligosaccharides to protein/amino acids is changed in, for example, a glycoprotein of this disclosure.
• a protein of this disclosure comprises a percentage by weight of glycosidic residues that is different from a suitable control.
• cells modified according to this disclosure have one or more characteristics, which may be improved relative to a control, or not diminished relative to a control, such as turbidity, viability during or after exposure to a shear stress, such as a shear stress that arises during mixing the cells in, for example, a bioreactor.
• a recombinantly produced protein as described herein comprises a change relative to a control, such as an unmodified protein, in the Core 1 O-glycan structure, Gal b1-3GalNac, and/or the amount of Core 1 derivatives of Gal b1-3GalNAc, and/or the amount of terminally substituted sialic acids therein, or a change in GalNAc (N- acetylgalactosamine) monosaccharide glycosylation.
• a protein described herein can comprise the Core 2 O-glycan, GlcNAc b1-6(Gal b1-3)GalNAc and/or the Core 2 derivatives of GlcNAc b1-6(Gal b1-3)GalNAc, which comprise at least 5 percent of all Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, and Cor e8 O-glycan structures.
• such a protein is produced by human cells that are cultured as further described herein, including but not necessarily limited to a suspension culture.
• Figure 3f provides a representative relative abundance of core O-glycan structures.
• the relative abundance is based on the frequency of the glycans (i.e numbers) and is given by the spectral intensity on the MALDI-MS mass chromatogram for O-glycans released from glycoprotein by beta-elimination.
• Relative abundance of O-glycans as summarized in Figure 3 is as follows: GalNAc monosaccharide 86.3%; Core 18.2%; representative example being GalNAc-Gal; Core 25.3%.
• Core III and IV, V, VI, VII, VIII were not detected. If present, they were present at very low levels that are below the detection limit of MALDI-MS.
• proteins of this disclosure may be in the form of monomers, dimers, multimers, and combinations thereof.
• monomer/dimer ratios, proportions, and/or concentrations are changed, relative to suitable controls.
• segments of proteins described herein can be separated by any suitable linking amino acids.
• linker can comprise from 1-20 amino acids, inclusive, and including all integers and ranges of integers there between.
• linkers are comprised of a glycine, serine, or serine and glycine.
• linking amino acids do not intereven tandem repeats.
• no linker is included in a polypeptide of this disclosure.
• any one or combination of proteins described herein can be associated with a cell membrane.
• the disclosure thus provides biopolymers with tunable sizes, O-glycan side-chain spacing, and distinct glycan types for glycocalyx editing.
• secreted forms of glycosylation mutants are provided.
• the disclosure provides proteins, and DNA sequences encoding the proteins, that have a polymer backbone, and at least one of the following elements, non- limiting examples of which are provided herein: a leader tag; an optical reporter, such as any fluorescent protein, a transmembrane domain; and a cytoplasmic motif. Any one or any combination of these elements can be excluded from the constructs presented herein.
• cell surface mucins include a cytoplasmic recycling motif on a transmembrane domain, which can alter glycosyolation and/or sialylation of the proteins, and further illustrated herein by the description and figures.
• the cytoplasmic recycling motif comprises or consists of the amino acid sequence CQC.
• Cytoplasmic recycling and motifs that facilitate the same are known in the art. Such motifs facilitate trafficking through the trans Golgi network (TGN), endosome, and/or from the endosome back to the plasma membrane.
• TGN trans Golgi network
• a polypeptide of this disclosure may have one or more modified amino acids that are, for example, conjugated to another moiety.
• a polypeptide of this disclosure is conjugated to at least one azido group such that they can be readily conjugated to other moieties, such as using click chemistry.
• a polypeptide of this disclosure is cyclized, or stapled.
• a tandem repeat sequence described herein is incorporated into any glycoprotein.
• the glycoprotein is any mucin or lubricin protein.
• the mucin is any of MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, and PODXL, the amino acid sequences of which are known in the art.
• the glycoprotein is Proteoglycan 4, also referred to in the art as lubricin, which comprises a protein that in humans is encoded by the PRG4 gene.
• SynMuc1 as described further below.
• a modified lubricin is provided as SynLubricin, as further described below.
• production of protein is increased using cells modified herein, wherein the cells are present in a cell culture container, including but not limited to any cell culture dish, and bioreactors.
• modified cells according to this disclosure are used in bioreactors to produce any desired protein, or combination thereof.
• the bioreactor comprises a suspended cell bioreactor.
• bioreactors have a volume of from 1-25,000 liters, inclusive, and including all numbers and ranges of numbers there between.
• the bioreactor is has a volume of at least 100 liters, or at least 1,000 liters.
• cDNA libraries are provided.
• the disclosure comprises providing a cDNA library as described herein, and selecting one or a combination of the cDNAs described or modifying cells by introducing the cDNA and/or an expression vector encoding the cDNA into a cell. Selection can be based upon an intended or actual use for the cells, such as for use in protein production, based on any particular protein and cell expression system. Kits encoding the proteins are also included.
• one or more proteins described herein can be combined with other agent(s), such as biodegradable polymer(s), nanoparticlespectin, alginate, cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides, hydroxypropyl methylcellulose, carboxymethylcellulose, lectins, rheology modifiers, plasticizers, chondroitin, glucosamine, and/or any hyaluronic acid.
• agent(s) such as biodegradable polymer(s), nanoparticlespectin, alginate, cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides, hydroxypropyl methylcellulose, carboxymethylcellulose, lectins, rheology modifiers, plasticizers, chondroitin, glucosamine, and/or any hyaluronic acid.
• compositions described herein can be administered in a
• compositions comprising the proteins described herein can be provided as liquids, tablets, powders, sprays, ointments, hydrogels, and aerosols.
• compositions comprising one or more proteins of this disclosure can be administered to an individual using any suitable route, including but not necessarily limited to topically, orally and parenterally, and as further described below.
• the proteins can be administered intravenously, by direct injection into synovial joints or other synovial structures (tendon sheaths, bursae), intraperitoneally, by direct injection into the pericardial sac, by direct injection into the pleural cavity, subdermally, subcutaneously, or by direct application to skin, mucous membranes, or the eye.
• the disclosure comprises methods, compositions, and devices for treating an ocular disease, disorder or condition in a mammal.
• proteins produced by cells as described herein are used for treatment of eye disease or condition using any method or device known to those of ordinary skill in the art.
• compositions comprising the proteins are used for intracameral, intravitreous,
• the proteins may be delivered directly to the eye (for example: topical ocular drops or ointments; slow release devices in the cul-de-sac or implanted adjacent to the sclera or within the eye) using techniques well known by those skilled in the art. It is further contemplated that the proteins described herein may be formulated in intraocular insert or implant devices.
• a pharmaceutical comprising one or more proteins described herein is used to treat an eye disorder that comprises one or more diseases or injury to the retina, including age-related macular degeneration (AMD), retinitis pigmentosa (RP), and diabetic retinopathy (DR).
• AMD age-related macular degeneration
• RP retinitis pigmentosa
• DR diabetic retinopathy
• the individual has dry, atrophic (nonexudative) age- related macular degeneration, defined as progressive age-related degeneration of the macular associated with retinal pigment epithelial changes including atrophy and drusen, which is a common cause of vision loss in adults for which therapy is limited.
• the disorder comprises one or more diseases or injury to the cornea.
• the individual has glaucoma, which may include primary, secondary and/or congenital glaucoma.
• proteins of this disclosure can be provided in the form of eye drops.
• the eye drops comprise any one or more of steroids, antihistamines, sympathomimetics, beta receptor blockers, parasympathomimetics, parasympatholytics, prostaglandins, nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, antifungal, or topical anesthetics.
• the eye drops are for use with any dry eye condition.
• the eye drops are for use in lubrication of eyes, including but not necessarily for a contact lens wearer.
• the compositions are provided as lubricating eye drops.
• the lubricating eye drops comprise artificial tears.
• the eye drops may be free of medications, and thus function only as lubricating/tear-replacement compositions.
• the eye drops may be for treatment of ocular allergic reactions, and thus my also comprise antihistamines, and/or vasoconstriction agents.
• compositions comprising proteins described herein can be used in conjunction with contact lenses.
• the proteins are used in a contact lens solution.
• proteins described herein can be mixed with any suitable contact lens solution components, which include but are not necessarily limited to saline, mild abrasives, surfactants, anti-fungal and anti-bacterial agents, which include but are not limited to conventional amicrobial agents, or hydrogen peroxide or boric acid, and preservatives, such as ascorbic acid or edetate disodium.
• contact lens solution components include but are not necessarily limited to saline, mild abrasives, surfactants, anti-fungal and anti-bacterial agents, which include but are not limited to conventional amicrobial agents, or hydrogen peroxide or boric acid, and preservatives, such as ascorbic acid or edetate disodium.
• Contact lenses provided in a solution comprising one or more proteins described herein are included within the scope of this disclosure.
• compositions comprising proteins described here can be directed to a mucosal lining.
• the mucosal lining includes, for example, the upper and lower respiratory tract, eye, buccal cavity, nose, rectum, vagina, urogenital tract, periodontal pocket, intestines and colon.
• the compositions can be used for oral inhalations.
• the oral inhalation comprises nasal applications, and thus may include nasal sprays, nasal drops, and nasal ointments.
• oral inhalation may comprise bronchial sprays and inhalers.
• the proteins may be used to access mucosa through use of throat lozenges, chewing gum, mouthwashes or gargles, suppositories, or tampons.
• compositions comprising proteins described herein are used as surgical anti-adhesives (intraperitoneal lubricants to lubricate viscera and prevent post-op intestinal and visceral adhesions during intra-abdominal surgical procedures/manipulations; intrapleural lubricants to lubricate lungs and prevent postoperative pleural adhesions during intra-thoracic surgical procedures/manipulations; intrapericardial lubricants to lubricate the cardiac surface and prevent post-op pericardial adhesions during cardiac surgical
• an article of manufacture may be coated and/or impregnated with a composition comprising any of the proteins described herein.
• the article of manufacture is coated on any porous or non-porous surface.
• the article comprises a medical device, including but not necessarily limited to a surgical device, a dental or orthopedic device, sutures, catheters, an intubation device, an anesthesia delivery device, a dressing, bandage, etc.
• proteins described herein are used to coat cell culture devices, including, but not necessarily limited to, cell culture plates, multiwell plates, bioreactors, and any other surface, wherein an anti-adhesive property is desirable.
• the disclosure includes a supplement product, such as a nutraceutical product, a dietary supplement, a food ingredient, etc.
• the supplement product can be provided in the form of, for example, a liquid, capsules, tablets, softgels, powders, and the lie.
• a pharmaceutical and/or nutraceutical product comprising one or more proteins described herein is provided in a container, such as any suitable closed or sealable container which may be sterile.
• the product comprises printed material.
• the printed material can be provided as a product insert, label, or as a component of packaging. The printed material provides an indication that composition comprising the polypeptides is to be used for treating any disease, disorder, or condition as described herein, or for producing an anti-adhesive effect for any purpose.
• polypeptides described herein are used as a supplement for treating a condition of joints, including, but not necessarily limited to joint pain, arthritis, including, but not necessarily limited to, osteoarthritis, rheumatoid arthritis, injuries to joints, menisci or cartilage, such as sports injuries, or in conjunction with joint/ligament repair surgeries.
• administering compositions described herein for the purposes of improving the health or well-being of an individual are included within the disclosure.
• compositions of this disclosure can be injected directly into a joint and/or synovial fluid.
• compositions of this disclosure can be also be used for injection directly into the tendon, tendon sheath, ligament or bursa following a tendon, ligament or bursal injury, trauma or infection.
• This Part I of the disclosure provides non-limiting and representative examples of sequence-specific mucins with controllable glycosylation patterns, and data and discussion of the same.
• this Part I relates to the understanding that, prior to the present disclosure, few design guidelines existed for encoding customized mucin glycoproteins with tunable glycosylation patterns.
• Part I accordingly provides a library of swappable DNA bricks for mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized native mucin repeats and new, rationally designed domains for synthetic mucins.
• this Part I provides a library of over 50 mucins, each with unique chemical, structural, and optical properties. The library is applied to develop general guidelines for the design and engineering of mucins, which form a part of this disclosure.
• Cell-surface mucins are a family of membrane-anchored biopolymers that are defined by their unstructured polypeptide backbone with a high density of sugar side chains(1). While historically viewed as simple structural molecules that protect the cellular surface and resist pathological cell deposition(2), cell-surface mucins are now recognized to have more sophisticated roles in regulating cellular life. In the cellular glycocalyx, mucin ensembles present bio-active glycan epitopes that mediate adhesion and communication between cells and with their external world. For instance, mucin sialic acids can modulate immune cell function through ligation of SIGLEC receptors on natural killer cells and other cell types in the microenvironment(3).
• Mucins can also physically regulate the spatiotemporal dynamics of receptor activation and signaling responses(4). Dense crowding of mucins in the glycocalyx is proposed to control the diffusion and activation of receptors on the cell surface, and to have a sieving effect that controls the passage of soluble factors from the
• mucins have molecular architecture can change dynamically through modulation of the types and frequencies of glycan side chains that are appended along the polypeptide backbone. For instance, the charge, size, and arrangement of glycans are proposed to control the extension and rigidity of the mucin backbone(6, 7). Glycosylation often changes dramatically with cell-state transitions, including differentiation and transformation(8, 9). As such, both the chemical and physical character of mucins is intimately coupled to cellular state, contributing to the diverse modulatory roles that mucins can play in cellular adhesion, communication, and signaling. However, how precise backbone sequences and glycosylation patterns contribute to the function of individual mucins and the collective behaviors of mucins in the glycocalyx is largely unresolved.
• Part I results demonstrate a modular biology-by-parts approach for combinatorial mucin cDNA construction.
• Each functional motif in the mucin coding sequence was flanked by restriction sites, so that unique cDNA“bricks” for mucin leader sequences, tandem repeats, optical reporters, transmembrane domains, and cytoplasmic domains could be readily swapped to construct mucins of altered functionality ( Figure 1a, b).
• the cDNA parts catalogue included 13 unique tandem repeats for mucin biopolymers of varying size, backbone chemistry, and frequency of serine and threonine (S/T) glycosylation sites ( Figure 1d).
• the cDNAs for the mucin polymer domains were fabricated through custom gene synthesis following codon optimization ( Figure 1c). For optimization, codon redundancy was exploited to find synonymous gene sequences that coded the desired polypeptide with minimal codon repetition.
• The“codon-scrambled” cDNA sequences were synthesized through standard custom gene synthesis services offered by commercial vendors.
• the tandem repeats that form the mucin polymer backbone were adapted from native mucins or newly designed (Figure 1d).
• the repeats PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8) and KEPAPTTP (SEQ ID NO:1) have similarity to native Muc1 and Proteoglycan 4 (Lubricin), respectively.
• Three repeats were designed based on statistical analysis of mucin O-glycosylation sites (PPASTSAPG) (SEQ ID NO:4) or analysis of O-GalNAc transfer efficiency (DAATPAP (SEQ ID NO:2) and DAATPAPP) 20 .
• the base Muc1 repeat was further modified through alanine substitutions to create Muc1-like tandem repeats with altered frequencies of S/T potential glycosylation sites (Muc1_21S, D, T). Across the library, the percentage of S/T sites in the mucin backbones varied from 10% to 33% ( Figure 1d). Constructing and Validating the Surface Expression of Sequence-Specific Mucins
• codon-scrambled mucin cDNAs were the potential to improve the stability of the nucleotide sequence during some DNA processing operations. Slippage during replication, transcription, reverse transcription and other nucleotide processing operations on repetitive nucleotide sequences often results in deletions or amplifications of cDNAs and mRNAs 24 .
• tandem repeats of native mucins are often polymorphic in number in humans, resulting in a variation of mucin size amongst individuals 25 and short alleles of Muc1 have been shown to be associated with gastric cancer 26 .
• the polymorphic cDNAs expressed well on the cell surface and displayed the expected differences in size and extent of glycosylation. As expected based on previous reports 27 , the larger mucins formed a glycocalyx that was substantial enough to dislodge epithelial cells from their substrate.
• cDNAs for the desired Muc1 mutants with 21 repeats each were optimized through codon scrambling and fabricated through custom gene synthesis.
• the single (Muc1_21S), double (Muc1_21D), and triple (Muc1_21T) glycosylation mutants had 21, 42, and 63 total S/T to alanine substitutions, respectively, and varied in potential glycosylation frequency at 20%, 15% and 10%.
• An IgK signal peptide and 6x-His-SUMOStar tag was fused to the 21 copies of the wild-type Muc1 repeat or the three mutant repeats (Figure 3a). No transmembrane protein anchor was included, so that the IgK signal peptide would direct secretion of the recombinant mucin protein.
• the secreted mucins were harvested from the media supernatant of HEK293 cells and analyzed by Western and lectin blot.
• the wild-type and glycosylation mutants had a considerably higher apparent molecular weight than the theoretical molecular mass of the undecorated peptide backbones ( Figure 3b, c and 8).
• the potential glycosylation site mutants migrated faster in SDS-PAGE, indicating that they had fewer glycan chains or that their glycans were shorter and, thus, less obstructive to their electrophoretic mobility (Figure 3c).
• glycotransferases for glycan extension we used Cellular O-glycome Reporter/Amplification (CORA), a method which allows protein-free profiling of the overall cellular O-glycome 28 .
• CORA Cellular O-glycome Reporter/Amplification
• the backbones varied in frequency of glycosylation sites (S/T) from 12-33%.
• Sialylation of O-glycans has occurs at least partially in the endosome and trans-Golgi network following endocytosis of cell-surface mucins 29 .
• endocytosis and trafficking we created cDNA“bricks” for mucin cytoplasmic tails with different endocytosis and trafficking signals.
• the Muc1 cytoplasmic domain can signal for clathrin-mediated endocytosis, while the Muc1 sequence CQCRRK (SEQ ID NO:11) at the boundary of transmembrane and cytoplasmic domain signals for Muc1 recycling back to the plasma membrane 30 .
• TM21 21-amino-acid transmembrane anchor
• anti-Human MUC1 CD227) (clone HMPV; 555925, BD Biosciences), mouse anti- b-Actin (clone C4; 47778, Santa Cruz), chicken anti- SUMO/SUMOstar (AB7002, LifeSensors), mouse 6xHis (552565, BD Biosciences), mouse anti- a-tubulin (clone B-7; 5286, Santa Cruz), mouse anti-GFP (clone 4B10; 2955, Cell Signaling Technology), m-IgG ⁇ binding protein– horseradish peroxidase (HRP; 516102, Santa Cruz), goat anti-mouse IgG (Alexa FluorTM 647 conjugated, A-21235; Alexa FluorTM 488 conjugated, A-11001; Alexa FluorTM 568 conjugated, A-11004; ThermoFisher) and goat anti-chicken IgY (Alexa Fluor 488TM conjugated; A-11039, Thermo
• Lectins used were: unconjugated Arachis hypogaea lectin/ peanut agglutinin (PNA; L0881, Sigma), biotin- conjugated PNA (B-1075, Vector Laboratories), biotin-conjugated Maackia amurensis lectin (MAA; BA-7801, EY Lab), fluorescein-labeled succinylated Wheat Germ Agglutinin(s- WGA; FL-1021S, Vector Lab), and biotin-conjugated Vicia villosa lectin (VVL,VVA; B- 1235, Vector Lab).
• Fluorescent dyes used were: Alexa FluorTM 647 NHS Ester (A20006, Invitrogen), Alexa FluorTM 568 NHS Ester (A20003, Invitrogen) and AFDye 568
• Biotinylated lectins were detected using ExtrAvidin-Peroxidase (E2886, Sigma) or NeutrAvidin Protein (Dylight 650 conjugated; 84607, ThermoFisher).
• E2886 ExtrAvidin-Peroxidase
• NeutrAvidin Protein Density Protein
• doxycycline was used for induction (204734, Santa Cruz).
• Streptavidin Sepharose® beads 3419, Cell Signaling Technology
• cDNAs for cytoplasmic-tail-deleted human Muc1 (Muc1 dCT) and Muc1 tandem- repeat fusion with the synthetic membrane domain TM21 (Muc1 TM21) were generated and cloned into the tetracycline-inducible piggybac expression vector with Puromycin resistance cassette (pPB tetOn Puro) as previously described 27 .
• cDNA of Muc1 TM21 was also inserted into the pcDNA3.1 vector using BamHI and EcoRI restriction sites.
• the cDNA for mOxGFP (Addgene #68070) was first amplified with primers: 5’- GGCAGCTCAGCTATGGTGTCCAAGGGCGAGGAGCTGT- 3’ (SEQ ID NO:12) (forward) and 5’- GGCAGCTGAGCCCTTATACAGCTCGTCCATGCCGTGAGT-3’ (reverse) (SEQ ID NO:13).
• the PCR product was then cloned into pJET1.2 and subcloned non-directionally into the BlpI site of pPB Muc1 dCT TetOn Puro.
• cDNAs for mutant and rationally designed mucins tandem repeats were generated through custom gene synthesis following codon optimization.
• the least repetitive gene sequence for the desired mucin repeats was found using Codon Scrambler
• Synthetic oligos for the desired tandem repeats were then synthesized by custom gene synthesis (General Biosystems and Genscript) and cloned in place of the Muc1 tandem repeats in either pPB Muc1 mOxGFP dCT TetOn Puro using the BamHI and Bsu36I restriction sites, pcDNA3.1 Muc1 TM21 using the BsrGI and Bsu36I restriction sites, or pPB 6x His Sumostar Muc1 using BsrGI and Bsu36I restriction sites (See Supporting Information for cDNA sequences).
• the Muc1 construct with 0 tandem repeats was generated through deletion of the tandem repeats in pcDNA3.1 Muc1_10 TM21 through Q5 site-directed mutagenesis with 5’- TGGAGGAGCCTCAGGCATACTTTATTG-3’ (forward) (SEQ ID NO:14) and 5’- CCACCGCCGACCGAGGTGACATCCTG-3’ (reverse) (SEQ ID NO:15) primers.
• pcDNA3.1 Muc1_10 TM21 CQC was generated from pcDNA3.1 Muc1_10 TM21 through Q5 site-directed mutagenesis with 5’- CCGAAAGTAGGAATTCGGGCCCGTTTAAACCCGC-3’ (forward) (SEQ ID NO:16) and 5’- CGGCACTGACATCTAGAGTACCACAACAAAGCCAGGC-3’ (reverse) (SEQ ID NO:17) primers.
• the cDNA of native CT was subcloned into the XbaI and EcoRI site of pcDNA3.1 Muc1_10 TM21 CQC.
• the 40 tandem repeats of DAATPAP (SEQ ID NO:2) and DAATPAPP (SEQ ID NO:3) mucin cDNAs in pcDNA3.1 were doubled in size to 80 repeats using Golden Gate Assembly.
• Two pairs of custom primers for tandem repeats and complete mucin vector were designed to attach BsmbI recognition sites with unique 4bp overhangs so that the PCR products of the 40 tandem repeats and complete mucin expression vector would ligate in a Golden Gate Assembly reaction to amplify the tandem repeat number (Table S2).
• Golden Gate Assembly reaction was conducted as previously reported 47 .
• MCF10A human mammary epithelial cells and HEK293T SV40-transformed human embryonic kidney cells were obtained from ATCC. MCF10A cells were cultured in
• DMEM/F12 media (ThermoFisher) supplemented with 5% horse serum (ThermoFisher), 20 ng/mL EGF (Peprotech), 10 mg/ml insulin (Sigma), 500 ng/mL hydrocortisone (Sigma), and 100 ng/mL cholera toxin (Sigma).
• HEK293T cells were cultured in DMEM (ThermoFisher) supplemented with 10% fetal bovine serum (ThermoFisher). Cells were maintained at 37 °C, 5% CO 2 , and 90% Relative humidity (RH).
• FreeStyleTM 293-F cells were cultured in suspension in FreeStyleTM 293 Expression Medium (ThermoFisher).
• HEK293T cells were transiently transfected with the calcium phosphate method according to standard protocols.
• FreeStyleTM 293-F cells were transiently transfected with PEI as previously described 49 .
• CRISPR/Cas9 mediated knockout of COSMC in MCF10A Muc1 dCT cells were generated as previously reported 50 .
• HEK293T cells were plated at 55,000 cells/cm 2 and transfected with calcium phosphate for 24-36 hrs before lysis with cell lysis buffer.
• MCF10A cells were plated at 20,000 cells/cm 2 and induced with 0.2 mg/mL doxycycline for 24 hrs before lysis with cell lysis buffer. Lysates were separated on NuPAGE 3-8% or 7% Tris-Acetate gels and transferred to PVDF membranes.
• Primary antibodies were diluted at 1:1000 and fluorophore- conjugated or biotinylated lectins were diluted to 2 mg/mL in 5% BSA TBST and incubated overnight at 4 °C.
• ExtrAvidin-HRP or Neutravidin-Dylight 650 were diluted at 1:2000 or 1 mg/mL in 5% BSA TBST and incubated for 1 hr at room temperature. Blots were either imaged on a ChemiDoc MP Imaging System (Bio-Rad) or after being developed in LumiGLO ® reagent and peroxide. Integrated blot intensity was quantified with the FIJI distribution of ImageJ 51,52 . The statistical significance of the differences among the data was calculated using a one-way ANOVA with repeated measures or two-tailed t-test. Periodate Labeling of Cell Surface Sialic Acids
• HEK293T cells were collected after 36hrs of transfection. Cells were washed with cold DPBS with Ca 2+ and Mg 2+ followed by a 10-minute incubation with 1 mM sodium periodate (Sigma) in DPBS. The periodate was quenched by 1 mM glycerol in cold DPBS and washed with cold DPBS. Samples were stained with 25 mM AFDye-568-hydroxylamine (Fluoroprobes) in the presence of 10 mM aniline (Sigma) in sterile filtered DPBS + 5% FBS pH 6.7 for 30 min at 4°C in the dark with gentle agitation.
• HEK293T cells were plated at 55,000 cells/cm 2 and transfected with the calcium phosphate method for 24-36 hrs before lysis with cell lysis buffer.
• the lysates were incubated with 125 mg/mL biotinylated lectin PNA at 4 °C with gentle rocking overnight.
• Streptavidin Sepharose ® beads were added to the cell lysates following manufacturer’s instructions and the suspension was incubated at 4 °C for 3 hrs. The beads were washed 2 times with lysis buffer and then resuspended in 4x LDS loading buffer. The resuspension was subsequently analyzed by Western blot.
• HEK293T cells were plated at 45,000 cells/cm 2 and transfected with calcium phosphate for 24 hrs before being fixed with 4% paraformaldehyde.
• Antibodies were diluted at 1:100 in 5% normal goat serum in PBS and incubated overnight at 4 °C.
• Lectins were diluted to 2 mg/mL in 5% normal goat serum in PBS and incubated for 2 hrs at room temperature. Samples were imaged on a Zeiss LSM inverted 880 confocal microscope using a 40x water immersion objective (NA 1.1).
• the clarified culture media was bound to Ni-NTA agarose (Qiagen) at 4 °C overnight, washed (20 mM sodium phosphate pH 8.0, 0.5 M sodium chloride (NaCl), 20 mM imidazole), and eluted with imidazole (20 mM sodium phosphate pH 8.0, 0.5 M NaCl, 250mM imidazole).
• the eluted sample was diafiltrated into PBS with Amicon Ultra-4 Centrifugal Filter (10 kDa cutoff) and then desalted by using Zeba TM Spin desalting columns (7K MWCO).
• the salt-free protein solution was lyophilized and stored at -80 °C.
• the dried eluate was dissolved with dimethyl sulfoxide (DMSO) and methylated by using methyl iodide and NaOH-DMSO base (prepared by mixing DMSO and 50 % w/w NaOH solution).
• DMSO dimethyl sulfoxide
• NaOH-DMSO base prepared by mixing DMSO and 50 % w/w NaOH solution.
• the reaction was quenched with water and the reaction mixture was extracted with methylene chloride and dried.
• the permethylated glycans were dissolved in methanol and crystallized with a-dihydroxybenzoic acid (DHBA, 20 mg/mL in 50% v/v methanol: water) matrix.
• DHBA a-dihydroxybenzoic acid
• CORA was performed as previously reported 28 . Briefly, 500,000 HEK293T cells were plated in a 6 cm culture dish and transfected as above. Following transfection cultures were incubated in full media supplemented with 50 mM peracetylated BnGalNAc. After 48 hours the media was aspirated and loose cells and debris removed by centrifugation. The supernatant was then filtered (Millipore Amicon Ultra 4, 10 kDa MWCO) and benzyl glycans collected by gravity chromatography (Waters Sep-Pak C183 cc). The eluent was dried by speedvac before permethylation2.
• a sodium hydroxide slurry in DMSO was freshly prepared and 200 mL added to each dry sample followed by 100 mL methyl iodide (ACROS).
• the samples were mixed continuously for 10 mins then the reaction halted by the addition of 600 mL deionized water.
• Permethylated benzyl glycans were recovered by extraction with 200 mL chloroform then washed 4 times with 800 mL deionized water.
• the samples were further purified by C18 gravity chromatography (Waters Sep-Pak C181 cc) and dried by speedvac.
• This Part I provides a genetically encoded system to edit the mucin biopolymers, and can be used as a tool for glycocalyx engineering, among other significant utilities that are discussed above.
• Factors that are known to influence mucin glycosylation include the cellular repertoire of glycosyltransferases and their substrates 1,34 , frequency of O-glycosylation sites on the polypeptide backbone 35,36 , primary peptide sequences around the O-glycosylation sites 37–39 , and trafficking of the glycoprotein 32,40,41 .
• this Part I we modify signals and motifs in the mucin backbone sequences and cytoplasmic tails to encode mucins with varying physical features, backbone chemistries, and glycosylation patterns.
• repeat sequences are proceeded by the following sequence:
• pPB_Tet_Muc1_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR 19.
• pPB_Tet_Muc1_42_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR 20.
• sequences are representative amino acid sequences for mucin and lubricin constructs, as further described herein, and for which the entire sequences, including the N-terminal signal sequence, tandem repeat domain, fluorescent optical reporter (GFP in these sequence), the transmembrane domain to the cytoplasmic tail domain.
• GFP fluorescent optical reporter
• the GFP sequence may be, omitted or substituted by any other amino acid sequence, including but not limited to the sequence of other detectable proteins, or second polypeptides, as described above.
• the alphnuermic names given above each sequence are names of the sequences, rather than sequences themselves.
• This Part II of the disclosure illustrates mucin-coating technologies for protection and reduced aggregation of cellular production systems.
• the mucin coating technology directs the assembly of thick, highly hydrated barriers to strongly mitigate cell aggregation and protect cells in suspension against fluid shear stresses.
• the coating technology is demonstrated on suspension adapted human 293-F cells, which resist clumping even in media formulations that otherwise would induce extreme cell aggregation and show improved performance over commercially available anti-clumping agent.
• the stable biopolymer coatings do not show deleterious effects on cell proliferation rate, efficiency of transient transfection with cDNAs, or recombinant protein expression.
• the mucin coating technology and engineered cell lines described herein exhibit the ability to improve the single-cell growth and viability of suspended cells in bioreactors.
• Mucins are utilized to reduce adhesion and fouling at biological interfaces.
• Mucins are characterized by amino acid sequences rich in serine and threonine residues, which are post-translationally modified with O-linked pendant glycan structures (Thornton, Rousseau, & McGuckin, 2008).
• the bottlebrush molecular structure of mucins confers an anti-adhesive characteristic that is used by biological systems for diverse purposes, including antifouling coatings, lubrication, and modulation of cellular interactions (Jay & Waller, 2014; Kuo, Lucas, Zia, & Paszek, 2018; Paszek et al., 2014).
• Mucin-1 is recognized as an anti-adhesive protein that can interfere with integrin- and cadherin-mediated cell interactions (Klinken, Dekker, Buller, & Einerhand, 1995; Wesseling, Valk, & Hilkens, 1996; Wesseling, van der Valk, Vos, Sonnenberg, & Hilkens, 1995).
• the anti-adhesive properties of Mucl are conferred by its large ectodomain, which is heavily O-glycosylated during trafficking to the cell surface. Neutral and anionic sugar residues of the glycans can coordinate with water to form a highly hydrated barrier on the cell surface (Gendler & Spicer, 1995).
• novel mucin cDNAs and mucins encoded by them are described and used to create a genetically-encoded technology for reduction of aggregation of human-cell host production systems.
• the presently described mucin technology is improved, tested, and refined for use, for example, as an anti-adhesive coating on host-cell production systems.
• a tetracycline-inducible, transposon based Piggybac expression vector with an integrated, co-expressed reverse tetracycline transactivator gene (pPB tet rtTA NeoR) was used for stable line generation.
• the pPB tet rtTA NeoR plasmid was modified by the insertion of the internal ribosome entry site (IRES) of the encephalomyocarditis virus followed by the fluorescent protein copGFP into the NotI and XbaI sites (pPB tet IRES GFP rtTA NeoR).
• Synthetic cDNAs containing either 21 or 42 tandem repeats (TR) of the amino acid sequence PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8) were codon optimized with codon scrambler (Tang & Chilkoti, 2016), generated through custom gene synthesis (General Biosystems), and cloned in place of the native tandem repeats in pcDNA3.1 Muc1 TM21– previously described in (Paszek et al., 2014; Shurer et al., 2017)– using the BamHI and Bsu36I restriction sites.
• the Muc1 gene containing the engineered 21 or 42 tandem repeats was then cloned into the pPB tet IRES GFP rtTA NeoR plasmid using the BamHI and EcoRI sites to generate Muc142TR TM21 pPB tet IRES GFP rtTA NeoR and Muc121TR TM21 pPB tet IRES GFP rtTA NeoR plasmids used to make the Mucin-270 and Mucin-135 biopolymer cell lines, respectively.
• the native Muc1 tandem repeats were deleted from the pcDNA3.1 Muc1 TM21 through Q5 site directed mutagenesis with 5’-TGGAGGAGCCTCAGGCATACTTTATTG-3’ ((SEQ ID NO:14) forward) and 5’-CCACCGCCGACCGAGGTGACATCCTG-3’ ((SEQ ID NO:15) reverse) primers.
• the Muc1 gene with 0TR was then cut from the pcDNA3.1 Muc10TR TM21 and cloned into the pPB tet IRES GFP rtTA NeoR plasmid via the BamHI and EcoRI sites.
• the plasmid pLV puro mRuby2 was used for transient transfection experiments with cytoplasmic red fluorescent protein (RFP).
• RFP cytoplasmic red fluorescent protein
• SS-mScarlet-I pPB tet IRES GFP rtTA NeoR plasmid was used.
• the backbone was linearized using BamHI-HF and EcoRI-HF.
• a dsDNA oligo encoding the Muc1 signal sequence (MTPGTQSPFFLLLLLTVLTVVTGS (SEQ ID NO:26)) fused by a linker (four Glycines followed by a Serine) to mScarlet-I was ordered from Integrated DNA Technologies. This fragment was inserted into the linearized backbone via NEB HiFi Assembly.
• FreeStyle 293-F Cells were obtained from Thermo Fisher Scientific. Cells were cultured and maintained according to the manufacturer’s guidelines in an Eppendorf New Brunswick s41i incubator in Erlenmeyer flasks. Cells were maintained between 0.5 x 10 6 and 3 x 10 6 cells/mL at 120 rpm, 37°C, and 8% CO 2 in FreeStyle 293 Expression Medium (Thermo). Transfections were performed using polyethyleneimine (PEI) as previously reported (Durocher et al., 2002).
• PEI polyethyleneimine
• Cells are inoculated at 0.5 x 10 6 cells/mL and grown overnight, 16-18 hr. Biopolymer expression was then induced with 1 mg/mL doxycycline, and cells were grown with doxycycline for an additional 48 hr. After 48 hr, a sample was taken for each cell line, pelleted at 200 rcf for 5 min before the supernatant was separated, and the cell pellet was lysed by resuspending in RIPA lysis buffer (Abcam), vortexing the sample for 30 seconds, and heating to 98°C for 10 min. Lysates were frozen on liquid nitrogen and stored at -80°C.
• Lysates were separated on Nupage 3-8% Tris-Acetate gels (Invitrogen) and transferred to PVDF membranes. Membranes were blocked with 3% BSA TBST for 2 hr. Primary antibodies were diluted 1:1000 and lectins were diluted to 1 mg/mL in 3% BSA TBST and incubated on membranes overnight at 4°C. Secondary antibodies or ExtrAvidin were diluted 1:2000 in 3% BSA TBST and incubated for 2 hr at room temperature. Blots were developed in Clarity ECL (BioRad) substrate and imaged on a ChemiDoc (BioRad) documentation system.
• PCR amplification was performed with Q5 Hot start high-fidelity DNA polymerase (New England Biolabs Inc., Ipswich, MA) using extracted genomic DNA as the template.
• Genomic DNA was extracted with GeneJET genomic DNA purification kit (Thermo
• Cells were inoculated at 0.75 x 10 6 cells/mL and induced with 1 mg/mL doxycycline after overnight growth (16-18 hr). Cells were then grown to a high cell density for an additional 48 or 72 hr in the presence of 1 m/mL doxycycline. Cell density was quantified by collecting sample of the culture, mixing thoroughly to dissociate large clumps, and counting viable cells with a hemocytometer and trypan blue exclusion. For imaging, samples were drawn with wide-bore pipette tips to reduce dissociation of large clumps and diluted in PBS to approximately 6.75 x 10 4 cells/cm 2 for imaging in 2D. Phase contrast images were acquired on an Olympus IX81 microscope with a 10x objective.
• Fiji was used for image processing (Schindelin et al., 2012). Two independent samples were collected and prepared as technical replicates for imaging with three regions of interest imaged per technical replicate. Three biological replicates were performed. Automated image analysis was performed using custom analysis software adapted from a previous publication (Shurer et al., 2017). Briefly, the analysis software located the center of each circular object. The coordinates of each cell’s center were then used to calculate the Ripley’s K function in MATLAB. The percent of single cells was calculated by counting the total number of cells which do not have any neighboring cells within 19 mm and dividing by the total number of cells in the image. Similarly, the percent of cells in various cluster sizes was calculated by binning the cells into clusters based on the number of neighboring cells within 19 mm.
• cultures were inoculated at 0.5 x 10 6 cells/mL and induced with 1 mg/mL doxycycline after overnight growth (16-18 hr). After 48 hr, cells were resuspended at 4 x 10 6 cells/mL. The culture media was then supplemented with 2 mM CaCl 2 , 1:300 anti-clumping agent (Thermo Fisher, 0010057AE), or both. Still images and videos of the cell suspension were acquired after 24 hr of treatment by transferring the culture to a glass test tube. The concentration of cells in suspension was determined by collecting duplicate samples from each culture after allowing the largest aggregates to settle out of suspension for 20 seconds. Cell concentration was measured using a hemocytometer and Trypan blue.
• Cells were inoculated at 0.5 x 10 6 cells/mL, grown overnight (16-18 hr), and induced with 1 mg/mL doxycycline for 48 hr.
• Using a 5 mL syringe with a 16-gauge needle connected to 6.5 in of 1.02 mm silicon tubing cell suspensions were sheared by flowing through a 500 mm constriction (Teflon tubing) at a constant force generated by a 1 kg mass applied to a syringe with gravity. Samples were passed through the constriction five times. Cells were then stained with 1 mg/mL CF640R PNA for 15 min at 4°C.
• Cells were inoculated at 0.5 x 10 6 cells/mL, grown overnight (16-18 hr), and induced with 1 mg/mL doxycycline for 48 hr. Cells were then diluted to 2 x 10 6 cells/mL in fresh medium containing 1 mg/mL doxycycline and transfected with 1 mg DNA per 10 6 cells. The next day (16-18 hr post-transfection), cells were diluted 1:1 with fresh medium containing 1 mg/mL doxycycline. To measure transfection efficiency, cells were transfected with the pLV puro mRuby2 plasmid and transfection efficiency was calculated by flow cytometry as the fraction of cells expressing RFP 72 hr post transfection.
• This Part II demonstrates creation of cDNAs that encode Muc1-like biopolymers with transmembrane domains for anchorage to the cell surface.
• the biopolymer domains consisted of an unstructured protein backbone with 0– 42 perfect repeats of
• PDTRPAPGSTAPPAHGVTSA SEQ ID NO:8
• SEQ ID NO:8 which is recognized by the O-glycosylation machinery of the endoplasmic reticulum and Golgi apparatus and heavily glycosylated while trafficked to the cell surface.
• Each biopolymer was targeted to the extracellular space by the native Muc1 signal sequence.
• the biopolymers were anchored to the cell membrane with a 21-amino acid transmembrane domain (Mercanti et al., 2010; C. R. Shurer et al., 2017).
• the genetic modification of the 293-F cell line was performed non-virally with an “all-in-one plasmid” that contained all necessary elements for selection and tetracycline- inducible expression (Fig.12A).
• the vector included a tetracycline-responsive promoter for expression of the biopolymer coating and an additional cassette for constitutive expression of the reverse tetracycline transactivator (rtTA-M2) and neomycin-resistance gene (Gossen, Bender, Muller, al, & Freundlich, 1995).
• rtTA-M2 reverse tetracycline transactivator
• GFP bicistronic green fluorescent protein reporter was also included for visual confirmation of transcription of the mucin cDNA.
• the cDNA for the biopolymers was stably incorporated into the genome at random locations by transposon mediated integration (X. Li et al., 2013; Wilson, Coates, & George, 2007;
• Mucin-like genes with 0, 21, and 42 tandem repeats were constructed.
• the contour lengths of the polymers with 21 and 42 repeats were predicted to be 135 nm and 270 nm, respectively.
• Mucin-270 Coatings Outperformed Commercially Available Anti-Clumping Agent
• the Mucin-270 biopolymer coating could reduce cell aggregation even in extreme pro-clumping conditions.
• Suspension adapted cell lines have previously been shown to significantly aggregate under specific media conditions, such as high calcium concentrations that are known to promote engagement of cadherins (Dee et al., 1997; Han et al., 2006b; Kim, Tai, Mok, Mosser, & Schuman, 2011; Meissner et al., 2001; Peshwa et al., 1993; Sjaastad & Nelson, 1997; Tolbert et al., 1980; Yamamoto et al., 2000; Zanghi et al., 2000).
• the Mucin-270 biopolymer coated cells When cultured in high calcium conditions (2 mM CaCl 2 ), the Mucin-270 biopolymer coated cells showed qualitatively less aggregation than w.t. cells (Fig.15A). Notably, cultures with Mucin-270 biopolymer coatings retained their turbidity in the pro-clumping conditions, whereas unmodified cells assembled into large clusters easily visible to the naked eye (Fig.15A). Mucin-270-coated cells show a slight decrease in concentration of cells in suspension upon calcium treatment while w.t. cells have essentially no cells remaining in suspension (Fig.15B).
• the Mucin-270 coating outperforms a commercially available anti-clumping agent in highly aggregating conditions. Under high calcium conditions, anti-clumping agent had no discernable efficacy in mitigating cell clumping (Fig.15A). Addition of commercial anti-clumping agent to Mucin-270 coated cells did not further enhance their resistance to clumping in our assays (Fig.15B). Together, these results demonstrated the ability of the presently provided genetically-encoded biopolymer coatings to reduce cell aggregation in suspension.
• Biopolymer Coatings Provided Resistance to Shear Stress
• the sensitivity of suspension-adapted mammalian cells to shear stresses imposes a limit on the rate of mixing and mass transfer in typical bioreactors (Hu, Berdugo, &
• Biopolymer Coated Cell Lines can be Transiently Transfected and Produced
• This Part II demonstrates, among other features, that established cell lines can be genetically modified to express engineered mucin biopolymers for anti-adhesion. Expression of these biopolymers does not negatively impact the desirable characteristics of 293-F cells, including their fast proliferation rates (Fig.12E) and high transfection efficiencies (Fig.15A, B). Moreover, the expression of the biopolymers significantly reduces undesirable cell clumping (Fig.14, Fig.15, Fig.19) and enhances resistance of the cells to shear forces (Fig. 6). Mucin-135 coating and thicker Mucin-270 coatings performed similarly in head-to-head tests and are expected to be equally well-suited for the applications described herein.
• the described biopolymer coatings provide a significant reduction of cell aggregation in serum-free media formulations that are typically used for production in bioreactor formulations. Notably, the coatings could reduce aggregation further even in media formulations that were designed to minimize cell clumping (eg. Invitrogen Freestyle 293-F media).
• the disclosure includes biopolymer expression on cell aggregation in media formulations that have historically been avoided due to issues of cell aggregation. For example, highly efficient transient transfections have long been performed with DNA- calcium phosphate precipitates (Jordan & Wurm, 2004).
• the disclosure includes further improvements of the described mucin coating can be achieved through additional optimization of the engineered mucins and their regulated expression.
• excessive over-production of highly glycosylated mucin-like proteins could possibly compete with recombinant glycoproteins for the cellular glycosylation machinery and the nucleotide sugar building blocks of glycans.
• Shedding of the engineered mucins from the cell surface is mitigated by the described selection of a membrane anchor, which lacks a proteolytic cleavage site.
• the mucin approached described herein can be employed as a solution for suspension-adapted suspension systems that tend to aggregate in the bio-reactor.
• compositions can be expected to help overcome some of the significant barriers to suspension adaptation.
• this Part II presents a mucin coating technology for improved single- cell growth of cells in suspension.
• the system was largely successful in mitigating cell aggregation.
• the MUC1 SEA module is a self-cleaving domain.
• Transient gene expression recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnology and Bioengineering, 75(2), 197–203.
• mammalian cells as aggregates in bioreactors Effect of calcium concentration of spatial distribution of viability. Biotechnology and Bioengineering, 41(2), 179–187.
• MUC1 Episialin
• This Part III provides representative and non-limiting approaches to stable
• mucins are membrane-bound or secreted glycoproteins containing a variable number of tandem repeats that are defined by their densely clustered sites for O-glycosylation (Hang & Bertozzi, 2005). This extensive glycosylation gives rise to a bottlebrush molecular structure that confers mucins with remarkable physical properties (Kuo, Vogel, Zia, & Paszek, 2018). Mucins at biological interfaces can coordinate with water molecules to form hydrated layers that protect delicate cellular or tissue structures, deter biofouling, and resist pathological cellular deposition (Hattrup & Gendler, 2008).
• transmembrane mucins such as Muc1 and Muc16 are densely grafted on the ocular surface, where they maintain hydration, resist abrasion, and provide a selective barrier to macromolecules (Gipson, Spurr-Michaud, Tisdale, & Menon, 2014; Mauris & Argüeso, 2012)
• the secreted mucin-like glycoprotein called proteoglycan 4 (PRG4), or lubricin can bind to cells and tissue interfaces, including the articular cartilage and ocular surfaces, enabling low friction lubrication and protection from pathological cellular deposition and biofouling (Rhee et al., 2005; Schmidt, Sullivan, Knop, & et al., 2013).
• Camptodactyly-Arthropathy-Coxa Vara-Pericarditis (CACP) syndrome including early-onset polyarthropathy as a result of pannus formation and impaired joint lubrication (Bahabri et al., 1998; Marcelino et al., 1999).
• CACP Camptodactyly-Arthropathy-Coxa Vara-Pericarditis
• Decreased synovial fluid lubricin concentrations have also been observed in patients with anterior cruciate ligament injury, osteoarthritis, and rheumatoid arthritis (Elsaid et al., 2008; Kosinska et al., 2015).
• CHO cells prior to the present disclosure, most biologics, including mucins, have been produced in CHO cells due to their fast growth, adaptability to suspension culture, and capacity for glycosylation and other important post-translational modifications.
• CHO cells can generate glycan epitopes that are now suspected to elicit adverse
• cDNA optimization through codon scrambling is an effective strategy to achieve stable recombinant production of mucins and mucin-like glycoproteins, and that this strategy is viable in suspension-adapted human 293-F cells.
• this strategy is viable in suspension-adapted human 293-F cells.
• the synthetic tandem repeats were flanked by additional sequences encoding the native N- and C- termini of human PRG4. These sequences included the native somatomedin and hemopexin domains of lubricin. We also included an IgK leader sequence, 6x histidine tag, and N-terminal SumoStar tag to aid in protein secretion and purification (Fig.20B). We named the new semi-synthetic gene encoded by the codon- optimized cDNA“synonymous lubricin” or“SynLubricin.”
• the nucleotides encoding SynLubricin were significantly less repetitive than native PRG4.
• the detected repeats were aligned with the queried sequence through a Smith-Waterman style local alignment, and the overall repetitiveness was scored by assigning +2 for each nucleotide match and -7 for each mismatch or indel (Benson, 1999). Thus, a higher score was indicative of more nucleotide repetition.
• tandem repeats of SynLubricin had a modest score of 168, whereas the native PRG4 repeats had a much higher repetition score of 1001.
• the present disclosure encompasses such sequences, wherein the overall repetitiveness score of a polynucleotide is compared to a suitable control.
• SynLubricin and the native repeats of human PRG4-A have similar compositions of alanine, glutamic acid, lysine, and threonine, while proline content is slightly higher in the SynLubricin repeats (37% vs 30.5%; Part III Supplemental Table 1).
• the native repeats contain small amounts of asparagine (0.2%), aspartic acid (0.4%), glycine (0.8%), isoleucine (0.2%), leucine (1.4%) and serine (2.6%), which are not contained in SynLubricin (Part III Supplemental Table 1).
• the amino acid sequence of SynLubricin is distinct from that of human PRG4.
• SynLubricin in transiently transfected mammalian cells were successful.
• the SynLubricin cDNA was fused to a bicistronic copGFP reporter and transiently transfected into adherent human embryonic kidney 293-T cells.
• the protein product of the SynLubricin gene was highly glycosylated, as desired, and exhibited the anti- adhesive properties that we predicted.
• Transfected cells maintained large gaps between cells in the monolayer, particularly at locations where visible copGFP fluorescence reported high expression levels of the bicistronic mRNA (Fig.26A). We noted that these observations were consistent with the known anti-adhesive functionality of native lubricin (Rhee et al., 2005).
• a non-viral transposon vector for“all-in- one” inducible expression of mucins contained a tetracycline-responsive promoter for inducible expression of the desired gene and a bicistronic copGFP reporter.
• the vector also contained a second cassette under control of an EF1alpha promoter for expression of the rtTA-M2 tetracycline transactivator and a bicistronic neomycin resistance gene for selection (Fig.20E).
• mucin-type cDNAs We tested whether the described strategy for mucin-type cDNAs was generalizable and could be applied to other mucins.
• mucin Muc1 which is important in the hydration and protection of the cornea and other epithelial surfaces (Mantelli & Argüeso, 2008).
• Muc1 the native tandem repeats of Muc1 are polymorphic, with 42 perfect repeats being most frequent in humans (Nath & Mukherjee, 2014).
• codon optimization strategy was applied to design a cDNA for 42-perfect Muc1 repeats,
• PDTRPAPGSTAPPAHGVTSA SEQ ID NO:8.
• the optimized sequence was fused to the codons for the native N-terminus of human Muc1.
• the optimized coding sequence for SynMuc1 was synthesized through standard CGS services, whereas efforts to synthesize the extremely repetitious sequence of the native Muc1 cDNA were not able to be carried out by commercial vendors.
• the custom synthesized SynMuc1 cDNA was transfected into 293-F cells.
• the recombinant protein was purified from the media supernatant via immobilized metal affinity chromatography (IMAC) and detected by Western blot with an antibody against the native human tandem repeats (Fig.28C).
• IMAC immobilized metal affinity chromatography
• the recombinant mucin was extensively O-glycosylated, as indicated by the strong signal when probed with peanut agglutinin (PNA), a lectin that is specific for a core-1, mucin-type disaccharide (Fig.28D).
• PNA peanut agglutinin
• Fig.28D mucin-type disaccharide
• genomic DNA was extracted from modified 293-F cells after two months of continuous culture.
• the SynLubricin cDNA was then amplified by polymerase chain reaction (PCR) using primers that were specific to SynLubricin (Fig.22).
• the amplified gene was approximately 4 kb in length, as expected for full-length lubricin, and indistinguishable in size from similarly amplified genes obtained using the original SynLubricin plasmid as the template or DNA extracted from transiently transfected cells (Fig.22).
• PCR polymerase chain reaction
• VPA histone deacetylase inhibitor
• SynLubricin is a functional biolubricant
• SynMuc1 we reasoned that the N-terminal histidine-tag could be buried in the large, random coil of the SynLubricin tandem repeats and abandoned the IMAC approach.
• SynLubricin bound to the anion-exchange resin strongly and eluted continuously over high salt concentrations ranging from approximately 350 mM to 1.5 M (Fig.24A, B).
• the continuous elution of SynLubricin was likely explained by a varying frequency of anionic sialic acids in the O-glycans of the recombinant SynLubricin (Estrella et al., 2010).
• a stringent wash step of approximately 500 mM NaCl could remove most protein contaminants detectable by silver stain, although some SynLubricin was inevitably lost to this high-salt wash (Fig.24C, D).
• SynLubricin was purified via anion exchange chromatography using the stringent 500 mM NaCl wash step to eliminate most protein contaminants (Fig.24D). Following purification, SynLubricin was dialyzed in saline and diluted to physiological concentrations. Lubrication was tested on bovine articular cartilage explants where the native lubricin boundary layer had been extracted using a custom linear reciprocating tribometer (Jones et al., 2007). Compared to a saline control, we found that SynLubricin-containing solutions, as well as control synovial fluid, significantly reduced the boundary friction of cartilage explants (Fig.25; p ⁇ 0.001 and 0.0001, respectively).
• SynLubricin can be expected to be suitable for diverse applications ranging from injectables for osteoarthritis to topical treatments for chronic dry eye. Moreover, given the speed and low cost of CGS, the approach described herein can be expected to be applied to rapidly prototype designer mucins with new or modified functional domains.
• mouse anti-human CD227 (555925, BD
• a tetracycline-inducible, transposon based Piggybac expression vector with an integrated, co-expressed reverse tetracycline transactivator gene (pPB tet rtTA NeoR) was used for stable line generation.
• the pPB tet rtTA NeoR plasmid was modified by the insertion of the internal ribosome entry site (IRES) of the encephalomyocarditis virus followed by the fluorescent protein copGFP into the NotI and XbaI sites of the plasmid (pPB tet IRES copGFP rtTA NeoR).
• Synthetic cDNA for a lubricin analog with 78 perfect repeats of KEPAPTTP (SEQ ID NO:1), native N-and C-terminal domains, and an N-terminal SumoStar tag (lifesensors) were generated through custom gene synthesis (General
• cDNA for a soluble, codon-scrambled Muc1 having 42 perfect repeats of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8) and a native human Muc1 N-terminus with SumoStar tag was generated by custom gene synthesis in the pcDNA3 plasmid.
• an mCherry2 cDNA was isolated by EcoRI and NotI digestion of pmCherry2 N1 and cloned into the EcoRI and NotI digested pPB tet IRES copGFP rtTA NeoR vector to create pPB tet mCherry2 IRES copGFP rtTA NeoR.
• FreeStyle 293-F (293-F) cells were obtained from Thermo Fisher Scientific. Cells were cultured and maintained according to the manufacturer’s guidelines in 100-ml Wheaton Celstir glass spinner flasks. Cells were maintained between 0.5 x 10 6 and 3 x 10 6 cells/mL at 120 rpm, 37°C, and 8% CO2 in FreeStyle 293 Expression Medium (Thermo).293-F transfections were performed using polyethyleneimine (PEI) as previously reported
• Stable cell lines were created by co-transfection of the pPB tet IRES copGFP rtTA NeoR plasmids described above with a hyperactive transposase plasmid (Shurer et al., 2018) and subsequently selected with 750 mg/mL of G418 for two weeks.
• Human embryonic kidney cells transformed with the SV40 large T antigen (293-T; ATCC) were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin.293-T cells were transfected through a standard calcium phosphate transfection protocol. Cell proliferation was quantified by cell counting on a hemocytometer with trypan blue exclusion.
• 293-F cells with stable incorporation of PRG4 IRES copGFP were expanded and induced at 1 x 10 6 cells/mL with 1 mg/mL doxycycline for 24 hours.
• the top 5% of copGFP- expressing cells were collected through Fluorescence Activated Cell Sorting (FACS) on a FACSAria Fusion (BD Biosciences). Cells were subsequently expanded in the absence of doxycycline to 1 x 10 6 cells/mL. Cells were induced with 1 mg/mL doxycycline for 24 hours and sorted a second time, collecting the top 10% of copGFP-expressing cells.
• FACS Fluorescence Activated Cell Sorting
• Protein in culture supernatants or purified samples were separated on NuPAGE 3-8% Tris-Acetate gels (Invitrogen) and transferred to PVDF membranes. Membranes were blocked with 3% BSA TBST for 2 hours. Primary antibodies were diluted 1:1000 and lectins were diluted to 1 mg/mL in 3% BSA TBST and incubated on membranes overnight at 4°C. Secondary antibodies or ExtrAvidin were diluted 1:2000 in 3% BSA TBST and incubated for 2 hours at room temperature. Blots were developed in Clarity ECL (BioRad) substrate and imaged on a ChemiDoc (BioRad) documentation system. Fiji was used for image processing (Schindelin et al., 2012).
• a custom sandwich ELISA was used to assess the concentration of SynLubricin, similarly to previous descriptions.
• a 96-well plate (Costar) was incubated overnight at 4°C with 10 mg/mL peanut agglutinin (Sigma) in 50mM sodium bicarbonate buffer, pH 9.5. Plates were blocked with 3% BSA PBS for 1 hour at room temperature. Serial dilutions of FPLC- purified bovine synovial fluid lubricin were used as standards. Samples were loaded at 1:200 dilution in DPBS for 1 hour at room temperature, followed by three washes in PBS + 0.1% Tween20.
• the primary antibody used (Millipore MABT401) binds to the native PRG4 tandem repeats of human and bovine lubricin, which have approximately 90% sequence similarity to the repeats of SynLubricin.
• Primary antibody and secondary antibody (Millipore AP126P) were diluted 1:5000 and 1:2000, respectively, and each incubated for 1 hour at room temperature, with three washes with PBS-T in between antibody incubations and following the secondary antibody incubation.
• the ELISA was developed at room temperature with 1-Step Ultra TMB (ThermoFisher) for 9-12 minutes or until a royal blue color appeared, at which point the reaction was stopped with 2N H2SO4.
• 293-F cells were transiently transfected using the PEI protocol previously described. After 24 hours, the media supernatant was collected. The media supernatant was diluted 1:4 in 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4 and incubated with 100 mL Ni Sepharose excel resin (17371201, GE) overnight at 4°C. Sample flow through was collected using a gravity column (29922, Thermo). The resin was washed with 5 mL 20 mM sodium
• SynMuc1 was eluted with 5 mL of 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4. SynMuc1 was desalted into PBS using a Zeba Spin Desalting Column (87766, Thermo).
• SynLubricin was purified from PRG4 IRES copGFP positive 293-F cell culture supernatant by fast protein liquid chromatography (FPLC) with Q Sepharose® resin (GE). The supernatant was diluted 1:10 with 50mM Tri-HCl buffer, pH 7.5, and loaded onto the column. The column was washed with 50mM Tris-HCl, 525mM NaCl, pH7.5. Purified SynLubricin was collected by eluting with 50mM Tris-HCl, 1M NaCl, pH 7.5. The purified SynLubricin was dialyzed into PBS using a Tube-O-Dialyzer (G-Biosciences) overnight at 4°C. The final purified product was obtained by concentrating with a SpeedVac on the low setting.
• FPLC fast protein liquid chromatography
• GE Q Sepharose® resin
• Valproic acid a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol Bioeng, 101(1), 182–189.
• CACP encoding a secreted proteoglycan
• glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles.
• results presented in this Part IV suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in diverse modes of communication between cells and with the extracellular matrix.
• T-cells of the adaptive immune system generate a high density of tubular microvilli to engage antigen presenting cells, and such structures may be similarly important for the recognition of tumor cells by engineered immune cell therapies (D’Aloia et al., 2018; Jung et al., 2016).
• Membrane projections also enable cell-to-cell communication over long ranges and at precise three- dimensional locations in tissues.
• cytonemes pinpoint delivery of morphogens from‘sender’ cells to specific‘receiver’ cells up to 40-microns away (Bischoff et al., 2013; Kornberg and Roy, 2014).
• Stem cells, immune cells, and many other cell types are also known to bend their plasma membranes into spherical microvesicles that are directly shed and can deliver macromolecular cargoes over long distances (Tricarico et al., 2017).
• curved membrane features are ubiquitous in physical cell behaviors, including migration and mechanotransduction.
• blebs are generated by primordial germ cells, tumor cells, and other cell types for protrusion and frictional coupling with the tissue matrix during migration (Paluch and Raz, 2013).
• Deregulation of membrane-shape generating processes can contribute directly to disease progression.
• aggressive tumor cells frequently extend numerous microvilli for adhesion and rolling in the vasculature (Kramer and Nicolson, 1979; Liu et al., 2018). Aggressive tumor cells can also project blebs for amoeboid migration (Bergert et al., 2015; Friedl and Wolf, 2010). Microvesicles often bud from the plasma membrane of tumor cells at abnormally high rates (Antonyak et al., 2011; Becker et al., 2016). Cargoes carried by these particles are now recognized to have diverse modulatory roles, including reprogramming of other cell types in the stroma and the preparation of distant metastatic niches for colonization (Becker et al., 2016).
• cytoskeletal dynamics Forces originating from cytoskeletal dynamics are posited to generate membrane curvature for the diverse spherical and tubular structures on the cell surface.
• Polymerizing cytoskeletal filaments are envisioned to push out at discrete points along the plasma membrane for extension of microvilli, cilia, filapodia and other finger-like projections (Footer et al., 2007; Gupton and Gertler, 2007; Peskin et al., 1993). Contraction of the cytoskeleton generates the hydrostatic pressure for spherical expansion of the membrane during bleb formation (Charras et al., 2005).
• the physical dynamics that bend sub-regions of the plasma membrane into microvesicles remain poorly understood; however, reports have implicated the actin cytoskeleton in their biogenesis (Tricarico et al., 2017).
• polypeptide and sugar co-polymers called mucins are frequently anchored at high densities on the surfaces of epithelial microvilli (Hattrup and Gendler, 2008; Kesavan et al., 2009; Kesimer et al., 2013), cilia (Button et al., 2012), and filapodia (Bennett et al., 2001); while hyaluronan polymers densely coat the microvilli of oocytes and mesothelium (Evanko et al., 2007; Makabe Sayoko et al., 2006); and long chains of sialic acid and hyaluronan decorate the highly curved surfaces of neuronal axons (Fowke et al., 2017; van
• T-cells and dendritic cells express cell-surface mucins upon activation or maturation, which coincides often with the dramatic changes in membrane tubularization and microvilli generation (Agrawal et al., 1998; Cloosen et al., 2004; Jung et al., 2016; Pilon et al., 2009). Aggressive tumor cells frequently produce an abundance of mucins and hyaluronan on their cell surface (Kufe, 2009; Turley et al., 2016), and the expression of these polymers has been anecdotally linked to their unique membrane features, such as extensive microvilli (Polefka et al., 1984).
• Mucins and hyaluronan polymers are also densely arrayed on the surfaces of enterocytes, reactive astrocytes, dendritic cells, and tumor cells that are known to secrete high levels of microvesicles (Cloosen et al., 2004, 2004; Gangoda et al.; McConnell et al., 2009; Paszek et al., 2014; Pelaseyed et al.; Tricarico et al., 2017). While the ubiquity of these correlations suggests a possible causal relationship between glycocalyx polymer composition and plasma membrane morphologies, a specific mechanism of action has not been delineated. The present disclosure contributes to an understanding of this mechanism of action.
• Mucins and long-chain polysaccharides are anchored to the membrane in such a way that long polymer chains or loops are expected to extend from the cell surface (Hattrup and Gendler, 2008; Lee et al., 1993).
• the ensemble resembles a well-studied structure in polymer physics called a brush, where polymers are grafted on one end to a surface (Chen et al., 2017).
• Polymer brush theory has long recognized that steric interactions in a densely crowded brush restrict the number of molecular configurations each polymer can explore, thereby increasing the free energy of the system through reduced entropy (de Gennes, 1980). Similar to the thermodynamic basis of gas pressure, the entropic penalty associated with molecular crowding can theoretically generate sufficient pressure to deform a flexible surface, like a membrane (Hiergeist and Lipowsky, 1996; Lipowsky, 1995). RESULTS
• Fig.30A and Fig.36A Each construct encoded a mucin polymer domain comprised of an unstructured polypeptide backbone with a high density of serine and threonine sites for O-glycosylation. When expressed in cells, the mucin domains were post- translationally modified with O-linked sugar side chains to form a bottlebrush molecular structure that defines mucins (Fig.35A, B).
• Polymer domains in the library included the 42 native tandem repeats (TR) of Mucin- 1 (Muc1-42TR), the serine and threonine-rich polymer domain of Podocalyxin (Podxl; S/T- Rich), and a new synthetic mucin that we rationally designed and constructed through the tandem fusion of 80 perfect repeats based on a consensus of mucin O-glycosylation sequence, PPASTSAPGA (Rational) (Fig.30A and Fig.35A).
• Each polymer domain was fused to the native Muc1 transmembrane anchor with the cytoplasmic tail deleted ( ⁇ CT), or a 21-amino acid synthetic transmembrane anchor (TM21), or a native mucin anchor with a membrane proximal green fluorescent protein for imaging (GFP- ⁇ CT) (Fig.30A and Fig.35A).
• ⁇ CT cytoplasmic tail deleted
• TM21 21-amino acid synthetic transmembrane anchor
• GFP- ⁇ CT membrane proximal green fluorescent protein for imaging
• each mucin polymer in our library triggered a dramatic tubularization of the plasma membrane, as observed by scanning electron microscopy (SEM) (Fig.30B, C and Fig.35B).
• SEM scanning electron microscopy
• Fig.30B, C and Fig.35B we concluded that this tubularization was likely a general consequence of polymer anchorage to the plasma membrane and did not require a specific biopolymer sequence or transmembrane anchor.
• the Muc1-42TR DCT was identical to native Mucin-1 except for the cytoplasmic tail, indicating that native glycocalyx constituents can influence plasma membrane morphology in addition to our rationally designed polymers. Mucin expression did not have a significant effect on endocytosis, arguing against lipid recycling and the regulation of membrane tension as a primary mechanism for the morphological changes (Fig.35C, D).
• the tubularization phenomenon was relatively insensitive to the length of the mucin polymer domain, provided that the polymers were expressed on the cell surface at moderate to high densities.
• cDNAs for 0, 10, or 42 Muc1 repeats were fused with a GFP- tagged transmembrane anchor to encode cell-surface mucins with expected contour lengths of 0, 65, and 270 nm, respectively (Fig.30D and Fig.35E).
• Cell lines expressing the constructs were sorted into populations with similar mucin surface densities using a nanobody that probed cell-surface GFP (Fig.30D).
• HAS3 hyaluronic acid synthase 3
• Fig.36A-D finger-like membrane extensions
• hyaluronidase HyA
• HyA hyaluronidase
• Molecular surface densities in the sorted populations ranged from 180 to ⁇ 50,000 mucins per mm 2 .
• the glycocalyx polymer model predicted a much different dependence of tubule projection on mucin density.
• the predicted point force required for maintaining an extended tubule decreased progressively with high mucin densities and exhibited no sharp transitions (Fig.32D).
• the frequency of cell-surface tubules observed in our sorted cell populations increased steadily with mucin density throughout the mushroom and brush regimes until the cell was fully saturated with tubes at very high mucin densities (Fig.33B- E).
• theory predicted that at these high densities, the required force for tubule extension is comparable to the polymerization force of a single cytoskeletal filament, ⁇ 1 pN (Footer et al., 2007).
• the polymer model also predicted that the spontaneous curvatures generated by high mucin surface densities exceeded the curvature of finger-like projections that we observed on the cell surface.
• the tubular membrane projections on our cells typically contained a filamentous actin (F-actin) core and did not contain microtubules (Fig.34A, B, Fig.41A-D).
• F-actin filamentous actin
• Fig.34A, B, Fig.41A-D Disruption of F-actin assembly with the drug Latrunculin A (LatA) led to a reduction in tubule diameter by approximately 30 nm (Fig.34C, D and Fig.41E, F), indicating that the mucin-induced spontaneous curvature exceeded the curvature of the stable, actin-filled projections.
• the conditioned media from Muc1-42TR-expressing cells contained massive concentrations of particles ranging in size from approximately 100-nm to 400-nm (Fig.5G), which is characteristic of microvesicles (Pol et al., 2016).
• Particle generation was further enhanced by LatA treatment to disrupt the supporting F-actin cores of surface projections and locally destabilize the plasma membrane (Fig.34H).
• Cryo-transmission electron microscopy confirmed that the secreted particles were indeed membrane vesicles and grafted with a distinct glycocalyx ultrastructure on their surfaces (Fig.34I).
• glycocalyx polymer extension correlates with cell surface density according to the classic scaling laws developed by de Gennes and others for polymer brushes (Gennes, 1979; Zhulina and Borisov, 1996).
• Rho family of GTPases are master regulators of cytoskeletal dynamics and cell-surface morphology (Hall, 1998).
• the description in this Part IV suggests that by modulating the barrier to membrane bending, the glycocalyx primes the membrane for expansion into specific types of spherical or tubular forms that are subject to regulation by Rho GTPases.
• This integrated view suggests that perturbation of normal cell-surface morphology could be achieved through deregulation of intracellular shape generating processes, glycocalyx polymer assembly, or both.
• Rho GTPase signaling For instance, deregulation of Rho GTPase signaling, cytoskeletal dynamics, and glycocalyx assembly are all common hallmarks of cancer cells (Paszek et al., 2014; Pinho and Reis, 2015; Porter et al., 2016; Yamaguchi and Condeelis, 2007) and may each contribute to the unique cell-surface dynamics that contribute to the lethality of metastatic cancer cells.
• Antibodies and reagents The following antibodies were used: FITC-Human CD227 (Muc1) (559774, BD Biosciences), Human CD227 (555925, BD Biosciences) (Muc1), Alexa Flour 488 Human Podocalyxin (222328, R&D Systems), Actin (sc1615, Santa Cruz), GFP (4B10, 2955S, Cell Signaling), 6xHis (9000012, BD Biosciences), Goat anti-Mouse IgG- HRP (sc-2005, Santa Cruz), Mouse anti-Goat IgG-HRP (sc-2354, Santa Cruz).
• Lectins used were: Biotinylated Peanut Agglutinin (PNA; B-1075, Vector Laboratories), CF568 PNA (29061, Biotium), CF640R PNA (29063, Biotium), CF633 Wheat Germ Agglutinin (WGA; 29024, Biotium). Biotinylated lectins were detected using ExtrAvidin-Peroxidase (E2886, Sigma). Hyaluronic acid (HA) was probed in blots with fluorescently labeled or biotinylated bovine nasal hyaluronic acid binding protein (HABP; Millipore).
• PNA Biotinylated Peanut Agglutinin
• CF640R PNA 29063, Biotium
• WGA 29024, Biotium
• Biotinylated lectins were detected using ExtrAvidin-Peroxidase (E2886, Sigma).
• Hyaluronic acid (HA) was probe
• Biotin-HABP was detected with horseradish peroxidase conjugated streptavidin (HRP-streptavidin; R&D Systems).
• HRP-streptavidin horseradish peroxidase conjugated streptavidin
• the DuoSet Hyaluronan kit was from R&D Systems. Actin depolymerization was induced through treatment with Latrunculin A (LatA; 76343-93-6; Cayman Chemicals).
• GUIs giant unilamellar vesicles
• DOPC 1,2-dioleoyl-sn-glycero-3- phosphocholine
• DOPC 1,2-dioleoyl-sn-glycero-3- phosphocholine
• DOGS-NTA-Ni nickel salt
• Bodipy-PC 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-2-phosphocholine
• GFP binding protein came from Chromotek. NHS-esters of Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647 were from Invitrogen. Electron microscopy-grade 16% paraformaldehyde, 10% glutaraldehyde, and 2% OsO4 for scanning electron microscopy (SEM) were obtained from Electron Microscopy Sciences.
• Podocalyxin S/T-Rich ⁇ CT
• pPB TetOn Puro tetracycline-inducible PiggyBac expression vector
• pcDNA3.1 mammalian expression vector
• the cDNA for human HAS3 (accession NP_005320) was obtained from R&D Systems and amplified via PCR with the forward primer, 5’- GGCACCTCGAGGATGCCGGTGCAGCTGACGACA-3’ (SEQ ID NO:88), and reverse primer, 5’-GGCAGAATTCTTACACCTCAGCAAAAGCCAAGCT - 3’ (SEQ ID NO:89).
• the PCR product was cloned into pJET1.2 (ThermoFisher) according to manufacturer’s protocol, and subcloned into the AbsI and EcoRI sites of pLV Hygro TetOn (Paszek et al., 2012).
• GFP cDNA for mOxGFP
• mOxGFP mOxGFP

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