Recombinant lubricin and compositions and methods of use thereof

Abstract

Provided are compositions and methods related to modified lubricin, methods of making the modified lubricin, and methods of using the modified lubricin to coat a variety of inanimate articles and to prevent and / or treat conditions in which enhanced lubrication of one or more parts of a human or non-human mammal is desired.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 792,660, filed January 15, 2019, the entire disclosure of which is incorporated by reference.

[0003] Government Funding

[0004] This application was made with government support under Grant Nos. 1DP2GM119133-01 and 1U54CA210184-01 awarded by the National Institutes of Health. The government has certain rights in the application. TECHNICAL FIELD

[0005] The present disclosure provides improved glycoproteins, as well as compositions and methods related thereto. BACKGROUND

[0006] Lubricin is a glycosylated protein found in several locations in mammalian anatomy. For example, lubricin is present in synovial fluid and on the surface of cartilage. Lubricin plays an important role in lubricating joints and maintaining proper joint environment.

[0007] Previous attempts have been made to provide recombinant forms of lubricin, but there remains a continuing and unmet need for new lubricin and lubricin-like glycoproteins that can be used in a variety of settings. The present disclosure relates to this need. SUMMARY

[0008] The present disclosure provides compositions and methods related to modified glycoproteins. Aspects of the disclosure relate to modified lubricin, pharmaceutical compositions containing modified lubricin, cDNAs and expression vectors encoding modified lubricin, eukaryotic cells expressing modified lubricin, and methods of using modified lubricin and compositions containing the same for a variety of purposes. The methods include using such agents to prevent and / or treat a variety of conditions in which improved lubrication of internal surfaces or fluids in a human or non-human mammal is desired. The disclosure also includes using the compositions to provide lubrication to a variety of inanimate items.

[0009] In certain embodiments, the modified lubricin comprises an alteration in the number of tandem repeats of a particular amino acid sequence relative to its naturally occurring counterpart, and / or one or more alterations in the amino acid sequence of the modified lubricin. In embodiments, the modified lubricin comprises an amino acid sequence derived from human, equine, or canine lubricin, but has different functional properties relative to recombinant forms of such sequences previously provided. In one embodiment, the modified lubricin has an extended half-life of more than 4 days, e.g., intra-articular half-life when injected into a mammal. In embodiments, the modified lubricin exhibits an intra-articular half-life of more than 15 days or at least 30 days. In embodiments, the modified lubricin has a modified glycosylation profile relative to unmodified lubricin.

[0010] In embodiments, the modified lubricin includes a contiguous repeat sequence that is one or a combination of KEPAPTTP (SEQ ID NO: 1), KEPAPTP (SEQ ID NO: 9), and KEPAPTTTP (SEQ ID NO: 10). In embodiments, the repeat sequence is repeated 10-120 times in succession. In one non-limiting embodiment, the repeat sequence is repeated 59 times.

[0011] In embodiments, the modified lubricin comprises an amino acid sequence of a derivative of lubricin produced by a human or non-human mammal. In embodiments, the contiguous repeat sequence is flanked on its N and C terminal segments by a lubricin amino acid sequence having at least 90% identity to a human, equine, or canine lubricin sequence.

[0012] In embodiments, the modified lubricin includes an additional component, e.g., an added secretion signal from a human or non-human mammal or other suitable source. BRIEF DESCRIPTION OF DRAWINGS

[0013] As described below, the figures and tables of the present disclosure are divided into four sections (Section I, Section II, Section III, and Section IV).

[0014] Section I Figures

[0015] Figure 1Combinatorial genetically encoded library of sequence-specific mucins. (a) Schematic of sequence-specific mucin combinatorial library. (b) Schematic showing the exchangeable bio-bricks and flanking restriction sites for the construction of full mucin constructs. (c) Workflow of design and manufacture of mucin tandem repeat backbone cDNA. (d) Overview of codon-scrambled mucin backbone in the library. Wild-type Muc1 sequence is SEQ ID NO: 8. Muc1 single mutant (Muc1_S) is SEQ ID NO: 5. Muc1 double mutant (Muc1_D) is SEQ ID NO: 6. Muc1 triple mutant (Muc1_T) is SEQ ID NO: 7. Synthetin 1 (Syn1) is DAATPAP, which is SEQ ID NO: 2. Synthetin 2 (Syn2) is SEQ ID NO: 3. Synthetin 3 (Syn3) is SEQ ID NO: 4. Lubricin consensus sequence (Syn4) is SEQ ID NO: 1.

[0016] Figure 2 Construction and validation of sequence-specific mucin expression. (a) Components and features of codon-optimized Muc1 variants with GFP reporter. Amino acid sequence in (a) is SEQ ID NO: 8. (b) Predicted molecular weight of polypeptide backbone. (c) Biosynthesis of Tn antigen, Core 1 and Core 2 glycans, and specificity of relevant lectins for their detection. (d) Western blot analysis of native Muc1 expression and glycosylation in wild-type and Core-1 β3-T specific molecular chaperone (COSMC) knock-out MCF10A cells. MCF10A cells were stably transfected with native Muc1. Surface sialic acids were labeled with AFDye 568 by periodate labeling, followed by collection of lysates. Blots were stained with MUC1 TR (CD227 HPMV) Ab-FITC and PNA-CF640 or biotinylated VVA (secondary: NeutrAvidin-Dylight 650) in multiple colors. (e) Western blot analysis of native and codon-scrambled Muc1 in extracts of transiently transfected HEK293T cells. (f) Immunofluorescence images of transiently transfected HEK293T cells expressing the indicated constructs and probed with PNA lectin (left), anti-Muc1 antibody (middle left), GFP (middle right) and Hoescht nuclear stain (right) (scale bar 10 μm). (g) PNA lectin blot analysis (left) and intensity distribution (right) of different sizes of mucins in extracts of transiently transfected HEK293T cells.

[0017] Figure 3Frequency modulation of O-glycan maturation in engineered Muc1 polymer backbones. (a) Components and features of secreted Muc1 and engineered variants each having 21 tandem repeats. (b) Molecular weight of the tandem repeat sequence and polypeptide backbone of secreted mucin mutants. Single, double, and triple glycosylation mutants (sMuc1S, sMuc1D, and sMuc1T) have one, two, or three serine / threonine (S / T) to alanine substitutions per repeat, respectively. Sequences of sMuc1 mutants (21 repeats) from top to bottom are: SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. (c) FreeStyle 293-F cell culture medium affinity-purified recombinant secreted mucins probed with anti-SUMOstar antibody and PNA, s-WGA, and VVA lectins. Lectin blots were co-stained with PNA-Alexa Fluor 568, s-WGA-FITC, and biotinylated VVA (secondary: neutravidin-Dylight 650) in multiple colors. (d) Representative fluorescence intensity electropherograms of blots in (c). (e) Ratio intensity analysis of PNA to VVA signal (top) and s-WGA to VVA signal (bottom) for the indicated mucins and the corresponding frequency of S / T glycosylation sites in their polymer backbones. Ratio fluorescence intensity was quantified along each lane and normalized to the signal from secreted mucin with wild-type Muc1 tandem repeats (sMuc1); data presented as mean and SEM from at least three independent experiments. *P < 0.05 **P < 0.01 ***P < 0.001 (f) Left panel: MALDI-TOF mass spectra recorded from permethylated glycan alditols of secreted mucins with wild-type Muc1 tandem repeats (sMuc1) and triple mutant (sMuc1T) from HEK293T cell culture medium. Ion signals are annotated according to the relative mass (m / z) of the detected molecular ions as sodium adducts and by assigning the corresponding core structures (core 1 in red, core 2 in black). Right panel: Schematic representation of O-linked glycans detected on secreted mucins. TM Representative Western blot analysis of affinity-purified recombinant secreted mucins from FreeStyle 293-F cell culture medium (three independent experiments). Lectin blots were co-stained with PNA-Alexa Fluor 568, s-WGA-FITC, and biotinylated VVA (secondary: neutravidin-Dylight 650) in multiple colors. (d) Representative fluorescence intensity electropherograms of blots in (c). (e) Ratio intensity analysis of PNA to VVA signal (top) and s-WGA to VVA signal (bottom) for the indicated mucins and the corresponding frequency of S / T glycosylation sites in their polymer backbones. Ratio fluorescence intensity was quantified along each lane and normalized to the signal from secreted mucin with wild-type Muc1 tandem repeats (sMuc1); data presented as mean and SEM from at least three independent experiments. *P < 0.05 **P < 0.01 ***P < 0.001 (f) Left panel: MALDI-TOF mass spectra recorded from permethylated glycan alditols of secreted mucins with wild-type Muc1 tandem repeats (sMuc1) and triple mutant (sMuc1T) from HEK293T cell culture medium. Ion signals are annotated according to the relative mass (m / z) of the detected molecular ions as sodium adducts and by assigning the corresponding core structures (core 1 in red, core 2 in black). Right panel: Schematic representation of O-linked glycans detected on secreted mucins.

[0018] Figure 4 Designer mucin domains reveal sequence-specific effects on glycosylation. Figure 4The sequences shown are KEPAPTTP (SEQ ID NO: 1), DAATPAP (SEQ ID NO: 2), DAATPAPP (SEQ ID NO: 3), and PASTSAPG (SEQ ID NO: 4). (a) Components and features of designer mucins. (b) Predicted molecular weights of mucin polypeptide scaffolds. (c) Representative Western blot analysis of extracts from transiently transfected HEK293T cells probed with anti-GFP antibody or co-stained with PNA and VVA lectins for the indicated constructs (from three independent experiments). (d) Representative fluorescence intensity electropherograms of the Western blots in (c) for the indicated constructs from three independent experiments. Dotted lines indicate peaks of the glycoforms visible in the PNA blots. Shaded boxes indicate the region between the bands with the highest and second-highest apparent molecular weight on the anti-GFP blots. (e) Ratio intensity analysis of PNA to VVA staining for the indicated mucins and the corresponding frequency of serine and threonine glycosylation sites in their polymer scaffolds. Fluorescence intensity was quantified along each lane of the double-probed lectin blots and normalized to the PNA:VVA ratio of KEPAPTTP (SEQ ID NO: 1) x 20 mucin; data presented as mean and SEM from three independent experiments. (f) Fold change in PNA:VVA ratio upon doubling the size of the indicated mucin scaffolds from 40 to 80 tandem repeats; data presented as mean and SEM from three independent experiments. *p<0.05.

[0019] Figure 5 : Engineering mucin glycosylation through cytoplasmic tail regions. (a) Components and features of cell surface mucins with a synthetic 21-amino acid transmembrane anchor (TM21) and engineered cytoplasmic motifs; native CT refers to the native cytoplasmic tail adapted from Muc1. (b) Lectin blot analysis of the indicated mucin isoforms from transiently transfected HEK293T cells to detect sialylated O-glycans by periodate oxidation and core-I structures by PNA; blots are representative of three independent experiments. (c) PNA-lectin blot analysis of the indicated mucin isoforms before and after sialidase treatment; blots are representative of three independent experiments. (d) Top: Representative MAA and PNA lectin blot analysis of immunoprecipitated of the indicated mucin isoforms from transiently transfected HEK293T cells (from four independent experiments). Bottom: Ratio intensity of sialic acid to core 1 glycan signal (MAA:PNA); data presented as mean and SEM from four independent experiments. *P<0.05.

[0020] Figure 6: Western blot analysis of MCF10A cells edited with lentivirus having native repeats (native_Muc1) and codon scrambled Muc1 cDNA (Muc1_42).

[0021] Figure 7 : Mucins having tunable size. Figure 7 The sequence shown in (a) is PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8). (b) Representative immunofluorescence images of transiently transfected HEK293T cells expressing the GFP-tagged Muc1 constructs shown in (a) and co-stained with PNA, anti-Muc1 antibody, and Hoechst nuclear stain (scale bar 10 μιη) from three independent experiments. (c) Components and features of a Mucin construct having a synthetic 21 amino acid transmembrane anchor (TM21) and codon scrambled Muc1 repeats. (d) Predicted molecular weights of the Mucin polypeptide backbone of the constructs shown in (c). (e) Representative Western blot analysis of the TM21 constructs shown in (c) from extracts of transiently transfected HEK293T cells and probed with PNA lectin or anti-Muc1 antibody (three independent experiments). (f) Representative phase contrast images of HEK293T expressing the indicated constructs in (c) from three independent experiments (scale bar 100 μιη).

[0022] Figure 8 : Western blot image of affinity purified recombinant secreted Mucin probed with anti-6xHis antibody and VVA lectin from FreeStyle TM 293-F cell culture media.

[0023] Figure 9 : Cell surface Mucin mutants derived from Muc1 tandem repeat sequences. Figure 9The sequences shown are PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8), PDTRPAPGATAPPAHGVTSA (SEQ ID NO: 5), PDTRPAPGATAPPAHGVTAA (SEQ ID NO: 6), and PDARPAPGATAPPAHGVTAA (SEQ ID NO: 7) from top to bottom. (a) Components and characteristics of mucins constructed with 21 native or engineered Muc1 repeats, a GFP reporter, and a native Muc1 transmembrane anchor. (b) Tandem repeats of native Muc1 (mMuc1) or engineered variants with single, double, or triple serine / threonine to alanine substitutions (mMuc1S, mMuc1D, or mMuc1T) and predicted backbone molecular weights. (c) Representative protein and lectin blot analyses of the indicated constructs in (a) from extracts of transiently transfected HEK293T cells and probed with an anti-GFP antibody or co-stained with PNA, VVA, and s-WGA lectins from three independent experiments. (d) Components and characteristics of mucins constructed with 21 native or engineered Muc1 repeats and a synthetic 21 amino acid transmembrane anchor (TM21). (e) Representative immunofluorescence images of transiently transfected HEK293T cells expressing the indicated constructs in (d) and co-stained with PNA lectin and Hoechst nuclear stain (scale bar 10 pm) from three independent experiments.

[0024] Figure 10 : MALDI-TOF MS spectra of mucin-type O-glycans as reported by the Cellular O-Glycome Reporter / Amplification (CORA) reporter. HEK293T cells were transiently transfected with the indicated synthetic mucin constructs or mock vehicle. Spectra were normalized to the matrix peak at m / z = 550.

[0025] Figure 11 : Mucins constructed with designer tandem repeats. Figure 11 The sequences shown are DAATPAP (SEQ ID NO: 2), DAATPAPP (SEQ ID NO: 3), and PPASTSAPG (SEQ ID NO: 4). (a) Components and characteristics of mucin constructs with designer tandem repeats, a GFP reporter, and a native Muc1 transmembrane anchor. (e) Representative immunofluorescence images of transiently transfected HEK293T cells expressing the indicated GFP-tagged constructs and co-stained with PNA lectin and Hoescht nuclear stain (scale bar 10 pm) from three independent experiments.

[0026] Figure 2

[0027] Figure 12 : Engineering of a biopolymer-coated cell line. A transposon-based approach was used to stably integrate DNA encoding an engineered biopolymer under a doxycycline inducible promoter. A, schematic representation of the all-in-one vector used to generate the biopolymer-coated cell line, showing key elements. To incorporate into the cell genome, the vector includes a tetracycline-responsive element (tetO), a minimal CMV promoter, a Muc1 signal sequence (Muc1 N-term), a tandem repeat of the biopolymer (0, 21, or 42 repeats of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8)), a transmembrane domain of Muc1 (Muc1 TM), a bicistronic green fluorescent protein reporter (IRES GFP), an EF-1a promoter, a reverse tetracycline transactivator (rtTA), and a second bicistronic neomycin resistance cassette (IRES NeoR). These elements are all flanked by the 5' and 3' inverted terminal repeat sequences (ITRs) required for transposon-mediated genomic incorporation. For vector replication and production in bacteria, there is also an ampicillin resistance cassette (AmpR) and an origin of replication (ori). B, schematic representation of the membrane-bound biopolymer expressed by the cell and localized to the cell surface. C, schematic representation of the relative size of the engineered biopolymer extracellular domain, named Mucin-0, Mucin-135, and Mucin-270, respectively, according to length in nm. The predicted molecular weights of these proteins are 42 kDa, 81 kDa, and 120 kDa, respectively.

[0028] Figure 13 : Validation of biopolymer coating. Expression and cell surface localization of biopolymer coating was validated for the new engineered 293-F cell line. A, representative confocal microscopy images of stably suspension-adapted human embryonic kidney 293 (293-F) cell lines - wild-type (w.t.) or stably expressing Mucin-0, Mucin-135, or Mucin-270 biopolymer. Images show cell membranes (shown in blue, CF633 wheat germ agglutinin, WGA), O-glycans covalently linked to Mucin-135 and Mucin-270 biopolymer (shown in red, CF568 peanut agglutinin, PNA), and green fluorescent protein co-expressed with Mucin-0, Mucin-135, and Mucin-270 biopolymer on a plasmid (shown in green, GFP). B, representative flow cytometry histograms showing the polydisperse population of the biopolymer-expressing cell line compared to w.t. cells, y-axis scaled to show population distribution of GFP-positive cells. Each histogram > 50,000 cells. C, quantification of the percentage of GFP-positive cells for each cell line. Figure 2GFP signal above the gray line in B was considered GFP positive. Mean and S.D. shown, >50,000 cells per sample, n=4. D, Representative immunoblots (left) and lectin blots (right) of whole cell lysates of each generated stable cell line compared to w.t. cells, n=3. E, Concentration of viable cells determined by trypan blue exclusion counted with a hemocytometer, n=3. F, GFP signal of Mucin-270 cells after induction of expression at t=0 hour measured by flow cytometry, n=3, >15,000 cells per sample. G, Agarose gel showing polymerase chain reaction (PCR) products of the Mucin-270 gene from DNA extracted from cells that were never transfected (mock), transiently transfected w.t. cells (transient), or cells in which the Mucin-270 gene was incorporated into the genome and cultured for 2 months (2 mo.) or 12 days (12 d) after gentamycin selection. Asterisk indicates the predicted molecular weight of the Mucin-270 PCR product. #1 and #2 are biological replicates. Mean and S.D. shown, ns - not significant.

[0029] Figure 14 Genetically encoded biopolymer coatings reduce 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 of w.t. and biopolymer cell lines. Images are of cells grown at a concentration of 3.8 ± 0.7 x 10 6 Figure 3 A. Quantification of the fraction of cells in various cluster sizes in the phase-contrast images shown in, e.g. Figure 3 A. Quantification of the fraction of cells in various cluster sizes in the phase-contrast images shown in, e.g. Figure 3 A. Ripleys K function calculated for the cell distribution acquired from the phase-contrast images shown in, e.g. Figure 3 Repeats described in B. ns - not significant; *p < 0.05; **p < 0.01; ***p < 0.005.

[0030] Figure 15 ​Mucin-270 reduces aggregation in high calcium media. Mucin-270 cell lines outperform commercial anti-clumping solutions in highly aggregated conditions. A, images of Mucin-270 and w.t. cultures grown in media containing 2 mM CaCl2(+Ca 2+ ). Mucin-270 expression significantly reduces cell aggregation even compared to a commercial anti-clumping reagent (+Anti-clumping agent). B, quantification of concentration of w.t. or Mucin-270 expressing cells in suspension for control cultures under the following conditions, no treatment (empty), addition of commercial anti-clumping reagent (+Anti-clumping agent), addition of 2 mM CaCl2(+Ca 2+ ) or both with anti-clumping reagent and 2 mM CaCl2(+Anti-clumping agent + Ca 2+ ). Statistical comparisons are to the empty condition for each cell line. Mean and S.D. are shown, n = 3. ns - not significant; * p < 0.05; ** p < 0.01; *** p < 0.005.

[0031] Figure 16 : Biopolymer coating enhances resistance to shear stress. Expression of stably incorporated biopolymers protects cells from shear stress. A, schematic representation of experimental set-up for shearing cells. Simply put, cells are sheared by flowing through a 500 pm Teflon tube under constant application of force by a 1 kg weight, followed by analysis of cells by flow cytometry with a live / dead cell stain. B, quantification of dead cell fraction after shearing cells for w.t. and biopolymer cell lines, mean and S.E.M. are shown, > 50,000 cells were measured per population, n = 6. ns - not significant; * p < 0.05; ** p < 0.01; *** p < 0.005.

[0032] Figure 17 : Biopolymer coated cells can be transfected. Transfection of biopolymer coated cell lines was determined by transfection with cytoplasmic red fluorescent protein (RFP). A, quantification of cell number for w.t. and biopolymer coated cells transiently transfected with cytoplasmic RFP. The count of transfected cells was normalised to the count of transfected w.t. cells per experiment to account for variable transfection efficiency between repeated transfections. > 50,000 cells were measured per population, n = 3. B, representative flow cytometry histograms showing expression distribution in the transfected cell population. The peak to the left of the grey line centred around zero represents the untransfected population of each cell line, which is further validated by the overlay histogram of untransfected w.t. cells (w.t. - empty). C, quantification of geometric mean of RFP for positively transfected cells from B. Mean and S.D. are shown, ns - not significant; * p < 0.05; ** p < 0.01; *** p < 0.005.

[0033] Figure 18 Quantification of secreted recombinant RFP from media supernatant of w.t. or mucin-270 expressing cultures transiently transfected with secreted RFP, n=3. Mean and S.D. shown, ns - not significant; *p<0.05; **p<0.01; ***p<0.005.

[0034] Figure 19 Additional data acquired 24 hours prior to Figure 14 A. A, Quantification of cell fraction in various cluster sizes of phase contrast images shown in Figure 3 A. For all plots, cells were grown for 48 hours at 3.2 ± 0.7 x 10 6 cells / mL. Center line shows the median; box limits indicate the 25th and 75th Figure 3 A. For all plots, cells were grown for 48 hours at 3.2 ± 0.7 x 10 6 cells / mL. Center line shows the median; box limits indicate the 25th and 75th Figure 3 A. Ripley’s K function with distance computed for cell distribution acquired for phase contrast images shown in Figure 3 B. Repeats described in B, n=3. ns - not significant; *p<0.05; **p<0.01; ***p<0.005.

[0035] Figure III

[0036] Figure 20: Design and synthesis of a synthetic lubricin (SynLubricin). A) Overview of the design and production strategy for the synthetic, codon- scrambled mucin. The DNA sequence of the desired protein product is optimized by global optimization to minimize repetitive DNA sequences by codon scrambling, followed by a second optimization that redistributes codons that are not commonly used in the host cell system. B) SynLubricin was constructed from 59 perfect repeats of KEPAPTTP (SEQ ID NO: 1) flanked by the N and C-termini of native human PRG4. An IgK signal sequence and SumoStar tag were fused to SynLubricin for secretion and purification. SynLubricin also retains two somatomedin B domains (SMB 1 and 2) and two heme-binding protein domains of the native protein. C) Repeat score calculated values for nucleotides encoding tandem repeats of human PRG4 isoform A (PRG4A) and SynLubricin. D) Alignment of the amino acid sequences of human PRG4 and SynLubricin. 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 showing the tetracycline inducible promoter, multiple cloning site (MCS) for cDNA of interest, bicistronic GFP reporter (IRES2 CopGFP), and second expression cassette for rtTA-M2 tetracycline transactivator and neomycin resistance gene.

[0037] Figure 21 : Sorting strategy to isolate stable polyclonal cell populations that produce high levels of SynLubricin. A) Strategy to isolate stable cell populations that express high levels of SynLubricin. B) Western blots of 293-F culture media supernatants showing relative SynLubricin production in unsorted and twice sorted (2x) cell populations; 1 and 2 indicate samples from two independent experiments; probed with anti-PRG4 (MABT401) and SUMO antibodies. C) Quantification of the relative signal intensities of the anti-PRG4 western blots in B. D) Phase contrast and fluorescent micrographs of unsorted and twice sorted 293-F cells expressing SynLubricin.

[0038] Figure 22Stable integration of SynLubricin cDNA in the cell genome. PCR amplification of the SynLubricin coding region in genomic DNA extracts of wild-type and stably integrated 293-F cells cultured for 2 months in succession. As positive controls, PCR amplification of SynLubricin plasmid and DNA extracts from SynLubricin transiently transfected 293-F cells (transient) are shown. The expected size of full-length SynLubricin is indicated by an asterisk.

[0039] Figure 23 Optimization of SynLubricin production. A) Western blot showing the relative production of SynLubricin in the medium of control cells and sorted 293-F cells induced with 1 pg / mL doxycycline for the indicated days in the absence or presence of the histone deacetylase inhibitor valproic acid (VPA; 3.5 mM) over time. B) Quantification of the signal relative intensity of the blots shown in A. C) Time course of glucose consumption in sorted 293-F cells induced with 1 pg / mL doxycycline at day 0 with or without 3.5 mM VPA. Mean and S.D. are shown, n = 3. D) Western blot showing lubricin in the medium collected from non-producing control cells (mock), cells transiently transfected with SynLubricin cDNA (transient), and two consecutive 1-L batch cultures of sorted 293-F cells (batch 1 and batch 2) induced with 1 pg / mL doxycycline and 3.5 mM VPA for three days; equine seminal fluid (ESF) was loaded as a control. E) Representative western blot of SynLubricin produced by stably expressing 293-F cells collected at the indicated time points after induction with 1 pg / mL doxycycline at day 0. F) Quantification of the western blot replicates represented in B, n = 3, ns - not significant.

[0040] Figure 24 Purification of SynLubricin by anion exchange chromatography. A) Silver staining and B) Western blot showing SynLubricin eluted from Q Sepharose®resin in a wide range of NaCl concentrations (concentrations are indicated in mM above the lanes). C) Silver staining and D) Western blot showing SynLubricin eluted from Q Sepharose®resin in a narrow range of NaCl concentrations (concentrations are indicated in mM above the lanes). The expected size of full-length SynLubricin is indicated by an asterisk. SynLubricin eluted from Q Sepharose®resin in a narrow range of NaCl concentrations (concentrations are indicated in mM above the lanes). The expected size of full-length SynLubricin is indicated by an asterisk.

[0041] Figure 25 Lubricin's function. Coefficient of friction of cartilage explants extracted in NaCl soaked in physiological saline (PBS), bovine synovial fluid or SynLubricin. SynLubricin was purified with DEAE Sepharose before the lubrication assay, eluted without washing or after a stringent 500 mM NaCl wash. Mean and S.D. are shown, indicating independent measurements. *** p < 0.001, **** p < 0.0001; NA: statistical test not applicable due to sample size.

[0042] Figure 26 : Transient expression of SynLubricin alters adherent cell morphology. A) Morphology of 293-T cells mock transfected or transfected with the cDNA of bi-cistronic SynLubricin IRES copGFP. Shown images are merged overlays of phase contrast and fluorescent micrographs. Note the inhibition of cell-cell adhesion in the vicinity of cells expressing high levels of the copGFP reporter. B) Western blot of equine synovial fluid (ESF) and culture supernatant from mock transfected and SynLubricin transfected cells probed with MAB T401 antibody against PRG4 tandem repeats.

[0043] Figure 27 : Validation of new transposon-based gene delivery vectors. Flow cytometry results showing correlation of levels of mCherry2 and copGFP reporter.

[0044] Figure 28 : Figure 28 The sequence shown above is PDTRPAPGSTAPPAHGVTSA (unmodified Muc1 repeat) (SEQ ID NO: 8). Application of codon- scrambling strategy to Muc1. A) Schematic of SynMuc1 with codon- scrambled tandem repeats. B) Repeat score calculated values of nucleotides encoding human Muc1 and SynMuc1 tandem repeats. C) Western blot of culture supernatant of 293-F cells transfected with SynMuc1 cDNA (+ cDNA) or untransfected cells (M), His affinity purified Ni-NTA resin by flow through (FT) and eluted protein (eluate) probed with Muc1 antibody. D) PNA-lectin blot of C. E) Western blot of C probed with SUMO antibody.

[0045] Figure 29 : SynLubricin has low affinity for immobilized metal affinity chromatography (IMAC) resin. A) Culture supernatant and Fe 3+ and Ni 2+IMAC purification with nitrotriacetic acid (NTA) resin loaded was probed by flow through, wash, and elution Western blots. Elution was performed at the indicated NaCl concentrations. No non-specific binding of sialic acid to multivalent Fe 3+ B) Western blots from flow through, wash, and elution fractions from non-NTA resin loaded.

[0046] Figure 30 : Images showing SynLubricin retention in vivo. Images depict SynLubricin localized at day 0 (injection), 2 weeks, and 7 weeks. Graph shows half-life for two rats over a period of about 40 days. For the graph, clearance kinetics of human SynLubricin injected into the left knee of adult male SD rats (n=2). Purified human SynLubricin was fluorescently labeled with sulfo-Cy7.5 near-infrared fluorescent dye, and 20 μL SynLubricin-Cy7.5 was injected into the healthy left knee via the patellar tendon approach. Total Lubricin fluorescence from the left knee was imaged, quantified on an IVIS Spectrum whole animal imaging system, and reported as total radiant efficiency. Data were fitted to a bi-exponential decay model to calculate alpha and beta decay constants. Half-life was reported as ln(2) divided by the beta decay constant.

[0047] Figure 31 : MALDI-MS spectra and graph and chart showing calculated relative percentages of O-glycans released from human SynLubricin.

[0048] Figure 32 : Coefficient of friction of NaCl extracted cartilage explants soaked in physiological saline (PBS) or SynLubricin at the indicated concentrations. For these experiments, SynLubricin was purified from 293-F culture media supernatant by cation exchange chromatography.

[0049] Figure IV Part IV

[0050] Figure 33: Glycocalyx polymers induce membrane tubulation. (A) Schematic and table showing genetically encoded biopolymers constructed and used throughout this work. Gene libraries encode natural and synthetic mucin polymers comprising a central polypeptide core, sugar side chains attached to serine (S) and threonine (T) residues, and a transmembrane anchor. (B) Quantification of membrane tubule density in epithelial cells showing that mucin polymers induce dramatic tubulation 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. (C) Scanning electron microscopy (SEM) images showing membrane morphology of cells expressing the indicated biopolymers. (D) (Left) Schematic of Muc1 GFP-ΔCT polymers of different lengths as indicated by the number of tandem repeats (TR). (Right) Flow cytometry data showing similar cell surface expression levels of the indicated mucins using GFP-binding nanobodies, n = 3, >40,000 cells per population. (E) Representative SEM images of cells described in (D). (F) (Left) Quantification of relative protein surface density on giant unilamellar vesicles (GUVs) with low density of membrane-anchored podocalyxin (Podxl), low density human serum albumin (HSA) (low HSA), or high density HSA (high HSA), n = 10-20. All GUVs were formulated with 10 mol% Ni-NTA-lipid for protein anchoring. (Middle) Quantification of 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).

[0051] Figure 34 : Membrane morphology of tissue synoviocytes is regulated by the glycocalyx. (A) Experimental workflow of excised horse synovial tissue. (B) Representative SEM images of primary synoviocytes expressing hyaluronan synthase 3 (HAS3) showing retraction of membrane tubules after 30 minutes of hyaluronidase (HyA) treatment to digest hyaluronic acid (HA). (C) Quantification showing that tubule density depends on the presence of HA. (D) Images of freshly excised synovial tissue showing nuclei (DAPI), surface-anchored HA (hyaluronan binding protein, HABP), and tissue collagen (second harmonic generation, SHG) of representative synoviocytes. Depth along the z-axis is color-coded according to the color bar. Note the HA-rich membrane extensions protruding from the synovial tissue surface. The lower right panel shows a schematic representation of the observed tissue synoviocytes. (E) Membrane tubules on synoviocytes are visible by SEM in freshly excised horse synovial tissue. Synoviocyte heads are pseudocolored in orange, protruding from the synovial tissue. HyA treatment to digest HA causes rapid retraction of synoviocyte tubules (right). ***p < 0.001 (post-hoc Student’s two-tailed t-test).

[0052] Figure 35 Polymer brush model of glycocalyx and generation of preferred membrane shapes. (A) Polymer model of membrane bending, showing simulated spontaneous membrane curvature induced by cellular glycocalyx. Low-density polymers are non-interacting and take on a tight structure in the "mushroom" system. In the "brush" system, polymers overlap (the average distance D between polymers is less than the radius of gyration R). G (A) Twice the length of the polymer brush and extend to avoid each other, increasing the height (H) of the polymer brush. Entropy pressure is the basis for the membrane curvature generated by the polymer mushroom and the brush. (B) Muc1 construct with SUMO and GFP tags attached to the polymer domains for visualization of polymer extension by dilatational microscopy (ExM). Polymer extension versus polymer fluorescence intensity is a proportional measure of surface density, showing the specified scaling relationship. Dots, squares, and triangles indicate measurements from three samples. The red line shows a linear regression through all data points. (C) Theoretical prediction of spontaneous curvature generated by the Muc1 polymer mushroom and polymer brush. Blue: Expected mushroom system (mushroom); Pink: Expected brush system (brush). The computational model here considers a 270 nm long mucin with a monomeric segment of 15 nm length (Kuhn length). These parameters are based on experimental characterization of the natural Muc1-42TR and were selected for comparison with the following experiments. (D) (Left) Theoretical prediction of the required pressure (Pa) as a function of mucin concentration for a bubble with radius = 250 nm. The illustration shows the minimum pressure near the mushroom-brush transition. (Right) Theoretical prediction of the point force (pN) required as a function of mucin concentration to maintain the membrane tubules.

[0053] Figure 36 Preferred membrane shape depends on the concentration of cell surface biopolymers. (A) A strategy using fluorescence-activated cell sorting (FACS) to sort cells into populations with different levels of cell surface mucin (Muc1-42TR-GFPΔCT). (B) Representative SEM images showing the membrane morphology transitions of sorted cell populations with specified mucin surface densities. Mucin density is selected to match specified points on the theoretical map ( Figure 3 (D) Mean radius of vesicle structures measured in the mushroom system and tube structures measured in the brush system. (D) Vesicle density observed on sorted cell populations with a specified mean mucin surface density. Significance is determined between the mushroom system and the brush system (*) or between the lowest brush system density and all other brush mucin densities (+). (E) Tube density observed on sorted cell populations with a specified mean mucin surface density. Symbols are as defined in (D). (F) From ( Figure 3D, the inverse of the predicted force, exhibited a linear relationship with the observed tube density from (E) with a Pearson correlation coefficient of 0.97. The number of measurements is shown on the x-axis of the box plot. Error bars indicate 95% confidence intervals. ns - not significant; * / + p < 0.05; ** / ++ p < 0.01; *** / +++ p < 0.001 (post-hoc Student’s two-tailed t-test).

[0054] Figure 37 : Glycocalyx-mediated membrane instability and extracellular vesicle biogenesis. (A) Representative confocal microscopy images of epithelial cells expressing Muc1-42TRACr, mucin stained with PNA (Wheat Germ Agglutinin staining), actin stained with phalloidin, n = 3. (B) Fluorescence intensity line traces from (A) (PNA image, red line). Values were normalized to the maximum intensity of phalloidin and PNA staining, respectively. (C) Average diameter of tubules in Muc1-42TRACr-expressing cells after treatment with DMSO (vehicle) or with 10 mM Latrunculin-A (+LatA) to interfere with actin assembly. (D) Representative SEM images of tubules in Muc1-42TRACr-expressing cells treated with vehicle or LatA. (E) (Left) Schematic sketch of a simulation model, where the actin core resists spontaneous membrane curvature driven by the glycocalyx brush. Upon actin depolymerization, the membrane tubules destabilize and the predicted relaxation to (right) various beaded structures and / or tubules representing minimal energy surfaces. These predicted sketches are shown together with representative pseudo-colored SEM images of Muc1-42TRACr-expressing cells. (F) Schematic sketch of a simulation mechanism, where beading and vesicle-like membrane instability (left) is disrupted and leads to microvesicle shedding (right). (G) Representative histograms showing the average concentration and size distribution of extracellular vesicles of wild-type (control) and Muc1-42TRACr-expressing cells, and (H) Muc1-42TRACr cells treated with DMSO (vehicle) or Latrunculin A (+LatA). For each plot, the particle concentration was normalized to the maximum peak. Shaded areas show 95% confidence intervals, n = 5, 5, 4, 7, respectively. (I) Representative cryogenic transmission electron microscopy (cryo-TEM) images of vesicles collected from Muc1-42TRACr-expressing cells. Red boxes indicate pseudo-colored areas of interest shown on the right. ***p < 0.001 (post-hoc two-tailed Student’s t-test).

[0055] Figure 38Validation of genetically encoded mucins. Sequences in (A) are PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8) and PPASTSAPG (SEQ ID NO: 4) from top to bottom. (A) Schematic representation of genetically encoded glycoproteins. Mucin-1 (Muc1) contains 42 repeats of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8) and the trans-Golgi protein (rich in S / T) has a rich region of serine and threonine for O-glycosylation. Engineered glycoproteins lack the native cytoplasmic tail signaling domain (ACT), while retaining the native transmembrane domain (TM) or exchanged with a synthetic 21 amino acid transmembrane anchor (TM21). A rationally designed mucin (Rational GFP- ACT) contains 80 repeats of PPASTSAPG (SEQ ID NO: 4) fused to a fluorescent tag (GFP) as well as the native stalk and TM, without the native cytoplasmic tail signaling domain (ACT). (B) Representative confocal microscopy images showing tubulation of the membrane induced by various engineered glycoproteins compared to wild-type (control) cells. Cell surface was visualized with the lectin WGA (wheat germ agglutinin). Mucin staining with the lectin PNA (peanut agglutinin) confirms glycoprotein O-glycosylation and surface localization on MCF10A cells, n = 3. (C) Quantification of endocytosis of Alexa Fluor 488-labeled transferrin (488TNF) after 0.5 or 1 h treatment. Quantified with flow cytometry, median signal reported minus background, >10,000 cells per population, n = 6, error bars are S.D. (D) Representative confocal microscopy images of endocytosed 488TNF after 0.5 h treatment. (E) Western blot showing the size of the polymer expressed in epithelial cells analyzed with an antibody against the green fluorescent protein (GFP) tag, n = 2. (F) Quantification of tubule density for the indicated mucin sizes. Number of cells analyzed is shown on the x-axis for each condition. Box notches indicate 95% confidence intervals. Statistical comparisons are to 42TR. ns - not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (post-hoc Student’s two-tailed t-test).

[0056] Figure 39: Hyaluronan localizes to the cell surface and induces cell surface protrusions. (A) (Left) Schematic of hyaluronan (HA) extruded by the transmembrane protein hyaluronan synthase 3 (HAS3). (Right) Blots of HA in lysates of wild-type (control) and human mammary epithelial cells (MECs, MCF10A) expressing hyaluronan synthase 3 (HAS3). Note that the expressed HA is a huge linear polymer in the MDa range. (B) ELISA quantification of HA secreted by MECs into their culture medium, normalized to the number of cells in the sample and to HA secretion by control cells, n = 3. (C) Representative confocal microscopy images of wild-type (control) or human MECs stably expressing HAS3. Cells were stained with Hoescht (nuclei) and Alexa Fluor 568 hyaluronan binding protein (HABP). (D) Representative SEM images showing highly elongated membrane tubules in human MECs expressing HAS3 (left) and a magnified region on the same cell (right). **p < 0.01 (post-hoc Student’s two-tailed t-test).

[0057] Figure 40 : Mucin causes tubulation of model lipid membranes. (A) Representative confocal images of DOPC giant unilamellar vesicles (GUVs) labeled with Bodipy-PC, increasing fraction of Ni-NTA lipids. Recombinant Alexa Fluor 568-labeled podocalyxin (Podxl) was associated with GUVs via a polyhistidine tag. Scale bar in each BODIPY-PC image is 5 pm. (B) (Left) Quantification of the fluorescence intensity (relative surface density) of Alexa Fluor 568-labeled human serum albumin (HSA) or Podxl on GUVs at different levels of Ni-NTA lipids, n = 10-20. Surface densities of HSA similar to that of mucin (low HSA) and high several-fold higher (high HSA) were used to control for protein crowding effects. (Right) Quantification of the fraction of GUVs with tubes at different levels of Ni-NTA lipids for each recombinant protein - low HSA, high HSA, and Podxl, error bars are standard deviations, n = 20-90 GUVs in 1-3 experiments. (C) Representative confocal images of Alexa Fluor 568-HSA on GUVs with high HSA forming tubes.

[0058] Figure 41Supporting information for physical characterization of mucin individual and mucin ensemble. (A) Schematic representation of recombinant Muc1 42 tandem repeat (Muc1-42TR) polymer fused to a 10x histidine tag. (B) Western blot validation of recombinant Muc1-42TR production (media + Muc1-42TR 10x His), Ni-NTA resin-bound protein (by flow), wash of non-specific proteins (wash), and purified recombinant Muc1-42TR polymer (eluate). Samples were probed with anti-Muc1 and anti-His antibodies and PNA that binds O-linked glycans (Peanut agglutinin). (C) SYPRO Ruby protein gel stain of samples described in B. (D) Quantification of epithelial microvilli diameter for the indicated relative mucin surface density. Box notches indicate 95% confidence interval. (E) (Left) Mucin construct (Muc1-42TR) with SUMO and GFP tags flanking the polymer domain for visualization of polymer extension with expansion microscopy (ExM). (Right) ExM sample workflow. First, samples are stained and fixed. Then, proteins are chemically linked (anchored) to monomers, which polymerize to form a gel. Proteins are then digested, and the gel is expanded to four times its original size. ns - not significant.

[0059] Figure 42 Theoretical predictions of additional polymer brush theory for curvature arising from intermolecular interactions in the glycocalyx. (A) Plot of predicted brush thickness as a function of biopolymer surface density in a brush regime. Brush thickness scales roughly as a power law in biopolymer concentration. (B) Plot showing energy contributions as a function of biopolymer density. In the mushroom regime, polymers have only elastic energy, while in the extended brush, excluded volume and electrostatic interactions contribute to the biopolymer free energy. (C) Plot depicting the change in spontaneous curvature arising from biopolymer density and molecular length. (D) Plot showing the spontaneous curvature trend as a function of biopolymer density and Kuhn length. Kuhn length is equal to twice the persistence length, proportional to the polymer bending stiffness, referred to in this manuscript as the length of a monomer segment. Curves in (A-D) are in log-log format. Curves in (A) and (B) use a biopolymer length, l = 270 nm, and a monomer segment length, l a = 15 nm. Curve (C) takes a polymer monomer segment size of 15 nm, and (D) uses a biopolymer length of 270 nm. (E) Predicted dependence of spontaneous curvature on biopolymer length at high density. This plot uses a polymer of l 2 = 15 nm packed at a density of 50000 number / m a

[0060] Figure 43 ​: Fluorescence activated sorting and quantification of Muc1 surface density. (A) Extended workflow for quantification experiments at different Muc1 surface densities. (B) SDS-Page calibration of Alexa Fluor 647 labeled nanobodies. (C) Calibration curve between the log value of the integrated density of fluorescence signal from a nanobody dilution series (shown in (B)) and the log value of the number of loaded molecules. Linear regression fit and R 2 value are shown. (D) Residuals of the linear regression fit shown in (C). (E) Fluorescence activated cell sorting (FACS) histograms showing nanobody fluorescence signal and the populations 'a' to 'e' collected for these experiments. (F) Representative scanning electron microscopic (SEM) images of wild type cells non-enzymatically detached from the substrate, then re-adhered for SEM imaging (detached control), and cells non-enzymatically detached from the substrate, collected by FACS, then re-adhered (FACS control). These images indicate that the FACS collection method does not affect the membrane shape Figure 2 shown in (F). (G) SDS-Page analysis of the fluorescence nanobody signal in each cell population a-e after the cells were collected and lysed. (H) Table describing the integrated density signal of the fluorescence images shown in (G), the number of molecules calculated based on the calibration curve in (C), and the number of cells loaded in the protein gel based on the number of cells collected with FACS for each population (E). (I) Calibration curve between the log of the average nanobody signal from FACS and the number of molecules calculated for each population. The number of molecules for each sample was normalized by the number of cells loaded and the approximate area of each cell. Linear regression fit and R 2 value are shown. (J) Residuals of the linear regression fit shown in (I).

[0061] Figure 44 : Tubular membrane shapes contain a filamentous actin core and resemble microvilli. (A) Representative confocal microscopic images of epithelial cells expressing Muc1-42TRACI showing indirect microtubule staining with an anti-microtubule primary antibody and Alexa Fluor 568 labeled secondary antibody. Mucins are labeled with Alexa Fluor 647 PNA (Peanut agglutinin). Bottom row shows the area of interest (yellow box) from the composite image, n = 3. (B) Fluorescence intensity line trace from (A) (bottom row, yellow line). Values are normalized to their respective maximum intensity. (C) Representative confocal microscopic images of epithelial cells expressing Muc1-42TRACI showing actin staining with Alexa Fluor 568 phalloidin. Mucins are labeled with Alexa Fluor 647 PNA. Bottom row shows the area of interest (yellow box) from the composite image, n = 3. This data is inFigure 5 A, B) Repeat and detail. (D) Fluorescence intensity traces from (C) (bottom row, yellow line). Values were normalized to their respective maximum intensities. (E) Representative confocal micrographs of the midplane of wild-type (control) or Muc1-42TRACT cells that had been treated with 10 μΜ latrunculin-A (LatA) for 1 h, n = 3. (F) Representative SEM images of LatA-treated Muc1-42TRACT cells. DETAILED DESCRIPTION

[0062] Unless expressly stated otherwise, each maximum numerical limitation given throughout this specification includes each and every narrower integer value within such numerical limits, as if such narrower numerical limitations were expressly stated herein. Each minimum numerical limitation given throughout this specification will include each and every narrower integer value as if such narrower numerical limitations were expressly stated herein. Each numerical range given throughout this specification will include each and every narrower numerical range that falls within such broader numerical limitation, as if such narrower numerical ranges were all expressly stated herein.

[0063] The present disclosure includes each of the amino acid sequences described herein, as well as each polynucleotide sequence encoding an amino acid sequence, including but not limited to cDNA sequences and mRNA sequences. Complementary and reverse complementary sequences are also included. The present disclosure encompasses expression vectors comprising such nucleotide sequences.

[0064] The present disclosure relates generally to improved glycoproteins, compositions comprising the proteins for different applications, and methods of making and using the glycoproteins. In embodiments, the glycoproteins are mucins and / or lubricins.

[0065] The present disclosure includes cells and cell cultures that express the proteins described herein. In certain embodiments, the present disclosure includes cell cultures that are modified to produce any of a variety of proteins due to a reduction in clumping, aggregation, and the like of the cells.

[0066] In embodiments, the cells used to express the proteins of the present disclosure are eukaryotic cells. In certain embodiments, the cells are eukaryotic cells, including but not limited to insect and mammalian cells. In embodiments, the mammalian cells are not Chinese hamster ovary (CHO) cells, although in certain instances CHO cells can be used. In embodiments, the cells are mammalian epithelial cells. In embodiments, the cells are human cells, and thus are more suitable for producing, for example, human biologies, than non-human mammalian cells. In embodiments, the cells are human 293 cells. In embodiments, the 293 cells are derived from 293 cells and stably express SV40 large T antigen. In embodiments, the cells are human 293 cells that are adapted to grow in suspension culture. In embodiments, the cells are human 293-F cells, which are commercially available from multiple suppliers.

[0067] In certain methods, such as therapeutic methods, the present disclosure includes modifying a heterologous cell or a cell obtained from an individual to express one or more glycoproteins described herein. Thus, in embodiments, a human or non-human cell can be modified to, for example, correct a deficiency in a mucin or mucin-like protein or in the production thereof. In embodiments, the cell modified according to the present disclosure is a totipotent, pluripotent, oligopotent stem cell or a multipotent stem cell. In embodiments, the cell is a hematopoietic cell. In embodiments, the cell is a chondrocyte. In embodiments, the cell is a mesenchymal stem cell or a bone marrow stromal cell. In embodiments, the cell is a synoviocyte. In embodiments, the cell is a chondrocyte precursor cell. In embodiments, the cell endogenously produces chondro-specific gene products, such as collagen type II and / or chondro-specific chondroitin sulfate proteoglycans (CSPGs). In embodiments, the cell is an epithelial cell or a precursor thereof, or is a goblet cell. In embodiments, the cell is an immune cell, and includes but is not necessarily limited to T cells, such as CD4+ and CD8+ T cells, and dendritic cells. The cell can be modified according to any established technique, including but not limited to using a viral expression vector, or by chromosomal editing, such as by any suitable CRISPR-based gene editing method. The modified cell can be administered to an individual in need thereof. In embodiments, a transgenic non-human animal has been created to express one or more modified proteins of the present disclosure, and such animals can be created and used to study a wide range of biological functions, disorders and conditions.

[0068] In embodiments, any of the glycoproteins described herein can be present in a fusion protein. Fusion proteins are recombinantly produced and contain segments of different proteins in a single continuous polypeptide. In embodiments, the fusion proteins described herein comprise a glycoprotein or segment thereof, and a second protein or segment that is not particularly limited. In embodiments, the second protein produces a detectable signal, and thus includes, for example, a fluorescent protein.

[0069] In certain embodiments, the compositions and methods of the present disclosure relate to recombinantly produced proteins having a repeating amino acid sequence, such as a tandem repeat sequence. In embodiments, the tandem repeat sequence is modified relative to its naturally occurring sequence, and the number of repeats can have been altered relative to the number of repeats in the naturally occurring protein. Combinations of different repeats can be included in the polypeptides described herein.

[0070] In embodiments, the present disclosure comprises introducing an expression vector described herein (which can be a codon-optimized expression vector) encoding one or more proteins described herein into a suitable cell / cell culture, allowing protein expression, and recovering the protein from the cell. In embodiments, cells in a cell culture are modified using any suitable expression vector to express at least a protein described herein.

[0071] The expression vector can be integrated into the chromosome of the cell, or can be maintained permanently or transiently as an epigenetic element. The expression vector can be configured to express the protein in a constitutive or inducible manner. In one non-limiting embodiment, a transposon-based expression vector can be used, or a lentiviral expression system can be used. In one non-limiting embodiment, a lentiviral system can be excluded as a tool to express a protein described herein. In embodiments, any protein described herein can or can not include a signal sequence. In embodiments, a polynucleotide, such as a cDNA encoding one or more proteins described herein, is randomly integrated into one or more chromosomes to produce a modified cell. In embodiments, random transposition into the genome using a cDNA is used.

[0072] In embodiments, a codon-optimized expression vector comprises a threshold number of altered codons, wherein the altered codons do not change the amino acid encoded by the particular codon. Thus, an optimized codon can contain, for example, a wobble base change. In embodiments, at least one codon is changed, from one codon to all codons encoding each amino acid in a particular protein can be changed. In embodiments, a codon-optimized cDNA reduces the repetitiveness of the cDNA sequence to improve the stability of the nucleotide sequence during DNA processing, including but not necessarily limited to replication, transcription, reverse transcription, and slippage during other nucleotide processing operations performed on repetitive nucleotide sequences, which often results in deletions or amplifications of the cDNA and mRNA. In embodiments, codons with a frequency of use below a predetermined threshold in a relevant cell type are replaced with codons having a high frequency of use. For example, in one embodiment, codons with a frequency of use less than or equal to 10% in human cells can be replaced.

[0073] In embodiments, the mucin / lubricin protein or protein for which production is desired to be improved can be modified for recovery using any suitable method, including but not limited to the inclusion of one or more purification tags, including but not limited to a His tag. In one embodiment, the His tag is a linear sequence of n histidine residues, where n is typically 6-10. The His tag enables purification by specific binding to nickel or cobalt ions, which can be attached to a matrix, such as any suitable bead, for example. The His tag or any other suitable purification tag can be placed at the N-terminus of the protein, at the C-terminus of the protein, or internally in the protein. In embodiments, a FLAG tag or FLAG octapeptide or FLAG epitope can be included in the proteins of the present disclosure. Suitable FLAG sequences are known in the art. In embodiments, a small ubiquitin-related modifier (SUMO) tag, such as a His-SUMO tag, can be included. In embodiments, a protease cleavage site, such as for protein identification, isolation, purification, etc. can be included. The protein can be purified to any desired degree of purity.

[0074] In non-limiting embodiments, the tandem repeat included in the proteins of the present disclosure comprises any one or any combination of the following amino acid segments: KEPAPTTP (SEQ ID NO: 1), KEPAPTP (SEQ ID NO: 9), and KEPAPTTTP (SEQ ID NO: 10), or combinations thereof.

[0075] In embodiments, 2-120 repeats are included in the proteins of the present disclosure. In non-limiting embodiments, 10, 21, 40, 42, 59, or 80 repeats are included. The repeat sequence can be entirely contiguous within the polypeptide. In embodiments, any of the amino acid sequences described herein can be a segment of a longer tandem repeat, and thus can have additional amino acid sequences at its N or C terminus. In embodiments, the amino acid sequence of the tandem repeat described herein comprises or consists of 7-80 amino acids. In embodiments, the tandem repeat described herein exhibits a predicted length of approximately 135 nm or 270 nm. In embodiments, the repeat is a perfect repeat, meaning the same sequence is repeated in the protein, unlike certain naturally occurring tandem repeats.

[0076] In embodiments, the disclosure includes all of the cDNA and amino acid sequences disclosed in Parts I-IV of the Examples, as well as variants thereof as described herein. At times, such representative sequences are referred to for convenience as "bio-bricks." In non-limiting embodiments, the disclosure provides polypeptides (such as glycoproteins) and codon-optimized expression vectors encoding glycoproteins, described herein as SynMuc1 and SynLubricin, Syn1_40, Syn1_80, Syn2_40, Syn2_80, Syn3_40, and other constructs for use with non-human mammals as further described below.

[0077] Polypeptides comprising amino acid sequences having at least 90% identity to the amino acid sequences of these sequences are included. In embodiments, the proteins comprise mutations relative to endogenous proteins. An "endogenous" protein is a protein that is normally encoded by an unmodified gene. Likewise, an endogenous gene or other polynucleotide comprises a DNA sequence that has not been modified as by recombination, gene editing, or other methods. As further described below, mutations can include amino acid insertions, deletions, and alterations, and can also include additional repeat sequences, or fewer repeat sequences, relative to the endogenous sequence.

[0078] In embodiments, tandem repeat amino acid sequences are introduced into glycoproteins at the N-terminus, C-terminus, or both the N-terminus and C-terminus thereof. In one exemplary embodiment, lubricin-like molecules are produced by fusing the natural N and C-termini of human lubricin or lubricin from a non-human mammal with repeats of KEPAPTTP (SEQ ID NO: 1), KEPAPTP (SEQ ID NO: 9), and KEPAPTTTP (SEQ ID NO: 10). In embodiments, the non-human mammal is a canine or equine or feline animal. Representative amino acid sequences of lubricin from equine and canine animals incorporated into modified lubricin are further described below. In embodiments, 10-120 repeats are included. In embodiments, 59 repeats are included. In embodiments, the repeat sequences or other sequences can be separated from one another by a sequence, or by a linker sequence, such as one to three amino acids.

[0079] In embodiments, recombinant lubricin polypeptides are provided in which the contiguous repeat sequences described below are located between an N-terminal amino acid sequence and a C-terminal amino acid segment that have at least 90% sequence identity to human, canine, or equine sequences. Accordingly, the disclosure includes the described tandem repeats flanked by sequences. The flanking sequences can comprise human lubricin N- and C-terminally derived amino acid sequences; canine lubricin N- and C-terminally derived amino acid sequences; or equine lubricin N- and C-terminally derived amino acid sequences.

[0080] In one non-limiting embodiment, wherein a contiguous repeat sequence is located between an N-terminal human lubricin-derived sequence and a C-terminal human lubricin-derived amino acid sequence, the N-terminal human lubricin-derived sequence has at least 90% sequence identity to the following human lubricin sequence:

[0081] QDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSSSTIRKIKSSKNSAANRELQKKLKVKDNKKNRTKKKPTPKPPVVDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTP (SEQ ID NO: 75)

[0082] the C-terminal human lubricin-derived amino acid sequence has at least 90% sequence identity to the following human lubricin sequence:

[0083] SEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPKSEDAGGAEGETPHMLLRPHVFMPEVTPDMDYLPRVPNQGIIINPMLSDETNICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNVVTSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVWYNCP (SEQ ID NO: 76)

[0084] In embodiments, the lubricin sequence flanking the contiguous repeat in the non- human animal can include an amino acid change, in non-limiting embodiments, the amino acid change is a change of 3-7 amino acids at the N or C terminus of the following sequence relative to the native sequence.

[0085] In one embodiment, the tandem repeat sequence is located between an N-terminal canine Lubricin sequence having at least 90% sequence identity to the following sequence: QDLPSCAGRCGEGYSRDAICNCDYNCQHYMECCPDFKKACTVELSCKGRCFESFARGRECDCDSDCKKYGKCCPDYEDFCGRVHNPTSPPSSKTAPPSPGASQTIKSTAKRSPKAPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSTIRKIKSSKNSAANKELKKKPKVKDNKKERTPKKKPPPEPPVVDEAGSGLDNGDIKLTPTPDIPTTQRNKVTTSPKFTTGKPINPKPSLPPNTDTSKETSSTPNKETTVKSKETLANKETSSKAKEKITSAKETRSAEKTPAKDFVPTTKAPVKSTPKAESTTKGPALTTP (SEQ ID NO: 77) (wherein, for example, the seven C-terminal amino acids can be changed from the native canine sequence, which is SPAPTTP (SEQ ID NO: 83)) and a C-terminal canine Lubricin-derived amino acid sequence having at least 90% sequence identity to the following canine Lubricin sequence:

[0086] the C-terminal canine Lubricin-derived amino acid sequence has at least 90% sequence identity to the following canine Lubricin sequence:

[0087] SEVTTTAKDKTTEKDIIPEITTAVPKITTQETATPTEETTTESKTSTTTQVTSTTSSKNTPKATTLAPKVMTATQKTTTTEETMNKPEETTAVPKDTATSTKVSTPRPRKPTKAPKKPASTKKPNTIPKRKKPKTTPTPPKMTTSTMPKLHPTSSVEAMLQTTTSPNQRPNSEIVEVNPNEDTDAAGKKPHMFPRPPVLTPIFIPGTDILVRGSNQDIAINPMLSDETNLCNGKPVDGLTTLRNGTMVAFRGHYFWMLSPSKPPSPPRKITEVWGIPSPIDTVFTRCNCEGKTFFFKGSQYWRFTNDIKDAGYPKQIVKGFGGLNGRIVAALSIAKYKDRPESVYFFKRGGSVQQYTYKQEPIKKCTGRRPAINYPVYGETTQVRRRRFERAIGPSQTHTIRIHYSPIRVSYQDKGFLHNEVKMSSQWRGFPNVVTSAIALPNIRKPDGYDYYAFSRNQYYNIDVPSRTARVVTTRFGRTLSNIWYNC (SEQ ID NO: 78) (wherein, for example, three N-terminal amino acids are changed relative to the corresponding canine sequence, which are PEM).

[0088] In embodiments, the contiguous repeat sequence is between an N-terminal equine lubricin sequence having at least 90% sequence identity to the following equine lubricin sequence: QDLSSCAGRCGEGYSRDATCNCDFNCQYYMECCPDFKKVCTSELSCKGRCFESFERGRECDCDADCKKYGKCCSDYESFCEEVHNPTSPPSSKTAPPPPGASQTIKSTAKRSPKSNKKKTKKVIESEEIIEEHSVSENQESSSSSSSSSSTIRKVKSSKNSAANRELKKKPKVKDSKKKRTPKKKPTPEPPVIDEAGSGLDNGDFMLIPTPKIPTTQRNKVTTSPKITTVKPINPKPSLPPNSDTSKETTSTPNKETTVETKETEITNKETSTSANEKTTSARKSTEKTSDKDFAPASEVPAKSTPKAETTTKGPALTTP (SEQ ID NO: 79), (wherein for example seven C-terminal amino acids can differ from the native equine sequence, which is SPSLTT (SEQ ID NO: 84)) and a C-terminal equine lubricin-derived amino acid sequence having at least 90% sequence identity to the following equine lubricin sequence:

[0089] the C-terminal equine lubricin-derived amino acid sequence has at least 90% sequence identity to the following equine lubricin sequence:

[0090] SEVSTTTTTMKPPTTPKNLAESTPEFPAEPTPKALENSPKEPAVPTTKAPEVTKPEVTTTAKDKVTGKDIHTIPEITTAAPKITTETATTTEEKTTESKVTSTIMQVTSTTEDTTTSSKITPKATTLAPKVMTATKTTTTQETINKLEETTAIPKDTATHSKVTTPKPKKPTKAPRKPTSTKKPKTPRKRKPKTTPIPPKITTPTTPKSNPTTLAEAMLQTTTSPNQTPNSAMIEVNPKNEDADAAEGEKPLVILRPHVLTPIVIPGPDFLVRGPNLGIGINPMLSDETNLCNGKPVDGLTTLRNGTLVAFRGHYFWMLRPFSPPSPPRRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKLISKGFGGLSGKIVAALSIATYKNRPESVYFFKRGGRIQQYIYKQEPIRKCPGRRPAIHYSVYGEAPQIRRRRFERAIGPSQTHTIRIHYSPVRVSYQDKVPSTDFLHNEVKVSTLWRGLPDTVTSAISLPNLRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVWYNCP (SEQ ID NO: 80) (wherein for example the three N-terminal amino acids can differ from the native horse sequence, which is SEA).

[0091] In embodiments, the recombinantly produced proteins described herein comprise a variant of a tandem repeat having any one or combination of the tandem repeat sequences described herein, wherein the variant comprises a modification of such sequence. Expression vectors encoding the variants are included. In embodiments, the modification comprises an amino acid segment having between 90.0-99.9% amino acid identity, inclusive, to the contiguous amino acid and polynucleotide sequences explicitly described herein, to the first decimal point, and including all numerical ranges therebetween. In embodiments, the recombinantly produced proteins of the present disclosure comprise a tandem repeat having 90, 95, 97, 98, 99, or 99.5% amino acid sequence identity to the amino acid sequences described herein over its full length. The recombinant protein is a protein expressed from a polynucleotide introduced into a cell that does not comprise a coding sequence for the protein prior to introduction of the polynucleotide. The same applies to recombinant cDNA sequences.

[0092] As is known in the art, to determine the percent identity of two nucleotide or amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced). The nucleotides or amino acids at each position of the aligned sequences are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = the number of identical positions / total number of positions x 100).

[0093] In certain embodiments, the tandem repeat variants described herein comprise 1, 2, 3, 4, or 5 amino acid changes. In embodiments, an amino acid can be deleted, added, or changed. In embodiments, the changed amino acid is a serine, threonine, or a combination of serine and threonine residues are changed. In embodiments, about 1-50% of the serine and / or threonine residues are changed. In embodiments, a serine or threonine residue present in the native protein sequence is changed to an alanine, or to another amino acid. In embodiments, the proteins of the disclosure comprise fewer or no amino acids present in the native (unmodified and / or endogenous protein). In embodiments, the native protein comprises one or any combination of asparagine, aspartic acid, glycine, isoleucine, leucine, and / or serine, which can be reengineered from a representative protein of the disclosure.

[0094] In embodiments, the amino acid changes introduced into the proteins of the disclosure result in altered glycosylation patterns. Accordingly, in embodiments, the disclosure provides for the production of recombinant proteins with controllable glycosylation patterns. In embodiments, the number of O-linked oligosaccharides present on the proteins of the disclosure is modified. In embodiments, the glycosylation pattern is altered relative to a control, such as a protein with no changes to the corresponding glycosylation sites. In embodiments, one or more properties of the protein and or the cell expressing the protein are altered. In embodiments, the stoichiometry of the oligosaccharide relative to the protein / amino acid is altered in, for example, the glycoproteins of the disclosure. In embodiments, the proteins of the disclosure comprise a different weight percentage of glycoside residues than a suitable control. In embodiments, the proteins of the disclosure exhibit a lubrication parameter, such as a coefficient of kinetic friction. In embodiments, the coefficient of friction can be determined using any suitable method, such as a cartilage friction test on cartilage. In embodiments, the proteins of the disclosure exhibit a different lubrication parameter than a suitable control.

[0095] In embodiments, a recombinantly produced protein as described herein comprises an amount of core 1 O-glycan structure, Galβ1-3GalNac, and / or a core 1 derivative of Galβ1-3GalNAc relative to a control, and / or a change in the amount of terminal substituted sialic acid, or a change in GalNAc (N-acetylgalactosamine) monosaccharide glycosylation. In embodiments, a protein described herein can comprise a core 2 O-glycan, GlcNAcβ1-6(Galβ1-3)GalNAc, and / or a core 2 derivative of GlcNAcβ1-6(Galβ1-3)GalNAc, comprising at least 5% of all core 1, core 2, core 3, core 4, core 5, core 6, core 7, and core 8 O-glycan structures. In embodiments, such a protein is produced from a human cell cultured as further described herein.

[0096] In embodiments, a protein of the present disclosure can be in monomeric, dimeric, multimeric, and combinations thereof. In embodiments, the monomer / dimer ratio, proportion, and / or concentration is altered relative to a suitable control.

[0097] In embodiments, a protein segment described herein can be separated by any suitable linking amino acid. In embodiments, a linker can comprise 1-20 amino acids, inclusive, and including all integers and integer ranges therebetween. Generally, a linker comprises glycine, serine, or serine and glycine. In embodiments, linking amino acids are not inserted into a tandem repeat. In embodiments, a secreted form of a glycosylation mutant is provided.

[0098] In embodiments, the modified lubricin lacks one or both of the cytoplasmic domain and the transmembrane domain. In embodiments, the lubricin of the present disclosure comprises a secretion signal, such as for production of the modified protein. The amino acid sequences of many suitable secretion signals are known in the art and can be used in this embodiment. In one embodiment, the human secretion sequence comprises or consists of MAWKTLPIYLLLLLSVFVIQQVSS (SEQ ID NO: 72). In one embodiment, the canine secretion signal comprises or consists of MQWKILPIYLLLLSVFLIQQVS (SEQ ID NO: 73). In one embodiment, the equine secretion signal comprises or consists of MEWKILPIYLLLLLSIFSIQEVSS (SEQ ID NO: 74), or another sequence as further described herein, including alterations to the N- and C-terminal amino acids. In embodiments, the native secretion signal is replaced with a segment of an immunoglobulin, such as an IgG kappa light chain sequence from a human or mouse or another mammal. In embodiments, the secretion sequence comprises a secretion sequence from any of IL-2, CD33, human IgG2 H, chymase, trypsinogen, Gaussia luc, influenza hemagglutinin, human insulin, or silk worm protein.

[0099] In embodiments, the polypeptides of the present disclosure can have one or more modified amino acids, which are, for example, conjugated to another moiety. In embodiments, the polypeptides of the present disclosure are conjugated to at least one azido group, such that they can be readily conjugated to other moieties, such as using click chemistry, such as by modification of O-glycans with azides. In embodiments, the polypeptides of the present disclosure are cyclized or stapled.

[0100] In embodiments, the tandem repeat sequences described herein are incorporated into any glycoprotein. In embodiments, the glycoprotein is any mucin or lubricin protein. In embodiments, the glycoprotein is proteoglycan 4, also known in the art as lubricin, which comprises a protein encoded in humans by the PRG4 gene. In a non-limiting embodiment, the present disclosure provides a modified mucin, termed SynMuc1, as further described below. In another non-limiting embodiment, a modified lubricin is provided as SynLubricin, as further described below.

[0101] In an embodiment, the use of the cells modified herein to increase protein production, wherein the cells are present in a cell culture vessel, including but not limited to any cell culture dish and bioreactor. In embodiments, the modified cells according to the present disclosure are used in a bioreactor to produce any desired protein or combination thereof. In non-limiting embodiments, the bioreactor comprises a suspension cell bioreactor. In embodiments, the bioreactor has a volume of 1-25,000 liters, inclusive, and including all numbers and ranges of numbers therebetween.

[0102] In embodiments, a cDNA library is provided. In embodiments, the present disclosure comprises providing a cDNA library as described herein, and selecting one or a combination of the described cDNAs, or modifying a cell by introducing the cDNA and / or expression vector encoding the cDNA into the cell. Selection can be based on the intended or actual use of the cell, such as for protein production based on any particular protein and cell expression system. Kits encoding the proteins are also included.

[0103] In embodiments, one or more proteins described herein can be combined with other agents, such as biodegradable polymers, nanoparticles, pectin, alginate, cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides, hydroxypropyl methylcellulose, carboxymethylcellulose, lectins, rheology modifiers, plasticizers, chondroitin, glucosamine, and / or any hyaluronic acid.

[0104] For use in preventing and / or treating diseases that can benefit from, for example, anti-adhesion agents, the compositions described herein can be administered in conventional dosage forms prepared according to known techniques by mixing with standard pharmaceutically acceptable carriers. Some examples of pharmaceutically acceptable carriers can be found in Remington: The Science and Practice of Pharmacy (2005) 21stEd., Philadelphia, PA. Lippincott Williams & Wilkins, the disclosure of which is incorporated herein by reference. In embodiments, pharmaceutical and other compositions comprising the proteins described herein can be provided in liquid, tablet, powder, spray, ointment, hydrogel, and aerosol forms.

[0105] In embodiments, pharmaceutical compositions comprising one or more proteins of the disclosure can be administered to an individual using any suitable route, including but not necessarily limited to topical, oral, and parenteral, and as further described below. For example, the proteins can be administered intravenously, by direct injection into synovial joints or other synovial structures (tendon sheaths or bursae), intraperitoneally, by direct injection into the pericardial sac, by direct injection into the pleural cavity, subdermally, subcutaneously, or by direct application to the skin, mucosa, or eye.

[0106] In embodiments, the disclosure includes administering an effective amount of one or more polypeptides described herein and / or compositions comprising such polypeptides. The effective amount can vary depending on the method of drug formulation, method of administration, age, weight, sex of the patient, time of administration, route of administration, and other factors that will be apparent to those skilled in the art. The composition can be administered once, or in a series of administrations. In embodiments, the disclosure includes a single dose or several doses. In non-limiting examples, for prophylactic and / or therapeutic uses in joints or analogous structures, a suitable concentration of the polypeptides is in the range of 250 pg / mL to 2 mg / mL, inclusive, and including all ranges of numbers therebetween, and all milligram, microgram ranges. In embodiments, approximately 1 to 3 mL is used for mammalian joint applications. The frequency of dosing can be adjusted on an individual basis. Given the surprising half-life polypeptides of the disclosure, a suitable frequency of dosing is once every 3-6 weeks.

[0107] In embodiments, the disclosure includes methods, compositions, and devices for treating ocular diseases, disorders, or conditions in a mammal. In embodiments, the proteins produced by the cells as described herein are used to treat ocular diseases or conditions using any method or device known to one of ordinary skill in the art. In embodiments, the compositions comprising the proteins are used for intra- anterior chamber, intravitreal, subconjunctival, sub-Tenon’s, subretinal, or topical application to the corneal surface. The proteins can be delivered directly to the eye using techniques well known to those of skill in the art (e.g.: topical eye drops or ointments; slow release devices in the cul-de-sac or implanted near the sclera or within the eye). It is further contemplated that the proteins described herein can be formulated in an intraocular insertion or implant device.

[0108] In embodiments, the medicament comprising one or more proteins described herein is used to treat an eye condition, including one or more retinal diseases or injuries, including age-related macular degeneration (AMD), retinitis pigmentosa (RP), and diabetic retinopathy (DR). In one embodiment, the individual has dry, atrophic (non-exudative) age-related macular degeneration, defined as progressive age-related macular degeneration associated with changes in the retinal pigment epithelium, including atrophy and drusen, which is a common cause of vision loss in adults for which there are limited therapies. In embodiments, the condition includes one or more corneal diseases or injuries. In embodiments, the individual has glaucoma, which can include primary, secondary, and / or congenital glaucoma.

[0109] In embodiments, the proteins of the present disclosure can be provided in the form of eye drops. In embodiments, the eye drops comprise any one or more of: a steroid, an antihistamine, a sympathomimetic, a beta blocker, a parasympathomimetic, a parasympatholytic, a prostaglandin, a non-steroidal anti-inflammatory drug (NSAID), an antibiotic, an antifungal, or a local anesthetic. In certain embodiments, the eye drops are for any dry eye condition. In embodiments, the eye drops are for lubricating the eye, including but not necessarily for contact lens wearers. In embodiments, the composition is provided as a lubricating eye drop. In embodiments, the lubricating eye drop comprises artificial tears. In embodiments, the eye drops can be free of pharmaceuticals and thus only act as a lubricating / tear replacement composition. In other embodiments, the eye drops can be used to treat ocular allergic reactions and thus can also comprise an antihistamine and / or a vasoconstrictor. In embodiments, the eye drop formulation comprises 250 pg / mL to 2 mg / mL, inclusive, and including all numerical ranges therebetween, as well as all milligram, microgram ranges. Such concentrations can be used in typical eye drop volumes, such as 1-2 drops per eye, approximately 0.05 to 0.01 mL per eye.

[0110] In embodiments, the compositions comprising the proteins described herein can be used in conjunction with contact lenses. In embodiments, the proteins are used in contact lens care solutions. Thus, the proteins described herein can be mixed with any suitable contact lens care solution components, including but not necessarily limited to physiological saline, mild abrasives, surfactants, antifungal and antibacterial agents, including but not necessarily limited to conventional microbiocides, or hydrogen peroxide or boric acid, and preservatives such as ascorbic acid or edetate disodium. Contact lenses provided in solutions comprising one or more proteins described herein are included within the scope of the present disclosure.

[0111] In embodiments, compositions comprising the proteins described herein can be directed to mucosal linings. Mucosal linings include, for example, the upper and lower respiratory tract, the eye, the oral cavity, the nose, the rectum, the vagina, the urogenital tract, the periodontal pocket, the intestine and colon. In certain embodiments, the compositions can be used for oral inhalation. In embodiments, oral inhalation includes nasal application, and thus can include nasal sprays, nose drops, and nose ointments. In embodiments, oral inhalation can include bronchial sprays and inhalants. In embodiments, the proteins can be used to access the mucosa by using a lozenge, a chewing gum, a mouthwash or gargle, a suppository, or a tampon.

[0112] In embodiments, compositions comprising the proteins described herein are used as surgical anti-adhesion agents (intra-peritoneal lubricants to lubricate the intestines and prevent post-operative intestinal and visceral adhesions during intra-abdominal surgery / manipulation; intra-pleural lubricants to lubricate the lungs and prevent post-operative pleural adhesions during intra-thoracic surgery / manipulation; intra-pericardial lubricants to lubricate the surface of the heart and prevent post-operative pericardial adhesions during cardiac surgery / manipulation). As post-operative synovial fluid replacement following any arthroscopic, tenoscopic or bursoscopic surgery, to maintain lubrication and prevent adhesion or pannus formation. In embodiments, the compositions are used to treat septic arthritis / infection in any of the mammals described herein. In certain embodiments, the compositions can be used in conjunction with wound healing, wound infection treatment, and systemic sepsis treatment.

[0113] In embodiments, the non-human mammals to which the compositions comprising the modified lubricins described herein are directed require any one or combination of the conditions described herein. Further, equine animals can require treatment for one or a combination of conditions to which equine animals are particularly susceptible. In one non-limiting embodiment, the equine animal requires treatment for osteochondritis dissecans (OCD). Other common equine animal embodiments that can be extended to canine and feline animals include treatment for intra-articular fractures, osteochondral fractures, meniscal injuries, cartilage injuries, synovitis, septic arthritis, and post-traumatic osteoarthritis (PTOA). Other embodiments include treatment for tendon and ligament injuries, including but not limited to: desmitomyositis / tenomyositis of the superficial and deep digital flexor tendons, desmitomyositis of the suspensory ligament, tenosynovitis, and scaphoid bursitis. Equine ophthalmic embodiments include: corneal ulceration, posterior lenticonus, and fungal keratopathy. In a non-limiting embodiment, canine animals require treatment for cranial cruciate ligament rupture (RCCL) (similar to anterior cruciate ligament injury in humans), elbow dysplasia, hip dysplasia, tendon / ligamentitis, and ophthalmic applications including keratoconjunctivitis sicca (KCS), immune-mediated keratopathy, and painless ulceration.

[0114] In embodiments, compositions comprising the modified proteins, such as the modified lubricin, can be administered to humans and non-human animals for therapeutic or prophylactic purposes. In embodiments, the modified lubricin is administered to a canine, feline, or equine animal to prevent or limit the severity of injuries that are prone to occur during sporting competitions or during the animal's work. For example, a composition can be administered to an equine animal to prevent or limit joint / cartilage injuries during equestrian competitions, or during work, such as police work or herding. Typical equestrian competitions include rodeo events, dressage, steeplechase, horse gymnastics, polo, horse racing, and many others where the risk of joint and related injuries is high, as will be apparent to one of skill in the art. In addition, it is believed that the compositions of the present disclosure will be useful in treating a variety of other non-human mammals, such as in veterinary hospitals and clinics, animal emergency facilities, and zoos. In embodiments, the compositions of the present disclosure are used to prevent and / or treat avian animals.

[0115] In embodiments, an article of manufacture can be coated and / or impregnated with a composition comprising any of the proteins described herein. In embodiments, the article of manufacture is coated on any porous or non-porous surface. In embodiments, the article includes a medical device, including but not necessarily limited to a surgical device, a dental or orthopedic device, a suture, a catheter, a cannula device, an anesthesia delivery apparatus, a dressing, a bandage, and the like. In embodiments, the proteins described herein are used to coat cell culture devices, including but not necessarily limited to cell culture plates, multi-well plates, bioreactors, and any other surface where anti-adhesion properties are desirable.

[0116] In another aspect, the present disclosure includes a supplement product, such as a health food product, a dietary supplement, a food ingredient, and the like, which can be provided, for example, in the form of a liquid, a capsule, a tablet, a gel capsule, a powder, and the like.

[0117] In embodiments, pharmaceutical and / or nutraceutical food products comprising one or more of the proteins described herein are provided in a container, such as any suitable closed or sealable container that can be sterile. In embodiments, the product comprises printed material. The printed material can be provided in the form of a product insert, a label, or in the form of a package component. The printed material provides an indication that the composition comprising the polypeptide will be used to treat any of the diseases, disorders, or conditions as described herein, or for any purpose to produce an anti-adhesion effect. In one embodiment, the polypeptides described herein are used as a supplement to treat joint conditions, including but not necessarily limited to joint pain, arthritis (including but not necessarily limited to osteoarthritis, rheumatoid arthritis), joint, meniscal or cartilage injury, such as sports injuries, or in conjunction with joint / ligament repair surgery. Thus, administration of the compositions described herein for the purpose of improving the health or well-being of an individual is included within the present disclosure. In embodiments, the compositions of the present disclosure can be injected directly into a joint and / or synovial fluid. In embodiments, the compositions are administered directly or indirectly to any synovial structure, including but not limited to synovial joints and tendon sheaths or bursae. In embodiments, the compositions of the present disclosure can also be used for direct injection into a tendon, ligament or bursa following injury, trauma or infection to the tendon, tendon sheath, ligament or bursa. In embodiments, the compositions can be in contact with mesothelial surfaces: for example, the compositions are administered so that they are in contact with the abdominal or pericardial contact surfaces, in order to prevent and or treat conditions associated with one or more such surfaces.

[0118] The present disclosure can be better understood by reference to the following non-limiting examples, which are offered as exemplary of the present disclosure, and are divided into four parts. The following examples are presented in order to more fully illustrate embodiments of the present disclosure, however, they should not be construed as limiting the broadest scope of the present disclosure. The list of references cited in the present disclosure does not indicate that any particular cited reference is important for patentability.

[0119] Examples

[0120] Part I

[0121] This Part I of the present disclosure provides non-limiting and representative examples of sequence-specific mucins with controllable glycosylation patterns, as well as data and discussion thereof.

[0122] In particular, this Part I relates to the understanding that prior to the present disclosure, there were few design guidelines for engineering customized mucin glycoproteins with tunable glycosylation patterns. Accordingly, Part I provides libraries of interchangeable DNA bricks: mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized natural mucin repeats and new rationally designed domains of synthetic mucins. From over 400 possible cDNA combinations, Part I provides a library of over 50 mucins, each with unique chemical, structural, and optical properties. The libraries were applied to develop general guidelines for designing and engineering mucins, which form part of the present disclosure. Surprisingly, it was found that the extension of immature a-GalNAc Tn antigens to core 1 and core 2 glycan structures strongly depends on the frequency of O-glycosylation sites along the mucin backbone. According to the present disclosure, it is apparent to one of skill in the art that sialylation of the glycan structures is readily modulated by recycling motifs on the cytoplasmic tail of the mucin. It was also demonstrated that the overall length of the mucin polypeptide backbone can have an unpredictable impact on glycosylation. Without wishing to be bound by any particular theory, it is expected that the partial list of mucins presented here, and the described design guidelines for making new mucins, can be broadly applied to the study of the glycocalyx and mucin-based biotechnology.

[0123] Part I Introduction

[0124] Cell surface mucins are a family of membrane-anchored biopolymers defined by a non-structured polypeptide backbone with a high density of glycan side chains (1). While cell surface mucins have historically been viewed as simple structural molecules that protect the cell surface and resist pathological cell deposition (2), it is now recognized that they have more complex roles in regulating cell life. In the cell glycocalyx, mucins collectively present bioactive glycan epitopes that mediate adhesion and communication between cells and the cell with its external world. For example, mucin sialic acids can modulate immune cell function by engaging SIGLEC receptors on natural killer cells and other cell types in the microenvironment (3). Mucins can also physically modulate the spatiotemporal dynamics of receptor activation and signaling responses (4). It is proposed that the dense crowding of mucins in the glycocalyx controls the diffusion and activation of receptors on the cell surface and has a sieving effect that controls the passage of soluble factors from the microenvironment to the cell surface (5).

[0125] A key feature of mucins is that their molecular architecture can be dynamically altered by modulating the type and frequency of glycan side chains attached along the polypeptide backbone. For example, it has been proposed that the charge, size, and arrangement of glycans control the extension and rigidity of the mucin backbone (6, 7). Glycosylation generally changes dramatically with cell state transitions, including differentiation and transformation (8, 9). Thus, both the chemical and physical features of mucins are tightly linked to cell state, contributing to the diverse regulatory roles that mucins can play in cell adhesion, communication, and signaling. However, how precise backbone sequences and glycosylation patterns contribute to the function of individual mucins and the collective behavior of mucins in the glycocalyx is largely unresolved.

[0126] One of the major obstacles in making progress toward such understanding is the lack of tools for precisely editing mucin molecular structure. Genetic approaches targeting glycosyltransferases can efficiently alter mucin glycosylation (10), but these approaches typically affect a broad class of glycoproteins, making it difficult or impossible to pinpoint any observed effects on cell behavior to specific mucins. To overcome the limitations of genetic approaches, libraries of biomimetic mucin polymers with plasma membrane anchors have been developed for glycocalyx editing (6, 11). While successful in elucidating some mechanistic details of mucin function, synthetic polymers are typically cleared from the cell surface within hours to days and must be continuously replenished by supplementation of the culture medium (12, 13). Thus, behavior studies over longer durations, especially in vivo, are largely unattainable with synthetic mucin mimics.

[0127] Prior to the present disclosure, a strategy for mucin engineering and glycocalyx editing that combines the important features of synthetic chemistry (defined backbone chemistry, customized glycan structures, and precise glycan placement) with the power of genomic encoding and long-term stability has not been developed. Advances in custom gene synthesis support the development of cDNA sequences at unprecedented speed and low cost. However, custom gene synthesis is not readily applicable to highly repetitive DNA sequences, which are characteristic of most mucins. Repetitive gene sequences hinder DNA fragment assembly in custom gene synthesis and are challenging to amplify by polymerase chain reaction (PCR) due to primer mismatches (14, 15).

[0128] As described in Part I, one solution is to employ codon redundancy to construct synonymous gene sequences with minimal codon redundancy, a method that has been successfully applied to elastin-like proteins (16, 17).

[0129] In this Part I, we exploit codon redundancy to develop an efficient strategy to design, genetically encode and manufacture cDNAs for the synthesis of sequence-specific mucins in cells. The combinatorial library of mucin moieties described in this invention enables facile construction of mucin biopolymers with tunable size, side chain spacing and glycan type for glycosome editing.

[0130] Part I - Results

[0131] Schematic representation of the combinatorial genetically encoded library of sequence-specific mucins

[0132] Part I results demonstrate a modular, compartmentalized biological approach for combinatorial mucin cDNA construction. Each functional motif in the mucin-encoding sequence is flanked by restriction sites, enabling easy exchange of unique cDNA "bricks" for mucin leader sequences, tandem repeats, optical reporters, transmembrane domains and cytoplasmic domains to construct functionally altered mucins Figure 1 a, b) The cDNA parts catalog includes 13 unique tandem repeats for mucin biopolymers of different size, backbone chemistry and frequency of serine and threonine (S / T) glycosylation sites Figure 1 d) cDNAs for mucin polymer domains are manufactured by custom gene synthesis after codon optimization Figure 1 c) To optimize, codon redundancy is exploited to find synonymous gene sequences that encode the desired polypeptide with minimal codon repeats. The "codon-scrambled" cDNA sequences are synthesized by standard custom gene synthesis services offered by commercial vendors.

[0133] Tandem repeats forming the mucin polymer backbone are adapted from natural mucins or newly designed Figure 1 d) The repeats PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8) and KEPAPTTP (SEQ ID NO: 1) have similarity to natural Muc1 and proteoglycan 4 (lubricin), respectively. Three repeats are 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 is further modified by alanine substitutions to generate Muc1-like tandem repeats with altered frequency of S / T potential glycosylation sites (Muc1_21S, D, T). Across the library, the percentage of S / T sites in the mucin backbone varies from 10% to 33% Figure 1 d).

[0134] Construction and validation of surface expression of sequence-specific mucins

[0135] We compared the expression of codon scrambled, synonymous mucin cDNAs to native mucin repeat cDNAs and evaluated the glycosylation of the protein products. We fused native and synonymous Muc1 tandem repeat cDNAs to signal / leader sequences, membrane anchors, and GFP reporters Figure 2 a). Each construct was transiently expressed in HEK293T. We analyzed the glycosylation pattern of the mucins by lectin blotting. Blots were probed with peanut agglutinin (PNA) to detect core 1 glycans, with Vicia villosa agglutinin (VVA) to detect unextended Tn antigens (a-GalNAc), and with Muc1 mAb (clone HMPV) to probe the MUC1 tandem repeat peptide core (Muc1TR) 21 We also labeled Muc1 sialic acids on our blots by mild periodate oxidation to generate aldehydes on sialic acids, followed by phenyl azide-catalyzed oxime ligation (PAL) with a hydroxylamine-AF568 probe 22 GFP reporters were also probed by western blot to detect expressed mucins. To validate the use of lectins PNA and VVA, Figure 2 c), we knocked out the core 1 b3-T specific molecular chaperone protein (COSMC) in MCF10A overexpressing native Muc1 to inhibit elongation of primary O-linked GalNAc 23 We compared the glycosylation pattern of overexpressed native Muc1 (native_Muc1) in wild type and knockout cells. In COSMC knockout cells, PNA reactivity of the mucin was reduced, while VVA binding was significantly increased, likely due to the elimination of glycan elongation Figure 2 d). The results confirmed that PNA can be a good indicator of elongated core 1 glycans, and VVA can be a good indicator of unelongated Tn antigens on mucins.

[0136] Western blot analysis of native and codon scrambled mucins confirmed that the molecular weight and glycosylation pattern of the codon scrambled synonymous Muc1 repeats (Muc1_42GFP) were comparable to native repeat Muc1 repeats (native_Muc1 GFP) Figure 2 e). The mucins ran as almost continuous smears in SDS-PAGE with the Muc1 TR antibody, indicating a heterogeneous mixture of glycoforms Figure 2 e; Muc1 TR). On the GFP blots, for each expression construct, dominant glycoforms with apparent molecular weights of approximately 470, 210, and 170 kDa were observed Figure 2 e; GFP). VVA staining was intense in the smear region between the upper and lower bands, while PNA and sialic acid signals were strongest near the 460 kDa band at the top of the smear Figure 2e) Based on these results, we concluded that the 460 kDa band is fully glycosylated Muc1, while the smear represents a heterogeneous mixture of Muc1 glycoforms containing unextended O-glycan structures. The lower bands observed on the Muc1 TR blot, but not on the GFP blot, in the absence of lectin or sialic acid probes, indicate that these bands can represent lowly glycosylated full-length Muc1. Both the native and codon- scrambled Muc1 were successfully trafficked to the cell surface and incorporated into the cell glycocalyx Figure 2 f).

[0137] One advantage of the codon-scrambled mucin cDNA is the potential to increase the stability of the nucleotide sequence during some DNA handling procedures. Slippage during replication, transcription, reverse transcription, and other nucleotide handling procedures often results in deletions or amplifications of cDNAs and mRNAs 24 We performed lentiviral stability analysis in which we assessed the fidelity of the cDNA incorporated into the cellular genome after viral delivery and reverse transcription. In cells transduced with the native, non-optimized Muc1 cDNA virus, the molecular weight of the Muc1 glycoprotein product was significantly lower than expected, consistent with a truncated cDNA. Cells transiently transfected with the native Muc1 cDNA, or modified with the codon-scrambled Muc1 cDNA virus, produced the glycoprotein of the expected size Figure 6 Although lentiviral analysis is not a direct test of genomic stability, the results indicate that non-repetitive mucin sequences are more stable during at least some types of nucleotide handling procedures.

[0138] The number of tandem repeats of native mucins in humans is often polymorphic, causing mucin size to vary between individuals 25 and short alleles of Muc1 have been shown to be associated with gastric cancer 26 Inspired by this natural variation, and to further validate our approach, we designed and constructed a series of synonymous mucins with variable numbers of tandem repeats (x42, x21, x10, x0; Figure 2 a). The polymorphic cDNAs were well expressed on the cell surface and showed the expected differences in size and degree of glycosylation. As expected based on previous reports 27 The larger mucins formed a glycocalyx of sufficient size to lift epithelial cells from their substrate.

[0139] Substitution of potential glycosylation sites with alanine in the mucin polymer backbone modulates O-glycan maturation

[0140] We next tested whether mucins with altered glycosylation patterns, including differences in glycan extension, could be encoded by mutating S / T sites in the mucin backbone. Our overall strategy was to generate secreted Muc1 tandem repeats with one, two, or three of the five potential glycosylation sites in each repeat substituted with alanine Figure 3 a, b). We envisioned that secreted mucins could then be collected from cell culture media for subsequent glycan analysis by lectin blotting and mass spectrometry.

[0141] cDNAs for the desired Muc1 mutants with 21 repeats were each codon- scrambled and manufactured by custom gene synthesis. 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 potential glycosylation frequencies varied at 20%, 15%, and 10% (Table 1). Figure 3 a). The transmembrane protein anchor was not included, so the IgK signal peptide would direct secretion of the recombinant mucins.

[0142] Secreted mucins were collected from the media supernatant of HEK293 cells and analyzed by protein and lectin blotting. The apparent molecular weights of the wild-type and glycosylation mutants were significantly higher than the theoretical molecular mass of the unmodified peptide backbone Figure 3 b, c, and 8). The potential glycosylation site mutants migrated faster in SDS-PAGE, indicating they had fewer or shorter glycan chains and thus less hindrance to their electrophoretic migration Figure 3 c).

[0143] We found that substituting S / T modulates O-glycan maturation. Secreted Muc1 glycoproteins were analyzed by blotting and probed for Tn antigens with VVA, core 1 glycans with PNA, and GlcNAc, the building block of core 2, 3, 4, and 6 glycans Figure 3 c). We constructed electropherograms by recording the fluorescence intensity of the glycan probes along each lane of the single co-stained blot Figure 3 d). Core 1 (PNA) and GlcNAc-containing (s-WGA) glycans were enriched in the mucin glycoforms with the highest apparent molecular weights. The lower apparent molecular weight glycoforms contained abundant VVA-reactive glycans and minimal core 1 and GlcNAc-containing glycans. Gradual alanine substitution clearly shifted the glycoform distribution toward mucins with more unextended VVA-reactive glycans and fewer extended core 1 and GlcNAc-containing glycans Figure 3d, e). Surprisingly, even a single serine substitution (see sMuc1S) significantly altered the glycosylation pattern, leading to the production of more non-fully extended glycoforms Figure 3 c, d).

[0144] To validate our lectin analysis and to classify specific glycan structures on the mucin, we performed mass spectrometry to profile the O-glycans on wild-type mucin repeat (sMuc1) and the mutant with three S / T alanine mutations in each repeat (sMuc1T). We identified similar core 1 and core 2 glycans in both samples Figure 3 f). However, the signal for extended glycans was much stronger in wild-type mucin (sMuc1) compared to the triple mutant (sMuc1T), consistent with our lectin blot. We also fused the glycosylation mutant cDNAs to a transmembrane anchor for cell surface expression and observed a similar trend of inhibition of glycan extension in the glycosylation site mutants Figure 9 c). To ensure that overexpression of the mucin constructs did not affect the functionality of glycosyltransferases for glycan extension, we used the cellular O-glycome reporter / amplification (CORA), a method that allows for protein-free analysis of the overall cellular O-glycome 28 Similar core 1 and core 2 glycan structures were detected in wild-type and HEK293T cells overexpressing Muc1, indicating that the activity of T synthase and other glycosyltransferases involved in mucin extension was not inhibited by mucin overexpression Figure 10 ) Overall, these data demonstrate that the extension of glycans in both cell surface and secreted mucins is sensitive to alanine substitutions along the polymer backbone.

[0145] Designer mucin domains reveal sequence-specific effects on glycosylation

[0146] We next tested whether new types of sequence-specific mucins could be generated for editing the glycan shield. The parallel goal was to further explore the influence of specific backbone features, including glycosylation site frequency and proline number, on mucin glycosylation patterns. We constructed cell surface mucin cDNAs with GFP reporters for our three designer mucin repeats DAATPAP (SEQ ID NO: 2), DAATPAPP (SEQ ID NO: 3), and PPASTSAPG, as well as KEPAPTTP (SEQ ID NO: 1), which has similarity to secreted human proteoglycan 4 Figure 4 a). Based on the in vitro results, we expected the three designer mucin repeats to be fully glycosylated 20The backbone glycosylation sites (S / T) frequency varied from 12-33%. We also amplified tandem repeats by PCR and reassembled with the original cDNA to multiply the repeat number to 80, yielding extended variants of DAATPAP (SEQ ID NO: 2) and DAATPAPP (SEQ ID NO: 3) mucins. All mucins were well expressed, properly trafficked to the cell surface, and fully modified with O-glycans Figure 4 c and Figure 10 b).

[0147] We analyzed the glycosylation pattern of the mucins by lectin blotting. On the anti-GFP blot, multiple bands were visible for each mucin, revealing a complex distribution of mucin glycoforms on and inside the cell Figure 4 c). Highly glycosylated mucins, as indicated by high PNA and VVA reactivity, typically run as a smear between the highest and second highest molecular weight bands on the anti-GFP blot Figure 4 c, d). These regions are shaded in grey on the electropherograms to aid visualization Figure 4 d). The highest molecular weight glycoforms are highly modified with core 1 glycans Figure 4 d; see PNA). Glycoforms rich in unextended O-glycans are heterogeneous in terms of apparent molecular weight and run in a smear below the core 1 -modified mucins Figure 4 d; compare VVA with PNA).

[0148] We then assessed whether the frequency of O-glycosylation sites could influence the maturation and extension of O-glycans. We quantified the relative ratio of core 1 to Tn antigens in our synthetic mucins by analyzing the ratio of integrated PNA and VVA signals on our lectin blots Figure 4 e). For mucins with 20 or 40 repeats, we saw a significant increase in core 1 structures compared to Tn antigens in the mucin backbone with higher S / T content. However, for backbones with higher S / T content, the glycoform distribution was broader, as indicated by more pronounced smearing on the lectin blots and increased width of the PNA and VVA peaks on the electropherograms Figure 4 c, d).

[0149] We also considered whether the proline content could influence the glycosylation of the mucin backbone, as prolines have been previously reported to promote the interaction of glycosyltransferases with the mucin backbone 7. We compared the glycosylation of DAATPAP (SEQ ID NO: 2) and DAATPAPP (SEQ ID NO: 3) mucins, which differ only in the single proline in each tandem repeat. For mucins with 40 copies of each repeat, the ratio of core 1 glycans to unextended Tn antigens was not significantly different between the two mucins Figure 4 e) However, for mucins with 80 copies of each repeat, the relative core 1 glycan content was significantly lower in the mucin with one additional proline in each repeat Figure 5 f) These results suggest that proline content can affect glycosylation in a way that depends on the overall size of the mucin backbone.

[0150] Engineering mucin glycosylation through cytoplasmic tail

[0151] O-glycan sialylation occurs at least in part in the endosomal and trans-Golgi network after cell surface mucin endocytosis 29 In attempting to use endocytosis and trafficking as potential tools to alter mucin glycosylation, we generated a cDNA "bricks" library of mucin cytoplasmic tails with different endocytosis and trafficking signals. We noted that the Muc1 cytoplasmic domain signals clathrin-mediated endocytosis, while the sequence CQCRRK (SEQ ID NO: 11) at the junction of the transmembrane and cytoplasmic domains signals Muc1 recycling back to the plasma membrane 30 We employed a synthetic 21 amino acid transmembrane anchor (TM21) that can anchor mucins to the plasma membrane without a cytoplasmic tail 31 or with two different cytoplasmic tails in our library to direct more efficient endocytosis 32 .

[0152] To test their function, we fused the TM21 anchor with or without a cytoplasmic tail to a codon scrambled Muc1 with 10 tandem repeats (Muc1_10) Figure 5 a) All mucin cDNAs were transiently transfected into HEK293T. We labeled sialic acids on the cell surface with PAL. On lectin blots, the PAL sialic acid signal was strongest at approximately 171 kDa, overlapping with the strong PNA signal, indicating that the PNA-reactive isoform was also sialic acid rich Figure 5b). To confirm, we treated the cell lysates with sialidase, followed by lectin blot analysis, and analyzed the PNA staining pattern to detect changes in electrophoretic mobility caused by removal of negatively charged sialic acids. Regardless of the cytoplasmic tail motif, the PNA-reactive bands in the mucs were higher and broader after sialidase treatment, indicating that the major PNA-reactive isoforms in all constructs were sialylated Figure 5 c).

[0153] To further analyze the sialylated isoforms, we pulled down the core-1 mucs glycotypes with PNA, followed by probing with Maackia amurensis lectin (MAA), which preferentially binds sialic acids with (a-2,3) linkages 33 Surprisingly, we did not see any MAA signal close to 171 kDa, but noted a very high molecular weight glycotype that was reactive to MAA Figure 5 d top). The MAA-reactive, very high molecular weight glycotype was facilitated by the recycling motif. We found that including the CQC motif caused a 2-fold increase in the MAA / PNA ratio compared to the TM21 anchor alone, and that the longer cytoplasmic tail of Muc1 caused a 3-fold increase in the MAA / PNA ratio Figure 10 d bottom).

[0154] Materials and Methods

[0155] Antibodies and Reagents

[0156] The following antibodies were used: anti-human MUC1 (CD227) (clone HMPV; 555925, BD Biosciences), mouse anti-β-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 kappa binding protein-Horse Radish Peroxidase (HRP; 516102, Santa Cruz), goat anti-mouse IgG (Alexa Fluor 647 conjugated, A-21235; Alexa Fluor 488 conjugated, A-11001; Alexa Fluor 568 conjugated, A-11004; ThermoFisher), and goat anti-chicken IgY (Alexa Fluor 488 conjugated, A-11038; ThermoFisher). TM 647 conjugated, A-21235; Alexa Fluor TM 488 conjugated, A-11001; Alexa Fluor TM 568 conjugated, A-11004; ThermoFisher), and goat anti-chicken IgY (Alexa Fluor 488 conjugated, A-11038; ThermoFisher). TMConjugated; A-11039, Thermo Fisher). Lectins used were: unconjugated Peanut (Arachis hypogaea) agglutinin / Peanut agglutinin (PNA; L0881, Sigma), biotin-conjugated PNA (B-1075, Vector Laboratories), biotin-conjugated Sophora japonica agglutinin (SJA / MAA; BA-7801, EY Lab), fluorescein-labeled succinylated wheat germ agglutinin (s-WGA; FL-1021S, Vector Lab), and biotin-conjugated Vicia villosa agglutinin (VVL, VVA; B-1235, Vector Lab). Fluorescent dyes used were: Alexa Fluor TM 647 NHS ester (A20006, Invitrogen), Alexa Fluor TM 568 NHS ester (A20003, Invitrogen) and AFDye 568 hydroxylamine. Biotinylated lectins were detected using ExtrAvidin-peroxidase (E2886, Sigma) or Neutravidin protein (Dylight 650 conjugated; 84607, Thermo Fisher). For the tetracycline-inducible system, doxycycline was used for induction (204734, Santa Cruz). Streptavidin Dynabeads (3419, Thermo Fisher) were used for immunoprecipitation analysis. Cell lysis buffer (9803) and Reagents and peroxidase (7003) were from Thermo Fisher. Normal goat serum (S-1000) for sample blocking was from Vector Lab. Polyethylenimine (PEI) (25 kDa linear PEI, 23966, Polysciences) was used for FreeStyle TM 293-F cells were transfected.

[0157] Gene design and assembly of MUC1 tandem repeat domains

[0158] cDNAs for cytoplasmic tail deletion human Muc1 (Muc1 dCT) and fusion of Muc1 tandem repeats with synthetic membrane domain TM21 (Muc1 TM21) were generated and cloned into the tetracycline-inducible piggybac expression vector (pPB tetOn Puro) with a puromycin resistance cassette as previously described 27BamHI and EcoRI restriction sites. To generate pPB Muc1 mOxGFP dCT TetOn Puro, the cDNA of mOxGFP (Addgene #68070) was first amplified with primers: 5'-GGCAGCTCAGCTATGGTGTCCAAGGGCGAGGAGCTGT-3' (forward) (SEQ ID NO: 12) and 5'-GGCAGCTGAGCCCTTATACAGCTCGTCCATGCCGTGAGT-3' (reverse) (SEQ ID NO: 13). The PCR product was then cloned into pJET1.2 and non- directionally subcloned into the BlpI site of pPB Muc1 dCT TetOn Puro. To make the cDNA of secreted mucin (sMuc1), a synthetic oligonucleotide containing an IgK signal peptide and a 6x-His-SUMOStar tag (6xHis Sumostar Muc1) was generated by custom gene synthesis (General Biosystems) and cloned into a tetracycline-inducible piggybac expression vector (pPBtetOn Neo) with a neomycin resistance cassette. The lentiviral vector pLV puro Muc1 dCT was generated as previously reported 4 manufactured.

[0159] The cDNAs of the mutant and rationally designed mucin tandem repeats were generated by custom gene synthesis after codon optimization. The minimal repeat gene sequence of the desired mucin repeat was generated using Codon Scrambler (chilkotilab.pratt.duke.edu / codon-scrambler) 18Findings. The scrambled DNA sequence was adjusted for human codon bias by exchanging any codon that is used less than 10% in humans with a randomly selected synonymous codon with higher usage. Synthetic oligonucleotides of the desired tandem repeat were then synthesized by custom gene synthesis (General Biosystems and Genscript) cloned to replace the Muc1 tandem repeat in pPB Muc1 mOxGFP dCT TetOn Puro using BamHI and Bsu36I restriction sites, in pcDNA3.1 Muc1 TM21 using BsrGI and Bsu36I restriction sites, or in pPB 6xHis Sumostar Muc1 using BsrGI and Bsu36I restriction sites (see Supporting Information for cDNA sequences). To generate Muc1 dCT with 42 codon-optimized tandem repeats, a lentiviral vector of the pLV Muc1_42dCT construct, the synthetic cDNA of the codon-optimized repeat was inserted into pLVpuro Muc1 dCT using BamHI and Bsu36I restriction sites. The tandem repeat in pcDNA3.1 Muc1_10TM21 was deleted by Q5 site-directed mutagenesis with 5'-TGGAGGAGCCTCAGGCATACTTTATTG-3' (forward) (SEQ ID NO: 14) and 5'-CCACCGCCGACCGAGGTGACATCCTG-3' (reverse) (SEQ ID NO: 15) primers to generate a Muc1 construct with 0 tandem repeats.

[0160] The cDNA pcDNA3.1 Muc1_10TM21 CQC with the recycling motif CQCRRK (SEQ ID NO: 11) was generated from pcDNA3.1 Muc1_10TM21 by 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 the native CT was subcloned into the XbaI and EcoRI sites of pcDNA3.1 Muc1_10TM21 CQC.

[0161] PCR and Golden Gate assembly to extend synthetic tandem repeats

[0162] Golden Gate assembly was used to size multiply the 40 tandem repeats of DAATPAP (SEQ ID NO: 2) and DAATPAPP (SEQ ID NO: 3) mucin cDNAs in pcDNA3.1 to 80 repeats. Two pairs of custom primers for the tandem repeats and full mucin vectors were designed to ligate the BsmBI recognition site with unique 4bp overhangs such that the PCR products of the 40 tandem repeats and full mucin expression vectors would ligate in the Golden Gate assembly reaction to expand the number of tandem repeats (Table S2). Golden Gate assembly reactions were performed as previously reported 47 were performed.

[0163] Cell lines, culture, and transfection

[0164] MCF10A human mammary epithelial cells and HEK293T SV40-transformed human embryonic kidney cells were obtained from ATCC. MCF10A cells were cultured in DMEM / F12 medium (Thermo Fisher) supplemented with 5% horse serum (Thermo Fisher), 20 ng / mL EGF (Peprotech), 10 pg / mL insulin (Sigma), 500 ng / mL hydrocortisone (Sigma), and 100 ng / mL cholera toxin (Sigma). HEK293T cells were cultured in DMEM (Thermo Fisher) supplemented with 10% fetal bovine serum (Thermo Fisher). Cells were maintained at 37°C, 5% CO2, and 90% relative humidity (RH). FreeStyle TM 293-F cells were cultured in suspension in FreeStyle TM 293 expression medium (Thermo Fisher). Suspension cultures were maintained in an orbital shaker at 37°C, 8% CO2, and 90% RH. Lentiviral transduction was performed in MCF10A cells with a stably integrated gene cassette expressing the tetracycline transactivator rtTA-M2 and neomycin resistance gene as previously reported 48 HEK293T cells were transiently transfected with the calcium phosphate method according to standard protocols. FreeStyle TM 293-F cells were transiently transfected with PEI as previously described 49 CRISPR / Cas9-mediated COSMC knockout was generated in MCF10A Muc1 dCT cells as previously reported 50 .

[0165] Western blot analysis

[0166] HEK293T cells were plated at 55,000 cells / cm 2Seeded and transfected with calcium phosphate for 24-36 hours, then lysed with cell lysis buffer. MCF10A cells were seeded at 20,000 cells / cm 2 Seeded and induced with 0.2 pg / mL doxycycline for 24 hours, then lysed with cell lysis buffer. Lysates were resolved on NuPAGE 3-8% or 7% Tris-acetate gels, transferred to PVDF membranes. Primary antibodies were diluted 1 : 1000 in 5% BSA TBST, fluorophore-conjugated or biotinylated lectins were diluted to 2 pg / mL, and incubated overnight at 4°C. Secondary antibodies ExtrAvidin-HRP or Neutravidin-Dylight 650 were diluted 1 :2000 or 1 pg / mL in 5% BSA TBST, incubated for 1 hour at room temperature. Bands were imaged on a ChemiDoc MP imaging system (Bio-Rad) or on a Typhoon FLA 9500 (GE Healthcare) using the appropriate laser and emission filters. Blots were imaged after development with reagents and peroxide. Quantification of band intensities was performed using the FIJI distribution of ImageJ 51,52 Statistical significance of differences in the data was calculated using one-way ANOVA with repeated measures or two-tailed t-tests.

[0167] Periodate labeling of cell surface sialic acids

[0168] HEK293T cells were collected 36 hours after transfection. Cells were washed with cold DPBS containing Ca 2+ and Mg 2+ , followed by incubation with a 1 mM sodium periodate (Sigma) solution in DPBS for 10 minutes. The periodate was quenched with a 1 mM glycerol solution in cold DPBS and washed with cold DPBS. Samples were stained with 25 pM AFDye-568-hydroxylamine (Fluoroprobes) in the presence of 10 mM aniline (Sigma) in sterile-filtered DPBS + 5% FBS pH 6.7 for 30 minutes at 4°C with gentle agitation in the dark.

[0169] Immunoprecipitation

[0170] HEK293T cells were seeded at 55,000 cells / cm 2 and transfected with the calcium phosphate method for 24-36 hours, then lysed with cell lysis buffer. Lysates were incubated with 125 pg / mL biotinylated lectin PNA at 4°C with gentle rocking overnight. Streptavidin beads were added to the cell lysate, and the suspension was incubated at 4°C for 3 hours. The beads were washed twice with lysis buffer, then resuspended in 4x LDS loading buffer. The resuspension was then analyzed by western blot.

[0171] Sialidase treatment of HEK293T

[0172] HEK293T cells were collected 24 hours after transfection and incubated with Arthrobacter ureafaciens sialidase (Roche, 10 mU, 100 μl final volume) in sialidase buffer at 37°C. 53 Incubate together for 30 minutes, then lyse with cell lysis buffer.

[0173] Immunofluorescence

[0174] HEK293T cells were injected at a rate of 45,000 cells / cm³. 2 The samples were inoculated and transfected with calcium phosphate for 24 hours, followed by fixation with 4% paraformaldehyde. The antibody was diluted 1:100 in PBS solution with 5% normal goat serum and incubated overnight at 4°C. The lectin was diluted to 2 μg / mL in PBS solution with 5% normal goat serum and incubated at room temperature for 2 hours. The samples were imaged using a 40× water immersion objective (NA 1.1) on a Zeiss LSM inverted 880 confocal microscope.

[0175] Expression and purification of secretory mucin

[0176] 16.25 μg pPB 6×His Sumostar Muc1 DNA was transfected into HEK293T cells in a 10 cm culture dish for 48 hours. 30 μg pPB 6×His Sumostar Muc1 DNA was transfected into 20 mL FreeStyle... TM 293-F cell cultures were cultured for 4 days. The culture medium was collected and clarified by centrifugation at 2000 rpm for 5 minutes. The clarified medium was incubated overnight at 4°C with Ni-NTA agarose (Qiagen), 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, 250 mM imidazole). The eluted sample was permeated into PBS through an Amicon Ultra-4 centrifuge filter (10 kDa cutoff), and then purified using Zeba. TM Desalting was performed using a purification and desalting column (7K MWCO). The salt-free protein solution was lyophilized and stored at -80°C.

[0177] O-glycan profile analysis of secretory mucin

[0178] All reagents were purchased from Sigma unless otherwise mentioned. Purified mucins (600 pg each) were denatured by heating at 100 °C for 5 min. The denatured proteins were then treated with 500 pL of 50 mM sodium hydroxide (NaOH) solution containing 19 mg of sodium borohydride (NaBH4) at 45 °C for 18 h 54 The samples were cooled, neutralized with 10% acetic acid, passed through a Dowex H+ resin column, and lyophilized under a stream of nitrogen to remove boronate. The previously reported method 55 was used to make the glycans fully methylated for structural characterization by mass spectrometry. Briefly, the dried eluate was dissolved in dimethyl sulfoxide (DMSO) and methylated by using iodomethane and NaOH-DMSO base (prepared by mixing DMSO with 50% w / w NaOH solution). The reaction was quenched with water, the reaction mixture was extracted with dichloromethane and dried. The fully methylated glycans were dissolved in methanol and crystallized with a-dihydroxybenzoic acid (DHBA, 20 mg / mL in 50% v / v methanol:water) matrix. The samples were analyzed for the presence of glycans by MALDI-TOF / TOF-MS in positive ion mode using an AB SCIEX TOF / TOF 5800 (Applied Biosystem MDS Analytical Technologies) mass spectrometer. The fully methylated glycans from the samples were infused onto an Orbitrap Fusion Tribrid mass spectrometer via an ESI probe with HCD and CID fragmentation options for further structural confirmation. MS1 and MS2 spectra of the glycans were acquired by simple parent ion scans at high resolution, and the corresponding ions were manually selected for further MS / MS scans. Glycan structures were assigned manually based on fragmentation patterns and common biosynthetic pathways and by using the Glycoworkbench software.

[0179] Cell O-glycome reporter / amplification (CORA)

[0180] All chemicals were purchased from Millipore Sigma unless stated otherwise. Solvents were HPLC grade or higher and included 0.1% (v / v) trifluoroacetic acid in all chromatography steps. Benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside (BnGalNAc) was fully acetylated by heating in a molar excess of 33% (v / v) acetic anhydride in dry pyridine at 65 °C for 1 h. The product was dried by speedvac (Thermo Scientific SPD1010) and used without further purification. Full acetylation was confirmed by LC-MS (Agilent 1100 series LC and G1956B MS, m / z calcd for: 438.18, obsd: 438.10 [M+H]+).

[0181] CORA was as previously reported 28were performed. Briefly, 500,000 HEK293T cells were seeded into 6 cm dishes and transfected as above. Following transfection, cultures were incubated in complete media supplemented with 50 mM peracetylated BnGalNAc. After 48 hours, media was aspirated and loose cells and debris were removed by centrifugation. Supernatants were then filtered (Millipore Amicon Ultra 4, 10 kDa MWCO) and benzyl glycans were collected by gravity chromatography (Waters Sep-Pak C18 3cc). Eluates were dried by speedvac, followed by permethylation 2. Fresh sodium hydroxide slurry in DMSO was prepared and 200 pL was added to each dried sample, followed by 100 pL iodomethane (ACROS). Samples were mixed continuously for 10 minutes, followed by reaction termination by addition of 600 pL deionized water. Permethylated benzyl glycans were recovered by extraction with 200 pL chloroform, which was then washed with 800 pL deionized water 4 times. Samples were further purified by C18 gravity chromatography (Waters Sep-Pak C18 1cc) and dried by speedvac. Dried samples were dissolved in 50% methanol and spotted with 10 mg / mL 2,5-dihydrobenzoic acid in 50% acetonitrile as matrix 1 : 1 (v / v). Benzyl glycans were analyzed using MicroFlex MALDI-TOF-MS (Bruker) in positive ion mode. Two external standards, per-methylated maltotetraose (Cayman Chemical, m / z calc: 885.43, obs: 885.65 [M+Na]+) and maltoheptaose (Cayman Chemical, m / z calc: 1497.73, obs: 1497.90 [M+Na]+) were included to confirm instrument performance and calibration. Benzyl glycan compositions were assigned based on the predicted mass of the sodium adduct ([M+Na]+) of known structures. Data were analyzed using Mnova (Mestrelab Research) and presentations were prepared with Prism 8 (GraphPad).

[0182] Discussion of Part I

[0183] O-glycosylation of mucins determines their physical and biochemical characteristics, and thus their biological functions. Part I provides a genetically encoded system to edit mucin biopolymers and can be used as a tool for glycosphere engineering, among other important utilities discussed above. Factors known to influence mucin glycosylation include the cellular lineage of the glycosyltransferases and their substrates 1,34 the frequency of O-glycosylation sites on polypeptide scaffolds 35,36 the primary peptide sequence surrounding O-glycosylation sites 37-39 and the trafficking of glycoproteins 32,40,41In this Part I, we modified the mucin backbone sequence and signals and motifs in the cytoplasmic tail to encode mucins with different physical characteristics, backbone chemistry, and glycosylation patterns.

[0184] Using codon degeneracy to design mucin cDNAs with minimal repeats, we were able to apply custom gene synthesis to construct 13 representative unique mucin repeats, each of which can be readily combined with other functional domains for cell surface anchoring and trafficking control. All test repeat sequences were manufactured successfully, with no failures. Thus, the disclosure includes the use of the described design strategy to generate other constructs as described herein. By combining these cDNAs with other functional cDNA "bricks" in a modular fashion, mucins with modified structure and function can be readily constructed with known molecular techniques given the benefit of the disclosure, including Gibson assembly, Golden Gate assembly, and other modern DNA assembly methods.

[0185] One observation in this Part I is that extension of O-glycans from Tn antigens to core 1 / 2 glycans is prevented by alanine substitution along the polymer backbone. Given the observed effects in both membrane-associated and secreted mucins, altered endocytosis and trafficking can not explain the difference in glycan maturation. It is also unlikely that the potential impact of mucin overexpression on the function of T synthase and other glycosyltransferases involved in early O-glycan extension explains the difference in glycosylation. Similar core 1 or core 2 glycan structures were observed in both overexpressing mucins and wild-type HEK293T as shown by cell O-glycome reporter / amplification analysis Figure 2 ).

[0186] The O-glycosylation analysis section in this Part 1 is based on lectin blotting. Controls were used to validate the lectin-based analysis. Knocking out COSMC to eliminate glycan extension caused PNA binding to decrease and VVA staining to increase, indicating the appropriateness of these lectins for detecting core 1 O-glycans and Tn antigens, respectively Figure 3 d) O-glycome analysis of purified mucins also validated the conclusions of the lectin analysis based on the types of glycan structures present on the mucins Index f).

[0187] We modified the mucin cytoplasmic tail for glycoengineering. Based on the change in electrophoretic mobility after sialidase treatment, we concluded that the recycling motif is not required for mucin sialylation. However, including the recycling motif promoted the production of a super high molecular weight mucin glycoform that reacted with MAA lectin. It is considered that swapping the mucin cytoplasmic tail can be a viable strategy to engineer at least some of the emerging glycoforms.

[0188] Table S1: Repeat analysis of mucin cDNA sequences

[0189] Tandem Repeat Finder algorithm 1 Repeat analysis was performed on native and codon scrambled cDNA. Nucleotide sequence matches were scored +2 and mismatches and indels were scored -7 to measure the identity between the query sequence and the detected tandem repeat. High alignment scores indicate high levels of repeat for the repeat.

[0190] Native_Muc1

[0191] Cycle size Copy number Common size Match percentage Indel percentage Score Index 6-2577 60 42.9 60 99 0 4982

[0192] Muc1_42

[0193] Cycle size Copy number Common size Match percentage Indel percentage Score Figure 12 146-468 60 5.4 60 75 3 220 146-468 120 2.7 120 80 2 328 149-513 120 3.0 120 79 5 294 728-897 60 2.8 60 80 1 171 746-984 60 4.0 60 75 4 169 1013-1233 60 3.7 60 77 0 208 794-1200 120 3.4 120 75 4 273 1205-1347 60 2.4 59 74 8 135 1097-1530 180 2.4 180 77 2 379 1304-1521 60 3.6 59 76 2 175 1514-1714 60 3.3 60 78 0 204 1709-1965 120 2.1 120 80 1 273 1781-2067 60 4.8 60 71 5 177 1733-2067 120 2.8 120 77 1 269 2150-2406 60 4.3 60 73 3 140 2222-2439 120 1.8 120 79 2 258

[0194] Table Explanation:

[0195] • Index of the repeat relative to the start of the sequence.

[0196] • Period size of the repeat.

[0197] • Number of copies aligned to the consensus pattern.

[0198] • Size of the consensus pattern (may differ slightly from the period size).

[0199] • Percent match between adjacent copies overall.

[0200] • Percent indels between adjacent copies overall.

[0201] • Alignment score.

[0202] References:

[0203] (1) Benson, G. Tandem Repeats Finder: A Program to Analyze DNA Sequences. Nucleic Acids Research 1999, 27 (2), 573-580.

[0204] Table S2: Golden Gate assembly primers.

[0205]

[0206]

[0207] Summary of cDNA “biobricks” as described in Part I

[0208] Summary of cDNA "biobricks" as described in Part I.

[0209] Leader tag

[0210] 1. Native FLAG

[0211] Amino acid sequence:

[0212] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNADYKDDDDLY (SEQ ID NO: 26)

[0213] cDNA sequence:

[0214] GGATCCATGACACCGGGCACCCAGTCTCCTTTCTTCCTGCTGCTGCTCCTCACAGTGCTTACAGTTGTTACAGGTTCTGGTCATGCAAGCTCTACCCCAGGTGGAGAAAAGGAGACTTCGGCTACCCAGAGAAGTTCAGTGCCCAGCTCTACTGAGAAGAATGCTGATTACAAGGATGACGACGACCTGTACA (SEQ ID NO: 27)

[0215] 2. His-SUMO

[0216] Amino acid sequence:

[0217] METDTLLLWVLLLWVPGSTGDGHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQADQAPEDLDMEDNDIIEAHREQIGGGSGSGHASSTPGGEKETSATQRSSVPSSTEKNADYKDDDDLY (SEQ ID NO: 28)

[0218] cDNA sequence:

[0219] GGATCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGGTCATCACCATCATCATCACGGGTCCCTGCAGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAACGTTCTTGTACGACGGTATTGAAATTCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGAGGTGGCTCCGGCTCCGGTCATGCAAGCTCTACCCCAGGTGGAGAAAAGGAGACTTCGGCTACCCAGAGAAGTTCAGTGCCCAGCTCTACTGAGAAGAATGCTGATTACAAGGATGACGACGACCTGTACA (SEQ ID NO:29)

[0220] In the representative polymer backbone segment sequences presented immediately below, the repeat sequence is preceded by the sequence LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV (SEQ ID NO:30), the relevant repeat sequence is designated with the relevant SEQ ID and its repeat number is designated with a subscript in parentheses, the subscript indicating the repeat number. The alphanumeric designation given above each sequence is the sequence name, not the sequence itself.

[0221] Polymer backbone

[0222] 1. Codon scrambled Muc1 x42 (Muc1_42)

[0223] 2. Amino acid sequence:

[0224] 3. LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [PDTRPAPGSTAPPAHGVTSA] 42 ASG (SEQ ID NO:30 [SEQ ID NO:8]42 ASG

[0225] cDNA sequence:

[0226]

[0227] 4. Codon scrambled Muc1 x21 (Muc1_21)

[0228] Amino acid sequence: LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV[PDTRPAPGSTAPPAHGVTSA] 21 ASG (SEQ ID NO: 30) [SEQ ID NO: 8] 21 ASG

[0229] cDNA sequence:

[0230]

[0231] 5. Codon scrambled Muc1 x10 (Muc1_10)

[0232] Amino acid sequence:

[0233] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV[PDTRPAPGSTAPPAHGVTSA] 10 ASG (SEQ ID NO: 30) [SEQ ID NO: 8] 10 ASG

[0234] cDNA sequence:

[0235] TGTACATGGACATGGTCGCTGTGAGTATGACCAGCAGCGTACTCTCCAGCCACAGCCCCGGTTCAGGCTCCTCCACCACTCAGGGACAGGATGTCACTCTGGCCCCGGCCACGGAACCAGCTTCAGGTTCAGCTGCCACCTGGGGACAGGATGTCACCTCGGTCCCAGATACAAGACCGGCCCCAGGATCTACGGCTCCTCCGGCTCATGGAGTCACTTCTGCTCCAGACACAAGGCCCGCGCCGGGTTCTACAGCACCGCCTGCTCATGGTGTTACTAGCGCACCCGATACGAGACCTGCTCCGGGATCAACGGCACCTCCTGCCCACGGGGTAACATCTGCACCGGACACTCGCCCTGCGCCCGGTTCAACCGCTCCACCCGCACACGGAGTGACAAGCGCTCCTGACACTAGACCAGCACCAGGTTCTACAGCCCCACCAGCCCATGGAGTTACCAGTGCACCAGATACTAGGCCAGCTCCAGGTAGTACTGCACCCCCAGCTCATGGGGTTACATCAGCTCCCGACACGCGACCAGCTCCTGGAAGCACTGCCCCTCCAGCTCACGGTGTGACCTCAGCACCTGATACACGCCCTGCACCTGGCTCTACTGCTCCCCCCGCTCATGGCGTAACTAGTGCCCCGGATACTCGACCCGCCCCTGGTTCCACAGCTCCGCCAGCACATGGTGTAACAAGTGCTCCTGATACCCGACCAGCGCCTGGAAGTACCGCACCACCTGCACATGGAGTAACTTCAGCCGCCTCAGG (SEQ ID NO: 52)

[0236] 6. Codon scrambled Muc1x0 (Muc1_0)

[0237] Amino acid sequence: LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVGGGGGASG (SEQ ID NO: 31)

[0238] cDNA sequence:

[0239] TGTACATGGACATGGTCGCTGTGAGTATGACCAGCAGCGTACTCTCCAGCCACAGCCCCGGTTCAGGCTCCTCCACCACTCAGGGACAGGATGTCACTCTGGCCCCGGCCACGGAACCAGCTTCAGGTTCAGCTGCCACCTGGGGACAGGATGTCACCTCGGTCGGCGGTGGTGGAGGAGCCTCAGG (SEQ ID NO: 99)

[0240] 7. Codon scrambled Muc1 monoglycosylation mutant x21 (Muc1_21S) amino acid sequence:

[0241] 8. LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV[PDTRPAPGATAPPAHGVTSA] 21 ASG (SEQ ID NO: 30) [SEQ ID NO: 5] 21 ASG

[0242]

[0243] 9. Codon scrambled Muc1 double glycosylation mutant x21 (Muc1_21D)

[0244] Amino acid sequence:

[0245] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV[PDTRPAPGATAPPAHGVTAA] 21 ASG (SEQ ID NO: 30) [SEQ ID NO: 6] 21 ASG

[0246]

[0247] 10. Codon scrambled Muc1 tri-glycosylation mutant x21 (Muc1_21T) amino acid sequence:

[0248] 11. LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV[PDARPAPGATAPPAHGVTAA] 21 ASG (SEQ ID NO: 30 [SEQ ID NO: 7] 21 ASG

[0249] cDNA sequence:

[0250]

[0251] 12. Lubricin consensus, KEPAPTTP x 20 (Syn4_20)

[0252] Amino acid sequence:

[0253] 13. LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [KEPAPTTP] 20 ASG (SEQ ID NO: 30) [SEQ ID NO: 1] 20 ASG

[0254] cDNA sequence:

[0255] TGTACATGGACATGGTCGCTGTGAGTATGACCAGCAGCGTACTCTCCAGCCACAGCCCCGGTTCAGGCTCCTCCACCACTCAGGGACAGGATGTCACTCTGGCCCCGGCCACGGAACCAGCTTCAGGTTCAGCTGCCACCTGGGGACAGGATGTCACCTCGGTCAAGGAACCTGCACCTACAACCCCGAAGGAGCCCGCACCGACCACCCCAAAAGAACCTGCGCCGACAACTCCAAAGGAGCCAGCTCCAACGACGCCAAAGGAACCAGCACCTACGACCCCCAAGGAACCCGCCCCGACGACTCCGAAGGAGCCTGCACCAACAACTCCTAAAGAACCAGCGCCTACTACGCCTAAAGAACCTGCTCCTACTACACCAAAAGAGCCAGCACCCACGACACCGAAAGAACCTGCCCCTACTACCCCTAAAGAACCCGCTCTACCACACCAAAGGAACCGGCTCCCACTACTCCCAAAGAACCAGCCCCAACTACACCTAAAGAACCGGCCCCCACCACTCCTAAAGAGCCGGCGCCAACTACTCCAAAAGAACCAGCTCCTACAACTCCCAAGGAGCCGGCACCTACTACTCCGAAAGAGCCCGCGCCCACAACACCCAAAGAGCCTGCTCCGACTACTCCTGCCTCAGG (SEQ ID NO: 55)

[0256] 14. Syn1, DAATPAP x 40 (Synl_40)

[0257] Amino acid sequence:

[0258] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [DAATPAP] 40 ASG (SEQ ID NO: 30) [SEQ ID NO: 2] 40 ASG

[0259] cDNA sequence:

[0260]

[0261] 15. Syn 1, DAATPAP x 80 (Synl_80)

[0262] Amino acid sequence:

[0263] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [DAATPAP] 80 ASG (SEQ ID NO: 30) [SEQ ID NO: 2] 80 ASG

[0264] cDNA sequence:

[0265]

[0266] 16. Synthesis 2, DAATPAPP x 40 (Syn2_40)

[0267] Amino acid sequence:

[0268] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [DAATPAPP] 40 ASG (SEQ ID NO: 30) [SEQ ID NO: 3] 40 ASG

[0269] cDNA sequence:

[0270]

[0271] 17. Syn2, DAATPAPP x 80 (Syn2_80)

[0272] Amino acid sequence:

[0273] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [DAATPAPP] 80 ASG (SEQ ID NO: 30) [SEQ ID NO: 3] 80 ASG

[0274] cDNA sequence:

[0275]

[0276] 18. Synthesis 3, PPASTSAPG x 40 (Syn3_40)

[0277] Amino acid sequence:

[0278] LYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSV [PPASTSAPG] 40 ASG (SEQ ID NO: 30) [SEQ ID NO: 4] 40 ASG

[0279] cDNA sequence:

[0280]

[0281] optical reporter

[0282] 1. mOxGFP

[0283] Amino acid sequence:

[0284] SGSASGSAMVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSA (SEQ ID NO: 31)

[0285] cDNA sequence:

[0286] CCTCAGGCTCTGCATCAGGCTCAGCTATGGTGTCCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCTCCGTGCGGGGCGAGGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCAGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGAGCTTCTCCCGCTACCCCGACCACATGAAGCGCCACGACTTCTTCAAGAGCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCCTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTTCAACTCCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACGTGGAGGACGGCTCCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGTCCACCCAGTCCAAGCTGTCCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTTCTGGAATTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTATAAGGGCTC AGC (SEQ ID NO: 61)

[0287] Membrane anchor

[0288] 1. Native TM

[0289] Amino acid sequence:

[0290] SASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRK* (SEQ ID NO: 32)

[0291] cDNA sequence:

[0292] GCTCAGCTTCTACTCTGGTGCACAACGGCACCTCTGCCAGGGCTACCACAACCCCAGCCAGCAAGAGCACTCCATTCTCAATTCCCAGCCACCACTCTGATACTCCTACCACCCTTGCCAGCCATAGCACCAAGACTGATGCCAGTAGCACTCACCATAGCTCGGTACCTCCTCTCACCTCCTCCAATCACAGCACTTCTCCCCAGTTGTCTACTGGGGTCTCTTTCTTTTTCCTGTCTTTTCACATTTCAAACCTCCAGTTTAATTCCTCTCTGGAAGATCCCAGCACCGACTACTACCAAGAGCTGCAGAGAGACATTTCTGAAATGTTTTTGCAGATTTATAAACAAGGGGGTTTTCTGGGCCTCTCCAATATTAAGTTCAGGCCAGGATCTGTGGTGGTACAATTGACTCTGGCCTTCCGAGAAGGTACCATCAATGTCCACGACGTGGAGACACAGTTCAATCAGTATAAAACGGAAGCAGCCTCTCGATATAACCTGACGATCTCAGACGTCAGCGTGAGTGATGTGCCATTTCCTTTCTCTGCCCAGTCTGGGGCTGGGGTGCCAGGCTGGGGCATCGCGCTGCTGGTGCTGGTCTGTGTTCTGGTTGCGCTGGCCATTGTCTATCTCATTGCCTTGGCTGTCTGTCAGTGCCGCCGAAAGTAGGGAATTC (SEQ ID NO: 62)

[0293] 2. Synthesis of TM TM21

[0294] Amino acid sequence:

[0295] ASGILYWRNPTESDSIVLAIIVPSLLLLLCLALLWYMRRRSM* (SEQ ID NO: 49) cDNA sequence:

[0296] CCTCAGGCATACTTTATTGGCGAAACCCAACGGAAAGTGATAGCATCGTTTTGGCAATTATCGTCCCCAGTCTGCTCCTCTTGCTCTGCCTGGCTTTGTTGTGGTACATGCGCCGACGAAGTATGTAGGAATTC (SEQ ID NO: 63)

[0297] Cytoplasmic motif

[0298] 1. Native CT

[0299] Amino acid sequence:

[0300] SRCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL* (SEQ ID NO: 33)

[0301] cDNA sequence:

[0302] TCTAGATGTCAGTGCCGCCGAAAGAACTACGGGCAGCTGGACATCTTTCCAGCCCGGGATACCTACCATCCTATGAGCGAGTACCCCACCTACCACACCCATGGGCGCTATGTGCCCCCTAGCAGTACCGATCGTAGCCCCTATGAGAAGGTTTCTGCAGGTAAtGGTGGCAGCAGCCTCTCTTACACAAACCCAGCAGTGGCAGCCGCTTCTGCCAACTTGTAGGAATTC (SEQ ID NO: 64)

[0303] 2. CQC

[0304] Amino acid sequence:

[0305] SRCQCRRK* (SEQ ID NO: 34)

[0306] cDNA sequence:

[0307] TCTAGATGTCAGTGCCGCCGAAAGTAGGAATTC (SEQ ID NO:65)

[0308] Construct List

[0309] Membrane-associated mucin

[0310] 1. pcDNA3.1+ Muc1_0_TM21

[0311] 2. pcDNA3.1+ Muc1_10_TM21

[0312] 3. pcDNA3.1+ Muc1_21_TM21

[0313] 4. pcDNA3.1+ Muc1_42_TM21

[0314] 5. pcDNA3.1+ Muc1_21S_TM21

[0315] 6. pcDNA3.1+ Muc1_21D_TM21

[0316] 7. pcDNA3.1+ Muc1_21T_TM21

[0317] 8. pcDNA3.1+ Muc1_10_TM21_CT

[0318] 9. pcDNA3.1+ Muc1_10_TM21_CQC

[0319] 10. pcDNA3.1+ Muc1_10_dCT

[0320] 11. pcDNA3.1+ Muc1_10_FL

[0321] 12. pcDNA3.1+ Muc1_Syn4_20_TM21

[0322] 13. pcDNA3.1+ Muc1_Syn1_40_TM21

[0323] 14. pcDNA3.1+ Muc1_Syn2_40_TM21

[0324] 15. pcDNA3.1+ Muc1_Syn3_40_TM21

[0325] 16. pcDNA3.1+ Muc1_Syn1_80_TM21

[0326] 17. pcDNA3.1+ Muc1_Syn2_80_TM21

[0327] 18. pPB_Tet_Muc1_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0328] 19. pPB_Tet_Muc1_42_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0329] 20. pPB_Tet_Muc1_21_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0330] 21. pPB_Tet_Muc1_10_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0331] 22. pPB_Tet_Muc1_0_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0332] 23. pPB_Tet_Muc1_21D_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0333] 24. pPB_Tet_Muc1_21T_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0334] 25. pLV_puro_teton_Muc1_42_dCT

[0335] 26. pLV_puro_teton_Muc1_dCT

[0336] 27. pPB_Muc1_mOxGFP_dCT_BlpI

[0337] 28. pPB_Muc1_42_mOxGFP_dCT_BlpI

[0338] 29. pPB_Muc1_21_mOxGFP_dCT_BlpI

[0339] 30. pPB_Muc1_10_mOxGFP_dCT_BlpI

[0340] 31. pPB_Muc1_0_mOxGFP_dCT_BlpI

[0341] 32. pPB_Muc1_21S_mOxGFP_dCT_BlpI

[0342] 33. pPB_Muc1_21D_mOxGFP_dCT_BlpI

[0343] 34. pPB_Muc1_21T_mOxGFP_dCT_BlpI

[0344] 35. pPB_Muc1_Syn4_20_mOxGFP_dCT_BlpI

[0345] 36. pPB_Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0346] 37. pPB_Muc1_Syn2_40_mOxGFP_dCT_BlpI

[0347] 38. pPB_Muc1_Syn3_40_mOxGFP_dCT_BlpI

[0348] 39. pPB_Muc1_Syn1_80_mOxGFP_dCT_BlpI

[0349] 40. pPB_Muc1_Syn2_80_mOxGFP_dCT_BlpI

[0350] Secreted mucin

[0351] 41. pPB_Tet_SumoStar_Muc1_42_rtTAsM2_IRES_NeoR

[0352] 42. pPB_Tet_SumoStar_Muc1_21T_rtTAsM2_IRES_NeoR

[0353] 43. pPB_Tet_SumoStar_Muc1_21D_rtTAsM2_IRES_NeoR

[0354] 44. pPB_Tet_SumoStar_Muc1_21S_rtTAsM2_IRES_NeoR

[0355] 45. pPB_Tet_SumoStar_Muc1_21_rtTAsM2_IRES_NeoR

[0356] 46. pPB_Tet_SumoStar_Muc1_0_rtTAsM2_IRES_NeoR

[0357] 47. pPB_Tet_SumoStar_Muc1_Synl_40_rtTAsM2_IRES_NeoR

[0358] 48. pPB_Tet_SumoStar_Muc1_Syn2_40_rtTAsM2_IRES_NeoR

[0359] 49. pPB_Tet_SumoStar_Muc1_Syn3_40_rtTAsM2_IRES_NeoR

[0360] 50. pPB_Tet_SumoStar_Muc1_Synl_80_rtTAsM2_IRES_NeoR

[0361] 51. pPB_Tet_SumoStar_Muc1_Syn2_80_rtTAsM2_IRES_NeoR

[0362] The following sequences are representative amino acid sequences of mucin and lubricin constructs as further described herein, as well as their complete sequences, including the N-terminal signal sequence, the tandem repeat domain, the fluorescent optical reporter (in some of these sequences, green fluorescent (GFP)), the transmembrane domain to the cytoplasmic tail domain. In embodiments, the modified lubricin omits the transmembrane domain, the cytoplasmic tail, the domain, and the optical reporter. It is recognized that the GFP sequence can be omitted or replaced by any other amino acid sequence, including but not limited to other detectable proteins or second polypeptides as described above. The alphanumeric name given above each sequence is the sequence name, not the sequence itself.

[0363] 1. PDTRPAPGSTAPPAHGVTSA_42

[0364] Muc1_42_mOxGFP_dCT_BlpI

[0365]

[0366] 2. PDTRPAPGSTAPPAHGVTSA_21

[0367] Muc1_21_mOxGFP_dCT_BlpI

[0368]

[0369] 3. PDTRPAPGSTAPPAHGVTSA_10

[0370] Muc1_10_TM21_CT

[0371] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNADYKDDDDLYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAASGILYWRNPTESDSIVLAIIVPSLLLLLCLALLWYSRCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL* (SEQ ID NO: 37)

[0372] 4. PDTRPAPGSTAPPAHGVTSA_0

[0373] Muc1_0_mOxGFP_dCT_BlpI

[0374] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNADYKDDDDLYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVGGGGGASGSASGSAMVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRK* (SEQ ID NO: 38)

[0375] 5. PDTRPAPGATAPPAHGVTSA_21

[0376] Muc1_21S_mOxGFP_dCT_BlpI

[0377]

[0378] 6. PDTRPAPGATAPPAHGVTAA_21

[0379] Muc1_21D_mOxGFP_dCT_BlpI

[0380]

[0381] 7. PDAR PAPGATAPPAHGVTAA_21

[0382] Muc1_21T_mOxGFP_dCT_BlpI

[0383]

[0384] 8.KEPAPTTP_20(Syn4_20)

[0385] Muc1_Syn4_20_mOxGFP_dCT_BlpI

[0386] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNADYKDDDDLYMDMVAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPKEPAPTTPASGSASGSAMVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRK*(SEQID NO:42)

[0387] 9.DAATPAP_40(Syn1_40)

[0388] Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0389] *(SEQ ID NO:43)

[0390] 10.DAATPAP_80(Syn1_80)

[0391] Muc1_Syn1_80_mOxGFP_dCT_BlpI

[0392]

[0393] 11. DAATPAPP_40 (Syn2_40)

[0394] Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0395] *

[0396] (SEQ ID NO:45)

[0397] 12.DAATPAPP_80(Syn2_80)

[0398] Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0399]

[0400] 13. PPASTSAPG_40 (Syn3_40)

[0401] Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0402] *(SEQ ID NO:47)

[0403] 14. PPASTSAPG_80 (Syn3_80)

[0404] Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0405]

[0406] List of constructs used in Part I

[0407] Membrane-associated mucins

[0408] 52. pcDNA3.1+_Muc1_0_TM21

[0409] 53. pcDNA3.1+_Muc1_10_TM21

[0410] 54. pcDNA3.1+_Muc1_21_TM21

[0411] 55. pcDNA3.1+_Muc1_42_TM21

[0412] 56. pcDNA3.1+_Muc1_21S_TM21

[0413] 57. pcDNA3.1+_Muc1_21D_TM21

[0414] 58. pcDNA3.1+_Muc1_21T_TM21

[0415] 59. pcDNA3.1+_Muc1_10_TM21_CT

[0416] 60. pcDNA3.1+_Muc1_10_TM21_CQC

[0417] 61. pcDNA3.1+_Muc1_10_dCT

[0418] 62. pcDNA3.1+_Muc1_10_FL

[0419] 63. pcDNA3.1+_Muc1_Syn4_20_TM21

[0420] 64. pcDNA3.1+_Muc1_Syn1_40_TM21

[0421] 65. pcDNA3.1+_Muc1_Syn2_40_TM21

[0422] 66. pcDNA3.1+_Muc1_Syn3_40_TM21

[0423] 67. pcDNA3.1+_Muc1_Syn1_80_TM21

[0424] 68. pcDNA3.1+_Muc1_Syn2_80_TM21

[0425] 69. pcDNA3.1+ Muc1 Syn3 80 TM21

[0426] 70. pPB_Tet_Muc1_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0427] 71. pPB_Tet_Muc1_42_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0428] 72. pPB_Tet_Muc1_21_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0429] 73. pPB_Tet_Muc1_10_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0430] 74. pPB_Tet_Muc1_0_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0431] 75. pPB_Tet_Muc1_21D_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0432] 76. pPB_Tet_Muc1_21T_TM21_IRES2_copGFP_rtTAsM2_IRES_NeoR

[0433] 77. pLV_puro_teton_Muc1_42_dCT

[0434] 78. pLV_puro_teton_Muc1_dCT

[0435] 79. pPB_Muc1_mOxGFP_dCT_BlpI

[0436] 80. pPB_Muc1_42_mOxGFP_dCT_BlpI

[0437] 81. pPB_Muc1_21_mOxGFP_dCT_BlpI

[0438] 82. pPB_Muc1_10_mOxGFP_dCT_BlpI

[0439] 83. pPB_Muc1_0_mOxGFP_dCT_BlpI

[0440] 84. pPB_Muc1_21S_mOxGFP_dCT_BlpI

[0441] 85. pPB_Muc1_21D_mOxGFP_dCT_BlpI

[0442] 86. pPB_Muc1_21T_mOxGFP_dCT_BlpI

[0443] 87. pPB_Muc1_Syn4_20_mOxGFP_dCT_BlpI

[0444] 88. pPB_Muc1_Syn1_40_mOxGFP_dCT_BlpI

[0445] 89. pPB_Muc1_Syn2_40_mOxGFP_dCT_BlpI

[0446] 90. pPB_Muc1_Syn3_40_mOxGFP_dCT_BlpI

[0447] 91. pPB_Muc1_Syn1_80_mOxGFP_dCT_BlpI

[0448] 92. pPB_Muc1_Syn2_80_mOxGFP_dCT_BlpI

[0449] Secreted mucin

[0450] 93. pPB_Tet_SumoStar_Muc1_42_rtTAsM2_IRES_NeoR

[0451] 94. pPB_Tet_SumoStar_Muc1_21T_rtTAsM2_IRES_NeoR

[0452] 95. pPB_Tet_SumoStar_Muc1_21D_rtTAsM2_IRES_NeoR

[0453] 96. pPB_Tet_SumoStar_Muc1_21S_rtTAsM2_IRES_NeoR

[0454] 97. pPB_Tet_SumoStar_Muc1_21_rtTAsM2_IRES_NeoR

[0455] 98. pPB_Tet_SumoStar_Muc1_0_rtTAsM2_IRES_NeoR

[0456] 99. pPB_Tet_SumoStar_Muc1_Syn1_40_rtTAsM2_IRES_NeoR

[0457] 100. pPB_Tet_SumoStar_Muc1_Syn2_40_rtTAsM2_IRES_NeoR

[0458] 101. pPB_Tet_SumoStar_Muc1_Syn3_40_rtTAsM2_IRES_NeoR

[0459] 102. pPB_Tet_SumoStar_Muc1_Syn1_80_rtTAsM2_IRES_NeoR

[0460] 103. pPB_Tet_SumoStar_Muc1_Syn2_80_rtTAsM2_IRES_NeoR

[0461] References cited in Part I - The references listed in any part of this disclosure are not an indication that any reference therein is important for patentability.

[0462] References

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[0518] Part II

[0519] This Part II of the disclosure describes a mucin-coating technology for protecting and reducing cell production system aggregation.

[0520] In connection with this Part II, there is a need to optimize host cell production systems for improved yield and production reliability in order to meet the increasing demand for biologies with complex post-translational modifications. Prior to the present disclosure, aggregation of suspension-adapted mammalian cells remained a significant problem that can limit cell density and volumetric productivity of bioreactors. This Part II provides a genetically encoded technology to direct synthesis of anti-adhesive and protective coatings on the cell surface. We genetically encoded a new cell surface coating by fusing engineered mucin domains to a synthetic transmembrane anchor. In combination with appropriate expression systems, the mucin-coating technology directs assembly of a thick, highly hydrated barrier that robustly reduces cell aggregation and protects suspension cells from fluid shear stress. The coating technology was demonstrated on suspension-adapted human 293-F cells that resist clumping even in media formulations that would otherwise induce extreme cell aggregation, and showed improved performance over commercially available anti-clumping agents. The stable biopolymer coating showed no deleterious effects on cell proliferation rate, cDNA transient transfection efficiency, or recombinant protein expression. Overall, the mucin-coating technology and engineered cell lines described herein demonstrate the ability to improve single-cell growth and viability of suspension cells in bioreactors.

[0521] This Section II, and other sections of this disclosure, relate to a biopolymer known in the art as a mucin, which is used to reduce adhesion and fouling at biological interfaces. Mucins are characterized by an amino acid sequence rich in serine and threonine residues, which are post-translationally modified with O-linked, pendant glycan structures (Thornton, Rousseau, and McGuckin, 2008). The bottlebrush molecular structure of mucins confers anti-adhesive properties, which are used by biological systems for a variety of purposes, including antifouling coatings, lubrication, and modulation of cell-cell interactions (Jay and Waller, 2014; Kuo, Gandhi, Zia, and Paszek, 2018; Paszek et al., 2014). Among the members of the mucin family, Mucin-1 (Muc1) is considered an anti-adhesive protein that can interfere with integrin- and cadherin-mediated cell interactions (Klinken, Dekker, Buller, and Einerhand, 1995; Wesseling, van der Valk, and Hilkens, 1996; Wesseling, van der Valk, Vos, Sonnenberg, and Hilkens, 1995). The anti-adhesive properties of Muc1 are conferred by its large extracellular domain, which is highly O-glycosylated during transport to the cell surface. The neutral and anionic sugar residues of the glycans can coordinate with water, forming a highly hydrated barrier on the cell surface (Gendler and Spicer, 1995).

[0522] In this Section II, novel mucin cDNAs and the mucins they encode are described, and used to establish genetically encoded techniques to reduce aggregation of human cell host production systems. In particular, the mucin technology described by the present invention is modified, tested, and improved for use as an anti-adhesive coating on, for example, host cell production systems. As a non-limiting demonstration, we developed a new 293-F cell line with a stable anti-adhesive coating, and evaluated its performance in terms of proliferation rate, cell aggregation, resistance to shear stress, and plasmid DNA transfection efficiency.

[0523] Materials and Methods

[0524] Antibodies and Reagents

[0525] The following antibodies were used: human CD227 (555925, BD Biosciences) (Muc1), β-actin (sc-4778, Santa Cruz), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz). The following lectins were used: 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). To induce transactivator cell lines, doxycycline (sc-204734, Santa Cruz) was used. For gentamicin selection, G418 (10131035, Thermo Fisher) was used.

[0526] Constructs

[0527] A tetracycline-inducible, transposon-based Piggybac expression vector with integrated co-expressed reverse tetracycline transactivator gene (pPB tet rtTANeoR) was used for stable line generation. The pPB tet rtTANeoR plasmid was modified by insertion of an encephalomyocarditis virus internal ribosome entry site (IRES) followed by insertion of the fluorescent protein copGFP at NotI and XbaI sites (pPB tet IRES GFP rtTA NeoR). Synthetic cDNAs containing 21 or 42 tandem repeats (TR) of the amino acid sequence PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8) were codon-optimized with a codon scrambler (Tang and Chilkoti, 2016), generated by custom gene synthesis (General Biosystems), and cloned using BamHI and Bsu36I restriction sites in place of the native tandem repeats in pcDNA3.1 Muc1TM21 previously described (Paszek et al., 2014; Shurer et al., 2017). The Muc1 genes containing the engineered 21 or 42 tandem repeats were then cloned into the pPB tet IRES GFP rtTA NeoR plasmid using BamHI and EcoRI sites to generate Muc1 42TR 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. To generate the Mucin-0 cell line, the native Muc1 tandem repeats were deleted from pcDNA3.1 Muc1TM21 by 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 excised from pcDNA3.1 Muc1 0TR TM21 and cloned into the pPB tet IRES GFP rtTA NeoR plasmid through BamHI and EcoRI sites. The plasmid pLVpuro mRuby2 was used for transient transfection experiments with the cytoplasmic red fluorescent protein (RFP). For the secreted RFP experiment, the SS-mScarlet-I pPB tet IRES GFP rtTANeoR plasmid was used. To construct this plasmid, the backbone was linearized using BamHI-HF and EcoRI-HF.A dsDNA oligo encoding the Muc1 signal sequence fused via a linker (four glycines followed by a serine) to mScarlet-I (MTPGTQSPFFLLLLLTVLTVVTGS (SEQ ID NO: 26)) was ordered from Integrated DNA Technologies. This fragment was inserted into the linearized backbone via NEB HiFi assembly.

[0528] Cell lines and culture

[0529] FreeStyle 293-F cells were obtained from Thermo Fisher Scientific. Cells were cultured and maintained in Eppendorf New Brunswick s41 i incubator in Erlenmeyer flasks according to the manufacturer’s guidelines. Cells were maintained at 0.5 x 106cells / mL in FreeStyle 293 expression medium (Thermo) at 120 rpm, 37 °C and 8% C02. Cells were passaged every 3-4 days and expanded to 1 x 106cells / mL. 6 between 3 x 106 6 cells / mL. Transfection was performed using polyethylenimine (PEI) as previously reported (Durocher et al., 2002). Genetically encoded stable cell lines were generated by co-transfecting the pPB tet IRES GFP rtTA NeoR plasmid described above with a highly active transposase plasmid (Shurer et al., 2017) and subsequently selected for two weeks with 750 pg / mL gentamycin. Cell proliferation was quantified by cell counting on a hemocytometer with trypan blue exclusion.

[0530] Confocal microscopy

[0531] Samples were collected, pelleted at 200 ref for 5 min and fixed in 4% paraformaldehyde for 10 min at room temperature. Samples were washed three times with PBS. Cells were labeled with 1 : 1000 CF568 PNA against O-glycans and 1 : 1000 CF633 WGA against cell membrane in PBS for 30 min at room temperature. Samples were washed three times with PBS and imaged on a Zeiss LSM800 at 63x water immersion objective.

[0532] Flow cytometry

[0533] All samples were measured using live cells unless otherwise indicated. Cells were collected from suspension cultures, pelleted at 200 ref for 5 minutes, resuspended in 0.5% BSA PBS. Samples were filtered through a 0.22 pm filter cap and analysed on a BD FACS Aria Fusion. For the doxycycline time course, cells were induced with 1 pg / mL doxycycline. Cell samples from cultures were taken at the indicated time points, pelleted at 200 ref for 5 minutes and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Samples were washed three times with PBS and stored at 4°C until flow cytometric analysis. Analysis of all flow cytometric data was performed using FlowJo software.

[0534] Immunoblot and lectin blot analysis

[0535] Cells were seeded at 0.5 x 10 6 cells / mL and grown overnight, 16-18 hours. Biopolymer expression was then induced with 1 pg / mL doxycycline, cells were grown with doxycycline for a further 48 hours. After 48 hours, samples were taken from each cell line, pelleted at 200 ref for 5 minutes, supernatant was then separated, cell pellets were lysed by resuspension in RIPA lysis buffer (Abcam), samples were vortexed for 30 seconds, heated to 98°C for 10 minutes. Lysates were frozen on liquid nitrogen and stored at -80°C. Lysates were separated on Nupage 3-8% Tris-acetate gels (Invitrogen), 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 pg / mL in 3% BSA TBST, 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 (Bio-Rad) substrate, imaged on a ChemiDoc (Bio-Rad) file system.

[0536] PCR amplification of mucin-270 transgene in transfected 293F cells

[0537] To test for amplification or deletion of the stable integrated mucin-270 cDNA in the 293F genome, PCR amplification was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs Inc., Ipswich, MA) using extracted genomic DNA as template. Genomic DNA was extracted using the GeneJET Genomic DNA Purification Kit (Thermo Scientific, Waltham, MA). A total of 60 ng of genomic DNA was used for PCR amplification. Primers: Mucin-270 FWD 5'-ATGACACCGGGCACCCAGTC-3' (SEQ NO: 85) and Mucin-270 REV 5'-CTACATACTTCGTCGGCGCATGTAC-3' (SEQ NO: 86). The amplicon was 2994 bp in size.

[0538] Cell clumping analysis

[0539] Cells were seeded at 0.75 x 10 6 cells / mL and induced with 1 pg / mL doxycycline after overnight growth (16-18 hours). Cells were then grown to high cell density in the presence of 1 pg / mL doxycycline for an additional 48 or 72 hours. Cell density was quantified by collecting samples of the culture, mixing thoroughly to dissociate large clumps, and counting viable cells using a hemocytometer and trypan blue exclusion. For imaging, samples were drawn up with wide-bore pipette tips to reduce dissociation of large clumps, diluted in PBS to approximately 6.75 x 10 4 cells / cm 2 for 2D imaging. 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, prepared as technical replicates for imaging, with three fields 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). In brief, the analysis software locates the center of each circular object. The Ripley's K function is then calculated using the coordinates of each cell center in MATLAB. The percentage of single cells is calculated by counting the total number of cells with no adjacent cells within 19 pm, divided by the total number of cells in the image. Similarly, the percentage of cells in various cluster sizes is calculated by binning cells into clusters based on the number of adjacent cells within 19 pm.

[0540] To assess resistance to calcium-induced cell aggregation, cultures were grown to 0.5 x 10 6cells / mL were seeded, grown overnight (16-18 hours), and induced with 1 pg / mL doxycycline for 48 hours. Cells were then diluted to 2 x 10 6 cells / mL. Media was then supplemented with 2 mM CaCl2, 1 :300 anti-clumping agent (Thermo, 0010057AE), or both. Static images and videos of cell suspensions were taken after 24 hours of treatment by transferring the cultures to glass test tubes. Cell concentration in suspension was determined by collecting duplicate samples from each culture after 20 seconds of allowing the largest aggregates to settle out of suspension. Cell concentration was measured using a hemocytometer and trypan blue.

[0541] Shear stress experiment

[0542] Cells were seeded at 0.5 x 10 6 cells / mL, grown overnight (16-18 hours), and induced with 1 pg / mL doxycycline for 48 hours. Cell suspensions were sheared by gravity generated constant force flow through a 500 pm constriction (Teflon tubing) using a 5 mL syringe with a 16 gauge needle attached to a 6.5 inch 1.02 mm silicon tube by applying a 1 kg mass to the syringe. The sample was passed through the constriction five times. Cells were then stained with 1 pg / mL CF640R PNA for 15 minutes at 4°C. Cells were washed three times with 0.5% BSA PBS and then stained with ethidium homodimer-1 (Dead Cell Stain, Thermo, L3224). Three biological replicates were performed with two technical replicates each. Percent dead cells was determined by measuring the fraction of cells that absorbed the dead cell stain on a BD FACS Aria Fusion. Control samples without shear were used to subtract background cell death for each cell line. For the Mucin-135 and Mucin-270 cell lines, only PNA positive cells were considered for analysis. Data analysis was performed using FlowJo software.

[0543] Transfection experiment

[0544] Cells were seeded at 0.5 x 10 6 cells / mL, grown overnight (16-18 hours), and induced with 1 pg / mL doxycycline for 48 hours. Cells were then diluted to 2 x 10 6 cells / mL in fresh media containing 1 pg / mL doxycycline, 1 pg DNA / 10 6Cell transfection. The next day (16-18 hours post-transfection), cells were diluted 1:1 with fresh media containing 1 pg / mL doxycycline. To measure transfection efficiency, cells were transfected with pLV puro mRuby2 plasmid and transfection efficiency was calculated by flow cytometry as the fraction of cells expressing RFP 72 hours post-transfection. For recombinant RFP production and secretion, cells were transfected with SS-mScarlet-I pPB tet IRES GFP rtTA NeoR. After 24 hours, secreted RFP fluorescence in the media supernatant was quantified using a Tecan M1000 Pro plate reader.

[0545] Statistical analysis

[0546] Statistical significance was determined using Prism (GraphPad) as ordinary one-way ANOVA or Student’s t-test (two-tailed) as appropriate. All plots were generated in Prism (GraphPad) except for the box plots generated in R.

[0547] Results

[0548] Genetically encoded biopolymers expressed on the surface of 293-F cell lines

[0549] This Section II demonstrates the generation of cDNAs encoding Muc1 -like biopolymers with a transmembrane domain for anchoring to the cell surface. The biopolymer domain consists of 0-42 perfect repeats of the unstructured protein scaffold with PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8), which is recognized by the O-glycosylation machinery of the endoplasmic reticulum and Golgi and highly glycosylated upon transport to the cell surface. Each biopolymer is targeted to the extracellular space by the native Muc1 signal sequence. The biopolymer is anchored to the cell membrane by a 21 amino acid transmembrane domain (Mercanti et al., 2010; C. R. Shurer et al., 2017). By replacing the native autocatalytic domain of Muc1 with an engineered 21 amino acid transmembrane domain (Levitin et al., 2005), we reduce the risk of ectodomain shedding from the cell surface. The engineered constructs described also lack a cytoplasmic tail to avoid inadvertent transduction by biochemical or physical stimuli of mucins.

[0550] Genetic modification of 293-F cell lines with “all-in-one” plasmids containing all elements necessary for selection and tetracycline-inducible expression Figure 12A) is performed non-virally. The vector includes a tetracycline-responsive promoter for expression of the biopolymer coating, and an additional cassette for constitutive expression of a reverse tetracycline transactivator (rtTA-M2) and a neomycin resistance gene (Gossen, Bender, Muller, al and Freundlieb, 1995). A bi- cistronic green fluorescent protein (GFP) reporter is also included for visual confirmation of transcription of the mucin cDNA. The cDNA for the biopolymer is stably incorporated into the genome at random locations via transposon-mediated integration (X. Li et al, 2013; Wilson, Coates and George, 2007; Woodard and Wilson, 2015). This approach avoids the use of any viral technology, which raises deep security concerns in biofabrication (Dumont et al, 2016). We predict that the modified cells will be coated on their surface with a dense inducible mucin biopolymer layer Figure 12 B).

[0551] We tested the effect of three different representative biopolymer sizes on 293-F cell aggregation. Mucin-like genes with 0, 21, and 42 tandem repeats were constructed. The contour lengths of the polymers with 21 and 42 repeats are predicted to be 135 nm and 270 nm, respectively. Thus, we named the biopolymers based on their relative lengths as Mucin-0, Mucin-135, and Mucin-270 Figure 13 C). Because the Mucin-0 construct lacks the large glycosylated biopolymer domain, it serves as a control for any effects related to expression of the transmembrane anchor of the biopolymer.

[0552] We confirmed expression and localization of the biopolymers to the cell surface. Fluorescence microscopy showed cDNA expression reported by the bi-cistronic GFP signal, and the presence of O-glycans on the membranes of cells expressing the Mucin-135 and Mucin-270 semi-synthetic genes Figure 2 A). We observed a wide range of distribution of biopolymer expression levels, which we attribute to random transposition of the cDNA into the genome without intending to be bound by any particular theory Figure 13 B). Despite the wide distribution, the majority of the cell population stably integrated the cDNA, as shown by the GFP reporter Figure 13 A-C). Expression and size of the biopolymers were further verified by Western blot Figure 13 D). Mucin-135 and Mucin-270 were detectable with an antibody against native Muc1 tandem repeats Figure 13 D, left). Wild-type (w.t.) cells have no detectable levels of endogenous Muc1 expression, and no significant O-linked mucin-like glycosylation Figure 13D). Mucin-135 and Mucin-270 are highly glycosylated when expressed. This is shown by the protein bands which are detected above the protein sequence molecular weight when probed with anti-Mucl antibodies Figure 13 D, left; the predicted molecular weights of Mucin-135 and Mucin-270 are 81 kDa and 120 kDa, respectively). O-glycosylation is further demonstrated by detecting the biopolymer with PNA that specifically binds to O-linked glycans, such as those found on Mucl (D, right). Figure 13

[0553] No significant differences in cell proliferation rates were observed for any of our biopolymer coated cell lines Figure 13 E). We infer that the additional protein load of our biopolymers did not adversely affect the fast growth rate of the parental 293-F cells. For the stable cell line, we used a well-characterized reverse tetracycline-inducible promoter (Gossen et al., 1995) that initiates gene transcription upon addition of doxycycline and interrupts transcription when doxycycline is deactivated. This cell line reacted to doxycycline induction as predicted, demonstrating temporal control over the expression of the mucin coating Figure 13 F).

[0554] Highly repetitive cDNAs, such as mucins, have been reported to have a higher frequency of amplification and deletion in the cell genome (Gemayel, Vinces, Legendre, and Verstrepen, 2010; Oren et al., 2016). The cDNAs of our Mucin-135 and Mucin-270 constructs were codon-optimized to minimize their repetitiveness. We found that the optimized cDNAs were stable when integrated into the host cell genome. Notably, no apparent amplification or deletion of the stably integrated Mucin-270, the largest and most repetitive of our biopolymer cDNAs, was observed after 2 months of cell culture Figure 14 G).

[0555] Biopolymer coating reduces cell aggregation

[0556] After establishing stable populations, we analyzed whether the biopolymer coating could reduce cell aggregation in suspension cell cultures. Phase-contrast images of the cell lines qualitatively showed more cell aggregates in the w.t. and Mucin-0 cell lines compared to the Mucin-135 and Mucin-270 cell lines Figure 12 A). Quantification of the fraction of single cells in the samples showed an increase in the percentage of single cells for the Mucin-135 and Mucin-270 coatings compared to the w.t. cells, while the Mucin-0 cell line did not show a difference compared to the w.t. cells Figure 19 B, Figure 14 ​A). Correspondingly, w.t. and mucin-0 coated cell lines were more likely to form clusters of two or more cells compared to mucin-135 or mucin-270 cell lines Figure 19 C, Figure 14 B).

[0557] Inspection of phase-contrast images of our mucin-135 or mucin-270 engineered 293-F cell lines revealed that most cells were single or doublets, with few detectable higher order aggregates Figure 14 B). Because there were no higher order aggregates, we inferred that the doublets in the mucin-135 and mucin-270 samples were likely actively dividing cells or cells that had not fully dissociated after cytoplasmic division. The appearance of doublets could also be caused by single cells that randomly settled out of suspension too close to one another to be resolved in the 2D plane of the images on our microscope. To approximate the frequency of single cells that might randomly settle out of suspension in this way, we created simulated data sets of randomly placed centroids and ran our cluster analysis on them. On average, simulated centroids were counted as singlets 66% of the time. In comparison, 57% of mucin-270 cells were singlets Figure 14 B).

[0558] To quantify the extent of cell clustering, we analyzed the spatial distribution of cells in the images using the Ripker K function, a spatial distribution statistic that counts the frequency of finding a neighboring particle within a given distance of any given particle. Using this statistical tool, we observed that the mucin-135 and mucin-270 biopolymers displayed reduced clustering compared to w.t. and mucin-0 cell lines Figure 19 D, Figure 15 C).

[0559] Mucin-270 coating outperforms commercially available anti-caking agents

[0560] We found that the mucin-270 biopolymer coating reduced cell aggregation even under extreme pro-caking conditions. Suspension-adapted cell lines have been shown previously to significantly aggregate under specific media conditions, such as high calcium concentrations known to promote cadherin engagement (Dee et al., 1997; Han et al., 2006b; Kim, Tai, Mok, Mosser, and Schuman, 2011; Meissner et al., 2001; Peshwa et al., 1993; Sjaastad and Nelson, 1997; Tolbert et al., 1980; Yamamoto et al., 2000; Zanghi et al., 2000). When cultured under high calcium conditions (2 mM CaCl2), mucin-270 biopolymer coated cells qualitatively displayed less aggregation compared to w.t. cellsFigure 15 A). Notably, cultures with the mucin-270 biopolymer coating retained their turbidity under clumping-promoting conditions, while unmodified cells assembled into large clusters easily visible to the naked eye Figure 15 A). Mucin-270 coated cells showed a slight decrease in cell concentration in suspension after calcium treatment, while w.t. cells had essentially no cells remaining in suspension Figure 15 B).

[0561] Furthermore, the mucin-270 coating outperformed a commercially available anti-clumping agent under highly clumping conditions. Under high calcium conditions, the anti-clumping agent had no discernible efficacy in mitigating cell clumping Figure 15 A). In our analysis, the addition of the commercial anti-clumping agent to mucin-270 coated cells did not further enhance their resistance to clumping Figure 16 B). In summary, these results demonstrate the ability of the genetically encoded biopolymer coating provided by the present invention to reduce cell aggregation in suspension.

[0562] Biopolymer coating provides resistance to shear stress

[0563] The sensitivity of suspension-adapted mammalian cells to shear stress imposes a limitation on the rate of mixing and mass transfer in typical bioreactors (Hu, Berdugo, and Chalmers, 2011). High-capacity bioreactors operated at high cell densities require increased mixing to overcome mass transfer limitations (Hu et al., 2011). Thus, the sensitivity of cells to shear imposes another limitation on bioreactor productivity. Because it is a physiological function of mucin to protect the ductal epithelium from shear stress, we considered whether our biopolymer coating would protect cells from shear stress as an additional benefit. To test this, we sheared suspension cells by passing them through a narrow constriction, then analyzed their viability after reintroduction into culture Figure 16 A). A 1 kg mass was applied to a vertically oriented syringe to create a constant and controlled pressure that drove the flow of suspension cells through a 7.6 cm length of 500 μιη diameter Teflon tubing. Cell death was analyzed by flow cytometry using a live / dead cell stain. We found that the mucin-135 and mucin-270 biopolymer coated cell lines had significantly greater viability after shearing compared to both the w.t. and mucin-0 cell lines Figure 17 B), indicating that the mucin coating can allow for higher mixing rates in bioreactors.

[0564] Biopolymer coated cell lines can be transiently transfected and produce comparable levels of recombinant protein

[0565] Recently, the use of transient cell transfection for recombinant protein production has attracted attention to avoid the excessively long time required for selecting and isolating stable cell lines for new drug development (Derouazi et al., 2004; Durocher et al., 2002; Swiech et al., 2011). Given the potential barrier effect of mucopolysaccharide coatings on cell surfaces, we tested whether the expression of the biopolymer provided by this invention would affect the transfection efficiency of cell lines. To test this, we transiently transfected cell lines with plasmids expressing cytoplasmic red fluorescent protein. We observed no statistically significant difference in transfection efficiency between mucin-0, mucin-135, or mucin-270 cell lines compared to wt cells. Figure 17 A). Single-cell analysis revealed similar distributions of recombinant protein production across engineered and parental cell populations. Figure 17 B). Furthermore, there was no significant difference in RFP signaling in transfected cells, indicating that the expression of transiently transfected proteins was comparable across different cell lines. Figure 18 C). We also tested the performance of engineered cells in producing secreted recombinant proteins. As a non-limiting example, we fused a signal peptide to the fluorescent protein mScarlet-I and measured the production of secreted proteins in the culture supernatant from transiently transfected cultures. Cells coated with mucin-270 produced the same amount of secreted recombinant protein as wt cells (C). Figure 12 Therefore, the described biopolymer coating did not adversely affect the transfection efficiency and high protein production rate of the 293-F cell system.

[0566] Part II Discussion

[0567] Among other features, this Part II demonstrates that the established cell lines can be genetically modified to express engineered mucin biopolymers for anti-adhesion. The expression of these biopolymers does not adversely affect the desired characteristics of 293-F cells, including their rapid proliferation rate. Figure 15 E) and high transfection efficiency ( Figure 14 A, B). Furthermore, the expression of biopolymers significantly reduced unwanted cell clumping (A, B). Figure 15 , Figure 19 , Figure 6 And enhances the cell's resistance to shear stress. Figure 15 The mucin-135 coating and the thicker mucin-270 coating performed similarly in head-to-head tests and are expected to be equally well-suited for the applications described herein.

[0568] The described biopolymer coating provides significantly reduced cell aggregation in serum-free media formulations that are typically used in production in bioreactor formulations. Notably, the coating can further reduce aggregation even in media formulations designed to minimize cell clumping, such as the Invitrogen Freestyle 293-F media. The present disclosure includes biopolymer expression on cells in media formulations that have historically been avoided due to cell aggregation issues. For example, high efficiency transient transfection with DNA-calcium phosphate precipitates has been used for long periods of time (Jordan and Wurm, 2004). However, at the high calcium concentrations required, 293-F cells are known to form large cell aggregates (Meissner et al., 2001; Peshwa et al., 1993). Based on the results of this Part II results Figure 20 ), cell aggregation was significantly reduced using mucin-135 or mucin-270 coating under conditions that improve protein production from transient transfection cultures.

[0569] The present disclosure includes further improvements to the described mucin coating that can be achieved through additional optimization of the engineered mucin and its regulated expression. Notably, overproduction of highly glycosylated mucin-like proteins can compete with the cell's glycosylation machinery and nucleotide sugar building blocks for glycan production. By the described membrane anchor selection reduces shedding of the engineered mucin from the cell surface, the membrane anchor lacks proteolytic cleavage sites.

[0570] The proximity mucin described herein can be used as a solution for suspension-adapted cell lines that tend to aggregate in bioreactors. But it should be recognized that the ability of these compositions to protect cells and strongly resist clumping can also benefit current biomanufacturing platforms, such as CHO cells, which can still aggregate under non-ideal reactor conditions or in non-optimal media formulations. As biomanufacturing seeks next-generation production platforms that go beyond CHO systems to reduce the risk of non-human glycan and other antigenic epitope, suspension-adapted cells remain a significant and time-consuming challenge for human, primate, and many other mammalian cell lines (Amaral et al., 2016; Rodrigues et al., 2013). By promoting cell viability and minimizing aggregation, the compositions provided by the present invention can be expected to help solve some of the significant hurdles for suspension-adapted cells.

[0571] In summary, this Part II presents a mucin coating technique for improving single-cell growth of cells in suspension. The described system is highly successful in reducing cell aggregation.

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[0624] Part III

[0625] This Part III provides representative and non-limiting methods for the stable recombinant production of codon-optimized Lubricin and Mucin in human cells, as well as characterization of modified Lubricins derived from human, equine, and canine sequences. This Part III demonstrates the use of codon redundancy to encode desired polypeptides with minimal nucleotide repeats. A codon-optimization strategy was applied to generate synonymous genes or “synDNAs” for two commercially relevant representative Mucins: Lubricin and Muc1. For the synonymous Lubricin cDNA, which is referred to herein at times as “SynLubricin,” stable long-term recombinant production in suspension-adapted human 293-F cells was demonstrated. Under optimal conditions, 293-F subpopulations produced recombinant SynLubricin at greater than 200 mg / L of media, and were stable throughout two-month continuous cultures. Functional tests confirmed that recombinant Lubricin was effective in inhibiting cell adhesion and lubricating cartilage explants. In sum, this Part III provides a viable workflow for cDNA design and stable Mucin production in mammalian host production systems, among other things.

[0626] Part III Introduction

[0627] As will be appreciated from the foregoing, mucins are membrane-bound or secreted glycoproteins that contain a variable number of tandem repeats defined by densely clustered O-glycosylation sites (Hang and Bertozzi, 2005). This extensive glycosylation gives rise to a bottlebrush molecular structure that imparts significant physical properties to mucins (Kuo, Gandhi, Zia, and Paszek, 2018). Mucins at biological interfaces can coordinate with water molecules to form a hydration layer that protects delicate cellular or tissue structures, repels biological contamination, and resists pathological cell deposition (Hattrup and Gendler, 2008). For example, transmembrane mucins, such as Muc1 and Muc16, are densely grafted at the ocular surface, where they maintain hydration, resist abrasion, and provide a selective barrier to macromolecules (Gipson, Spurr-Michaud, Tisdale, and Menon, 2014; Mauris and Argueso, 2012). Similarly, the secreted mucin-like glycoprotein known as proteoglycan 4 (PRG4) or lubricin can bind to cell and tissue interfaces, including articular cartilage and the ocular surface, enabling low-friction lubrication and protection from pathological cell deposition and biological contamination (Rhee et al., 2005; Schmidt, Sullivan, Knop, et al., 2013).

[0628] Alterations in mucin expression and glycosylation are observed in a variety of pathological conditions ranging from cancer and inflammatory bowel disease to ocular disease (Dhanisha, Guruvayoorappan, Drishya, and Abeesh, 2018). Patients with genetic mutations that prevent the synthesis of functional lubricin exhibit symptoms of Camptodactyly-Arthropathy-Coxa Vara-Pericarditis (CACP) syndrome, including early-onset polyarthropathy caused by pannus formation and impaired joint lubrication (Bahabri et al., 1998; Marcelino et al., 1999). Reduced synovial 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). Thus, there is considerable interest in developing recombinant lubricin and other mucins as injectable therapeutics for osteoarthritis and rheumatic diseases (Le Graverand-Gastineau, 2010) as well as topical treatments for chronic dry eye and other conditions requiring the application of exogenous lubricants (Schmidt et al., 2013).

[0629] Despite this commercial interest, recombinant production has proven challenging for Muc1, lubricin, and other mucins containing high numbers of tandem repeats. While high-producing clones of truncated Muc1 containing approximately 1 / 3 of the native tandem repeats have been isolated from Chinese hamster ovary (CHO) cells, similar attempts to isolate clones of full-length recombinant Muc1 have failed (Backstrom et al., 2003). Likewise, stable clones of recombinant lubricin with the full 76-78 native tandem repeats produced glycoprotein at low levels (Jones et al., 2007), but a modified recombinant lubricin protein construct containing only 1 / 3 of the tandem repeats (LUB:1) was more amenable to large-scale production (Flannery et al., 2009). Recently, production of full-length recombinant human lubricin expressed in suspension-adapted CHO cells has been reported and has demonstrated potential as an ocular lubricant for treating dry eye disease or hydration of contact lenses (Samsom et al., 2014). The precise details of how full-length lubricin achieves recombinant production remain unknown, and at the time of filing this application or patent, no published strategy for large-scale lubricin production was considered.

[0630] The exact biology of why mucins are difficult to produce at high levels remains unknown. However, long repeat DNA sequences, such as those commonly found in cDNAs of mucin tandem repeats, are relatively unstable in the cellular genome (Pearson, Edamura, and Cleary, 2005). The fidelity of nearly all DNA processing steps can be compromised by slippage and other errors associated with repetitive sequences (Lopez Castel, Cleary, and Pearson, 2010). Thus, repeats can be mutated by the addition or loss of their unit nucleotide sequence up to 100,000 times more frequently than point mutations in non-repetitive regions (Oren et al., 2016). Variation in the number of tandem repeats of Muc1 and other mucins in humans and mammals provides an evolutionary argument that these genomic cDNAs are hotspots for mutation (Gemayel, Vinces, Legendre, and Verstrepen, 2010). Recombination and truncation of Muc1 cDNAs exogenous to bacteria have also been reported, suggesting that these repeat sequences are also highly unstable in host microbial cells (Backstrom et al., 2003).

[0631] Now that advances in custom gene synthesis (CGS) enable rapid and cost-effective synthesis of long cDNAs (Kosuri and Church, 2014), this paper provides a new method to improve genomic stability of mucins and, in certain embodiments, utilizes codon redundancy to identify and use synonymous gene sequences that are less repetitive but encode the same desired polypeptide. Such codon optimization algorithms have been developed and successfully applied to elastin-like proteins and some other repetitive protein domains (Tang and Chilkoti, 2016). However, it is believed that, prior to the present disclosure, optimized synthetic cDNAs for the biomanufacturing of commercially relevant mucins have not been designed, synthesized, and tested.

[0632] Furthermore, prior to the present disclosure, most biologies, including mucins, are produced in CHO cells due to their fast growth, adaptability to suspension culture, and ability to perform glycosylation and other important post-translational modifications. However, CHO cells can produce glycan epitopes that are suspected to elicit adverse immunological reactions in humans (Butler and Spearman, 2014). That is, the a1,3-galactosyltransferase of CHO and other non-primate cells produces glycans with Galal,3-Gal residues that can be immunogenic to humans, apes, and other Old World monkeys that have lost a1,3-galactosyltransferase activity (Bosques et al., 2010; Brooks, 2004). CHO cells can also produce Neu5Gc, an end sialic acid that is common in most mammalian cells but has been lost in humans and primates (Ghaderi, Zhang, Hurtado-Ziola, and Varki, 2012). These glycans are of particular concern for recombinant mucins, which can consist of 75 mass % or more carbohydrates and are often highly sialylated (Estrella, Whitelock, Packer, and Karlsson, 2010). Recombinant production of glycoproteins in human cells would avoid the risk of Galal,3-Gal and Neu5Gc residues; however, it is believed that, prior to the present disclosure, no successful attempts at large-scale mucin production in human cell host production systems have been reported.

[0633] Thus, among other things, the present disclosure demonstrates that cDNA optimization by codon scrambling is an effective strategy to enable stable recombinant production of mucins and mucin-like glycoproteins, and this strategy is feasible in suspension-adapted human 293-F cells. Notably, several biologies produced in 293-F cells have recently been approved by the U.S. Food and Drug Administration (FDA), establishing the cell platform as a viable alternative to CHO and other non-human systems used to manufacture specialized therapeutics (Dumont, Euwart, Mei, Estes, and Kshirsagar, 2016). In the present disclosure, the codon scrambling approach is demonstrated for Muc1 and Lubricin, and further developed to produce a strategy to enable stable production of functional full-length recombinant Lubricin. Those skilled in the art will recognize that the methods described herein can be used to stably and robustly express other mucins and mucin-like proteins when given the benefit of the present disclosure.

[0634] Results from Part II

[0635] Design and synthesis of cDNA for synonymous Lubricin

[0636] As a method for recombinant mucin production, we applied a codon scrambling and optimization strategy to design and synthesize mucin cDNA within a minimal codon repeat Figure 20 A) Global codon optimization algorithms were applied to find the minimal repeat gene sequence encoding the desired mucin tandem repeat (Tang and Chilkoti, 2016). To tailor the sequence for production in a human host system, such as 293-F, subsequent optimization was performed to replace any codons that have a frequency of use in humans of less than 10% ( Figure 20 A) We envision that the optimized mucin cDNA can be synthesized by the rapid and low-cost service of CGS (Kosuri and Church, 2014; Tang and Chilkoti, 2016).

[0637] We first tested this approach against human Lubricin, which has approximately 59 tandem repeats with the consensus sequence KXPXPTTX (SEQ ID NO: 87), KEPAPTTP (SEQ ID NO: 1) being the most frequent repeat. For our synthetic Lubricin, we optimized the codons of the 59 perfect repeats of the KEPAPTTP (SEQ ID NO: 1) consensus sequence Figure 20 B) The protein sequence of the perfect repeat has approximately 88% similarity to the native human PRG4 repeat Figure 20C). The additional sequences that flank the natural N- and C-terminus of the coding human PRG4 were synthesized. These sequences include the natural growth-modulin and heme-binding protein domains of lubricin. We also included an IgK leader sequence, a 6x histidine tag, and an N-terminal SumoStar tag to aid protein secretion and purification Figure 20 B). We named the new semi-synthetic gene encoded by the codon-optimized cDNA "synonymous lubricin" or "SynLubricin".

[0638] The repetitiveness of the nucleotides encoding SynLubricin is significantly lower than that of natural PRG4. We analyzed the nucleotide sequence with an alignment algorithm that detects tandem repeats and scores their repetitiveness based on their repeat frequency and how closely the identified consensus sequence matches the nucleotides of the query sequence (Benson, 1999). The detected repeats are aligned to the query sequence by Smith-Waterman-style local alignment, and the overall repetitiveness is scored by assigning +2 for each nucleotide match and -7 for each mismatch or insertion-deletion (Benson, 1999). Thus, a higher score indicates more nucleotide repeats. The tandem repeats of SynLubricin have a moderate score of 168, while the natural PRG4 repeats have a much higher repeat score of 1001. The present disclosure encompasses such sequences, where the overall repetitiveness score of the polynucleotide is compared to a suitable control.

[0639] We also aligned the amino acids of the SynLubricin tandem repeats to the 59 tandem repeats of human PRG4 isoform A Figure 26 D). We noted that the perfect repeats of SynLubricin and the natural repeats of human PRG4-A have similar compositions of alanine, glutamic acid, lysine, and threonine, while the proline content is slightly higher in the SynLubricin repeats (37% vs. 30.5%; Table S1, Part III). We also noted that the natural repeats contain small amounts of asparagine (0.2%), aspartate (0.4%), glycine (0.8%), isoleucine (0.2%), leucine (1.4%), and serine (2.6%) that are not contained in SynLubricin (Supplemental Table 1, Part III). Thus, in addition to the different coding sequences, the amino acid sequence of SynLubricin differs from that of human PRG4.

[0640] The low repeat of nucleotides in the SynLubricin gene enabled the synthesis of the desired cDNA using available technology. We also obtained a cDNA with the native human lubricin / PRG4 sequence from a commercial supplier. However, our subsequent attempts to clone the native PRG4 cDNA sequence into a mammalian expression vector and recombinantly express the product in mammalian cells failed. Thus, we discontinued further efforts to recombinantly produce lubricin with the full-length native cDNA.

[0641] Efforts to produce 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 needed, and exhibited the anti-adhesion properties we predicted. Transfected cells maintained large intercellular gaps between monolayers of cells, especially at locations where high levels of expression of the copGFP fluorescent reporter mRNA were visible Figure 26 A). We noted that these observations were consistent with the known anti-adhesion function of native lubricin (Rhee et al., 2005). In contrast, mock-transfected cells grew as highly confluent monolayers in culture Figure 26 A). Western blots of culture supernatant from SynLubricin-transfected cultures exhibited a high molecular weight protein of approximately 460 kDa, which was similar in size to the native lubricin we detected in equine synovial fluid Figure 20 B). The expected molecular weight of the peptide backbone of SynLubricin was 145 kDa, indicating that SynLubricin was fully glycosylated.

[0642] We next developed a strategy for stable production of synthetic mucins in 293-F suspension cultures. In one embodiment, we generated a non-viral transposon vector for mucin "all-in-one" inducible expression. The vector contains a tetracycline-responsive promoter for inducible expression of the desired gene and a bicistronic copGFP reporter. The vector also contains a second cassette under the control of an EFla promoter for expression of an rtTA-M2 tetracycline transactivator as well as a bicistronic neomycin resistance gene for selection Figure 27E). To test the performance of the expression system, we cloned mCherry2 into the vector and transfected 293-F cells with cationic polyethylenimine (PEI) condensates following standard protocols (Boussif et al., 1995; delos Milagros Bassani Molinas, Beer, Hesse, Wirth, and Wagner, 2014; Sonawane, Szoka Jr., and Verkman, 2003). After two weeks of selection, stable cell populations were isolated and mCherry2 production was verified by flow cytometry. Based on flow cytometry analysis, we found that stable cells produced high levels of mCherry2, and fluorescence readout of the copGFP reporter was generally a good indicator of recombinant protein production Figure 28

[0643] Design and synthesis of cDNA for synonymous Muc1

[0644] We tested whether the strategy described for the mucin-type cDNA could be generalized and applied to other mucins. We chose the mucin Muc1, which is important for hydration and protection of the cornea and other epithelial surfaces (Mantelli and Argueso, 2008). We noted that the natural tandem repeats of Muc1 are polymorphic, with 42 perfect repeats being most common in humans (Nath and Mukherjee, 2014). We applied the codon optimization strategy to design a cDNA for the 42 perfect Muc1 repeats, PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8). The optimized sequence was fused to the codons at the natural N-terminus of human Muc1. We also added an IgK leader sequence, a 6x histidine tag, and a SumoStar tag, similar to SynLubricin Figure 28 A). We calculated a very high repeat score of 4997 for the nucleotide coding sequence of the natural human Muc1 tandem repeats. The repeat score was reduced to 220 in our synthetic cDNA, which we call SynMuc1 Figure 28 B).

[0645] The optimized coding sequence for SynMuc1 was synthesized by a standard CGS service, while the very repetitive sequence of the synthetic natural Muc1 cDNA could not be performed by commercial vendors. The custom-synthesized SynMuc1 cDNA was transfected into 293-F cells. The recombinant protein was purified from culture supernatant by immobilized metal affinity chromatography (IMAC) and detected by Western blot with an antibody against the natural human tandem repeats Figure 28 ​C). The strong signal as detected with Peanut agglutinin (PNA), which is a lectin specific for the core-1 mucin-type disaccharide, indicates that the recombinant mucin is fully O-glycosylated Figure 28 D).

[0646] During purification, we noticed that a considerable percentage of the mucin failed to bind the IMAC resin and was detected in the flow-through Figure 28 C, D). Western blots confirmed the presence of the 6x Histidine SumoStar purification tag on the recombinant protein in the flow-through and elution fractions, indicating that the N-terminus and purification tag are present but inaccessible to the immobilized IMAC cations, as in this case, for example, the tag is buried in the random coil of the mucin biopolymer Figure 21 E). Since the goal was to demonstrate the production of recombinant SynMuc1 and not to optimize its purification, alternative chromatography methods were not explored.

[0647] Stable host production of recombinant SynLubricin

[0648] Using the transposon system, we tested its application for SynLubricin production Figure 21 A). Unexpectedly, we found that after selection with G418, a relatively small number of cells exhibited high copGFP reporter levels after doxycycline induction Figure 21 B). To address the problem, we used the copGFP reporter to apply a two-round sorting strategy to isolate a subpopulation of cells expressing SynLubricin at high levels. The stable cells were expanded and sorted for the top 5% copGFP expressers, then expanded and sorted for the top 10% expressers a second time. We found that the sorting strategy improved SynLubricin production by 15-fold and did not affect the molecular weight of the glycosylated protein product Figure 22 B, C). After doxycycline induction, the sorted cell population clearly showed higher levels of the copGFP reporter, indicating successful isolation of a polyclonal population with higher gene expression levels.

[0649] To confirm the cDNA stability of the integrated SynLubricin gene in our stable 293-F cells, genomic DNA was extracted from the modified 293-F cells after two months of continuous culture. The SynLubricin cDNA was then amplified by polymerase chain reaction (PCR) using primers specific for SynLubricin Figure 22 ). The amplified gene was approximately 4 kb long, as expected for full-length lubricin, and was not distinguishable in size from a similarly amplified gene obtained using the original SynLubricin plasmid as a template or from DNA extracted from transiently transfected cells Figure 22). Even after 2 months of culture, the polyclonal cell population did not exhibit signs of SynLubricin gene application or loss, indicating high levels of genomic stability Figure 23

[0650] Optimization of SynLubricin production

[0651] We analyzed whether SynLubricin yield could be improved by adding valproic acid (VPA), a histone deacetylase inhibitor that has been shown previously to significantly increase production of some recombinant proteins in 293-F cells (Backliwal et al., 2008). Our sorted cell population was induced with doxycycline in the presence or absence of 3.5 mM VPA, and culture medium supernatants were sampled daily from the batch culture thereafter. The molecular weight of the protein product was similar, indicating that VPA did not significantly affect the overall glycosylation degree of the protein product Figure 23 A). Interestingly, recombinant protein levels peaked approximately 2-3 days after induction in cultures without VPA, after which they rapidly declined Figure 23 B). In VPA-treated cultures, SynLubricin levels in the medium did not significantly decline over time. We ruled out protein degradation as a possible explanation for the decline of recombinant protein in cultures without VPA, as we did not see significant lubricin degradation products on Western blots Figure 23 A). In fact, we considered the possibility that 293-F cultures can consume recombinant protein under conditions of reduced nutrient availability. Consistent with this possibility, we observed that the decline of recombinant protein levels coincided with the depletion of glucose in cultures without VPA Figure 23 C). As indicated by the sharp decline in glucose consumption, metabolic activity largely ceased in VPA-treated cultures after 3 days Figure 23 C). Thus, VPA can prevent loss of recombinant protein in batch cultures by slowing down 293-F cell metabolism.

[0652] We next scaled up production to 1 -liter bioreactors operated in batch mode, and performed two independent production runs with the addition of VPA. Each production run yielded abundant recombinant protein, which was comparable in molecular weight to recombinant protein isolated from transiently transfected cultures and to native lubricin detected in equine synovial fluid Figure 21 D). An ELISA using purified bovine lubricin as a standard reported approximately 200 mg / L of SynLubricin in batch runs of our stable 293-F cell line. Less than 50% of the stable cell population showed strong expression of the copGFP reporter in batch bioreactors, suggesting that yield improvement can be achieved with clonal expansion of the production cell line Figure 23 ​D). We note that a limitation of our ELISA-based quantification can be the use of bovine standards, which can over- or underestimate SynLubricin levels.

[0653] We tested whether regular medium exchange could be used to achieve stable protein production to avoid nutrient depletion. Conditioned medium was collected from doxycycline-induced cultures maintained in the absence of VPA for 10 consecutive days. Medium was exchanged every 48 hours in the batch culture to replenish nutrients and remove metabolic waste products. Every 48 hours, the viable cell concentration was also reduced to 1 x 10 6 SynLubricin production levels were stable over the 10-day culture, with constant SynLubricin molecular weight, indicating that glycosylation was also stable Figure 23 E). Although there appears to be a slight decrease in SynLubricin production over time, there is no significant difference in protein yield Figure 29 F).

[0654] SynLubricin is a functional bio-lubricant

[0655] Recombinant SynLubricin was efficiently purified from conditioned 293-F culture supernatant using anion exchange or cation exchange chromatography. Anion exchange chromatography followed the strategy we previously reported for isolating native Lubricin from horse synovial fluid, with slight modifications from using Q (Reesink et al., 2016). Successful purification using cation exchange was achieved on a POROS TM XS (Thermo Fisher) resin column with a linear elution gradient of 0.1 to 1 M NaCl in 50 mM phosphate buffer, pH 6.8, using a mobile phase of 50 mM phosphate buffer, 100 mM NaCl, pH 6.8. We also tried IMAC to purify native Lubricin, but recombinant SynLubricin had poor affinity for IMAC resin Figure 24 ). As with SynMuc1, we surmised that the N-terminal histidine tag can be buried in the large random coil of the SynLubricin tandem repeat, so we abandoned the IMAC approach. In contrast, SynLubricin bound strongly to anion exchange resin and eluted continuously over a high salt concentration ranging from approximately 350 mM to 1.5 M Figure 24A, B). The progressive elution of SynLubricin can be explained by the different frequency of the anionic sialic acids in the O-glycans of recombinant SynLubricin (Estrella et al., 2010). We found that a stringent washing step with approximately 500 mM NaCl removed most of the protein contaminants detectable by silver staining, although some SynLubricin was inevitably lost due to this high salt wash Figure 24 C, D).

[0656] To ensure the functionality of our recombinant SynLubricin, we tested its ability to lubricate cartilage and reduce friction. Recombinant SynLubricin was purified by anion exchange chromatography using a stringent 500 mM NaCl wash step to eliminate most protein contaminants Figure 25 D). After purification, SynLubricin was dialyzed into physiological saline and diluted to physiological concentrations. Lubrication was tested on bovine articular cartilage explants in which the native lubricin boundary layer had been extracted using a custom linear reciprocating tribometer (Jones et al., 2007). We found that SynLubricin-containing solutions, as well as control synovial fluid, significantly reduced the boundary friction of cartilage explants compared to physiological saline controls Figure 25 ; p < 0.001 and 0.0001, respectively).

[0657] We also tested a small sample of a second SynLubricin preparation that was purified without a stringent 500 mM NaCl wash of the anion exchange column. Notably, the cartilage friction coefficient of this SynLubricin preparation was greatly reduced compared to any of the friction coefficients measured for the more stringently washed SynLubricin preparations Figure 25 ). The sample volume of the unwashed SynLubricin preparation was low, precluding independent measurements sufficient for meaningful statistical comparisons Figure 26 . However, given the benefit of the present disclosure, further optimization of purification conditions using techniques that will be apparent to those skilled in the art is expected to yield recombinant lubricin fractions with improved biolubrication properties. For example, less negatively charged lubricin fractions eluted at lower salt concentrations (350-500 mM NaCl) can be important for cartilage biolubrication by independent action or synergistically with more negatively charged lubricin fractions. Alternatively, contaminants eliminated with the 500 mM NaCl wash can synergize with lubricin in cartilage lubrication.

[0658] This Part III embodiment provides a method for large scale biomannufacturing of mucins. The success of the design and synthesis of new semi-synthetic genes for Muc1 and Lubricin, and our isolation of a highly stable cell population expressing Lubricin, indicates that this method can be broadly applied to recombinant mucins with long repeat domains. Successful proof of recombinant production in a human cell system, which avoids the risk of immunogenic Galal,3-Gal and Neu5Gc epitopes. We found that the recombinant product of our SynLubricin gene is functional in its ability to resist cell adhesion Figure 25 A) and to lubricate biological surfaces, such as cartilage Figure 30 . Thus, SynLubricin is expected to be suitable for different applications ranging from injectable agents for osteoarthritis to topical treatments for chronic dry eye. Furthermore, given the speed and low cost of CGS, the method described herein is expected to be applied to rapid prototyper mucins with new or modified functional domains.

[0659] In addition to the foregoing, we tested various properties of SynLubricin.

[0660] As shown in Figure 30 , SynLubricin exhibits a remarkable and unexpected half-life in vivo. Figure 30 The results shown in . In particular, when injected into a mammal, SynLubricin exhibits an intra-articular half-life of over 4 days, which is the previously determined value for native Lubricin. (See, e.g., Hurtig et al. Two compartment pharmacokinetic model describes the intra-articular delivery and retention of rhprg4 following ACL transection in the Yucatan mini pig. J Orthop Res. Feb;37(2):386-396. doi: 10.1002 / jor.24191. Epub 2018 Dec 17). Thus, in embodiments, the recombinant proteins of the present disclosure exhibit a half-life of over four days, and can last for at least up to 30 days or more. In embodiments, the half-life is up to 50 days, at least 50 days or more.

[0661] Figure 31Results shown in the figures below were obtained as follows. All animal protocols were approved by the Cornell University Institutional Animal Care and Use Committee (Protocol Number 2017-0084). Male SD rats were purchased from Harlan Sprague-Dawley, Inc. (ENVIGO) at 10-12 weeks of age and housed in pairs under a standard 12-hour light / dark cycle beginning at 6 am. Animals were allowed free movement in their cages, fed a commercial diet without alfalfa to minimize background fluorescence (ENVIGO Teklad #2918), and allowed access to tap water. After a minimum of 3 days to acclimate upon arrival, animals were identified by ear notching and weighed. Under isoflurane anesthesia (1-1.5 L / min 2.5% isoflurane in O2), hair was shaved from the abdomen and flanks of each rat using a beard trimmer. After skin preparation with sterile povidone-iodine and 70% ethanol, the left knee was injected with 20 pL of a Dulbecco’s phosphate buffered saline (dPBS) solution of either 20 pL SynLubricin-Cy7.5 or 20 pL dextran 500kD-Cy7.5 through the patellar tendon approach using a 27g needle and 0.5 mL syringe (Becton Dickinson) and the knee bent at a 90° angle. Injection of ethanol removed any residual povidone-iodine. No injection was made in the right knee so that it could serve as an internal control for background fluorescence calculations.

[0662] Each rat was imaged using an IVIS Spectrum whole animal imaging system (PerkinElmer TM ) at 0, 6, and 12 hours after injection and at 1, 2, 3, 5, 7, 14, 21, 28 days up to 56 days. Both auto and 2 second exposure times were obtained. Animals were anesthetized under isoflurane anesthesia (1-1.5 L / min 2.5% isoflurane in O2) and the hair shaved at weekly intervals just prior to imaging, beginning at 7 days after injection. Two animals were injected with 20 pL of SynLubricin-Cy7.5 and four animals were injected with 20 pL of dextran 500kD-Cy7.5 as additional controls. Data were fit to a bi-exponential decay model to calculate the alpha and beta decay constants. A lubricin half-life of about 45 days was reported as ln(2) divided by the beta decay constant. In contrast, dextran was rapidly cleared from the rat knee.

[0663] As Figure 32As shown in Figure 6, SynLubricin produced in 293-F cells contained a mixture of Core I and Core II glycans. Glycan analysis was performed by MALDI-TOF / TOF-MS in positive ion mode and assignment of glycan structures was done manually by using the Glycoworkbench software. Of note, Core II glycans contained 20.3% of the detected Core O-glycans. Greater than 29% of the O-glycan structures were sialylated.

[0664] We performed additional tribological analysis to determine the dependence of cartilage-cartilage friction on SynLubricin concentration. As shown in Figure 7, a PBS solution of as little as 100 μg / mL SynLubricin was effective in reducing the coefficient of friction of cartilage (sliding velocity = 0.1 mm / s) compared to PBS control alone. Figure 33 As shown in Figure 6, SynLubricin produced in 293-F cells contained a mixture of Core I and Core II glycans. Glycan analysis was performed by MALDI-TOF / TOF-MS in positive ion mode and assignment of glycan structures was done manually by using the Glycoworkbench software. Of note, Core II glycans contained 20.3% of the detected Core O-glycans. Greater than 29% of the O-glycan structures were sialylated. Approximately 2 mm thickness) were used to obtain all data using a custom linear reciprocating tribometer. Endogenous cartilage-bound lubricin was extracted from explants using a 30 minute incubation in 1.5 M NaCl followed by a 1 hour equilibration step in PBS prior to testing. SynLubricin used for these studies was purified from conditioned 293-F media using cation exchange chromatography with POROS TM XS (Thermo) resin.

[0665] Materials and Methods

[0666] Antibodies and Reagents

[0667] The following antibodies were used: mouse anti-human CD227 (555925, BD Biosciences) (Muc1), mouse anti-human lubricin (MABT401, EMD Millipore), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz), mouse anti-SUMO (4G11E9, Kindsy). The lectin used was biotinylated peanut agglutinin (PNA; B-1075, Vector Laboratories). Biotinylated lectin was detected using ExtrAvidin-peroxidase (E2886, Sigma). To induce the transactivator cell line, doxycycline (sc-204734, Santa Cruz) was used. For neomycin selection, G418 (10131035, Thermo) was used. Valproic acid (VPA) was used as a histone deacetylase inhibitor (Sigma P4543-100G).

[0668] Constructs

[0669] A tetracycline-inducible, transposon-based Piggybac expression vector with 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 insertion of an encephalomyocarditis virus internal ribosome entry site (IRES) followed by insertion of the fluorescent protein copGFP at the Notl and Xbal sites (pPB tet IRES copGFP rtTA NeoR). Synthetic cDNA for a lubricin analog with 59 perfect repeats of KEPAPTTP (SEQ ID NO: 1), native N and C-terminal domains, and an N-terminal SumoStar tag (lifesensors) was generated by custom gene synthesis (General Biosystems) and cloned into the multiple cloning site of pPB tet IRES copGFP rtTA NeoR using BamHI and EcoRI restriction sites. Similarly, cDNA for a soluble, codon scrambled Muc1 with 42 perfect repeats of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8) and native human Muc1 N-terminus and SumoStar tag was generated in a pcDNA3 plasmid by custom gene synthesis. To construct a mCherry2 IRES2 copGFP expression plasmid, the mCherry2 cDNA was isolated from pmCherry2 Nl by EcoRI and Notl digestion and cloned into the EcoRI and Notl digested pPB tet IRES copGFP rtTA NeoR vector to generate pPB tet mCherry2 IRES copGFP rtTA NeoR.

[0670] Cell lines and culture

[0671] FreeStyle 293-F (293-F) cells were obtained from Thermo Fisher Scientific. Cells were cultured and maintained in 100-ml Wheaton Celstir glass spinner flasks according to manufacturer’s guidelines. Cells were maintained in FreeStyle 293 expression medium (Thermo) at 0.5 x 106cells / ml at 120 rpm, 37 °C, and 8% C02. Cells were passaged every 3-4 days. 6 At 3 x 106cells / ml 6between 1 and 5 cells per milliliter. 293-F transfection was performed using polyethylenimine (PEI) as previously reported (Durocher, Perret, and Kamen, 2002). Stable cell lines were generated by co-transfecting the pPB tet IRES copGFP rtTANeoR plasmid described above with a highly active transposase plasmid (Shurer et al., 2018) and then selected with 750 pg / mL G418 for two weeks. Human embryonic kidney cells transformed with 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 by standard calcium phosphate transfection protocols. Cell proliferation was quantified by cell counting on a hemocytometer with trypan blue exclusion.

[0672] Cell sorting and SynLubricin production

[0673] Expanded stable 293-F cells incorporating SynLubricin IRES copGFP or SynLubricin IRES mNeonGreen were induced at 2 x 105cells / mL for 24 hours with 1 pg / mL doxycycline. The first 5% of cells expressing GFP were collected by fluorescence-activated cell sorting (FACS) on a FACSAria Fusion (BD Biosciences). Cells were sorted a second time if needed, collecting the first 10% of cells expressing GFP. For SynLubricin production, cells were transferred to 1 L ProCulture glass spinner flasks (Corning) and induced at 2 x 105cells / mL with 1 pg / mL doxycycline and 3.5 mM VPA. Smaller scale lubricin production was also performed in 100-ml Wheaton Celstir glass spinner flasks in order to measure lubricin production rate and glucose consumption rate in the presence or absence of VPA. Glucose levels were recorded with a GlucCell glucose monitoring system (CESCO BioProducts). 6 6 Cell sorting and SynLubricin production

[0674] Immunoblot and lectin blot analysis

[0675] ​Proteins 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 in 3% BSA TBST, and lectins were diluted to 1 pg / mL, 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 (Bio-Rad) substrate and imaged on a ChemiDoc (Bio-Rad) documentation system. Fiji was used for image processing (Schindelin et al., 2012).

[0676] Enzyme-linked immunosorbent assay (ELISA)

[0677] Similar to previously described, a custom sandwich ELISA was used to assess SynLubricin concentration. 96-well plates (Costar) were incubated overnight at 4°C with a 50 mM sodium bicarbonate buffer, pH 9.5 solution of 10 pg / mL peanut lectin (Sigma). Plates were blocked with 3% BSA PBS for 1 hour at room temperature. Serial dilutions of FPLC purified bovine lubricin were used as standards. Samples were loaded at a 1 :200 dilution in DPBS for 1 hour at room temperature, followed by three washes in PBS + 0.1% Tween 20. The primary antibody used (Millipore MABT401) binds the native PRG4 tandem repeats of human and bovine lubricin, which have approximately 90% sequence similarity to the repeats of SynLubricin. Primary and secondary antibodies (Millipore AP126P) were diluted 1 :5000 and 1 :2000, respectively, and each incubated for 1 hour at room temperature, with three washes in PBS-T between antibody incubations and after secondary antibody incubation. ELISAs were developed with 1 -step Ultra TMB (Thermo) for 9-12 minutes at room temperature, or until royal blue appeared, at which point the reaction was stopped with 2N H2SO4. Plates were read on a 3M Microplate Reader at 450 nm with 540 nm background subtraction, and concentrations were calculated using Magellan software with a four-parameter Marquardt fit. Absorbance at 450 nm was measured on a 3M Microplate Reader at 540 nm background subtraction, and concentrations were calculated using Magellan software with a four-parameter Marquardt fit.

[0678] Purification of recombinant SynMuc1

[0679] Transient transfection of 293-F cells was performed using the previously described PEI protocol. After 24 hours, the culture supernatant was collected. The culture supernatant was diluted 1:4 in 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4 and incubated with 100 μL of Ni Sepharose excel resin (17371201, GE) at 4 °C overnight. The sample was collected by flow using a gravity column (29922, Thermo). The resin was washed with 5 mL of 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4. 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 Zeba Purification Desalting Columns (87766, Thermo).

[0680] Purification of recombinant SynLubricin

[0681] SynLubricin was purified from SynLubricin IRES copGFP or SynLubricin IRES mNeonGreen positive 293-F cell culture supernatant by fast protein liquid chromatography (FPLC) using Q resin (GE) or POROS TM XS (Thermo Fisher) resin. For anion exchange, the supernatant was diluted 1:10 with 50 mM Tris-HCl buffer, pH 7.5 and loaded onto the column. The column was washed with 50 mM Tris-HCl, 525 mM NaCl, pH 7.5. Purified SynLubricin was collected by elution with 50 mM Tris-HCl, 1 M NaCl, pH 7.5. Purified SynLubricin was dialyzed into PBS using a Tube-O-Dialyzer (G-Biosciences) at 4 °C overnight. The final purified product was obtained by concentration with a SpeedVac at low setting. For cation exchange, supernatant from SynLubricin IRES mNeonGreen positive 293-F suspension cell culture was first passed through a 0.8 μm pore size cellulose acetate filter (Sartorius), then desalted and captured by fast protein liquid chromatography (FPLC). Desalting was performed on Sephadex G-25 (GE) fine resin with a mobile phase of 50 mM phosphate buffer, 100 mM NaCl, pH 6.8. For the capture operation, the desalted sample was injected into POROS TMSynLubricin was identified in fractions eluting between 0.46 and 0.64 M NaCl and these fractions were pooled and used without further purification.

[0682] Glycan profiling of SynLubricin

[0683] Unless otherwise mentioned, all reagents were purchased from Sigma. Recombinant SynLubricin was denatured by heating at 100 °C for 5 min. The denatured protein was then treated with 500 μΐ, of 50 mM sodium hydroxide (NaOH) containing 19 mg sodium borohydride (NaBH4) at 45 °C for 18 h. The sample was cooled, neutralized with 10% acetic acid, boronate removed by Dowex H+resin column, lyophilized under nitrogen stream. Glycans were fully methylated using previously reported method for structural characterization by mass spectrometry. Briefly, the dry eluate was dissolved in dimethyl sulfoxide (DMSO) and methylated by using iodomethane and NaOH-DMSO base (prepared by mixing DMSO with 50% w / w NaOH solution). The reaction was quenched with water and the reaction mixture was extracted with dichloromethane and dried. The fully methylated glycans were dissolved in methanol and crystallized with a-dihydroxybenzoic acid (DHBA, 20 mg / mL in 50% v / v methanol:water) matrix. The glycans present in the sample were analyzed by MALDI-TOF / TOF-MS in positive ion mode using an AB SCIEX TOF / TOF 5800 (Applied Biosystem MDS Analytical Technologies) mass spectrometer. The fully methylated glycans from the sample were infused onto an Orbitrap Fusion Tribrid mass spectrometer through an ESI probe with HCD and CID fragmentation options for further structural confirmation. The MS1 and MS2 spectra of the glycans were acquired by simple parent ion scans at high resolution and the corresponding ions were manually selected for further MS / MS scans. Glycan structures were assigned manually and by using Glycoworkbench software based on fragmentation patterns and common biosynthetic pathways.

[0684] Tribology

[0685] SynLubricin was evaluated as a boundary lubricant using a custom linear reciprocating tribometer as previously described (Gleghorn and Bonassar, 2008). Briefly, cylindrical cartilage explants (6 mm diameter x 2 mm thickness) were harvested from femoral condyles of neonatal bovine stifle joints. Endogenous cartilage-bound lubricin was extracted using a 30 minute incubation in 1.5 M NaCl followed by a 1 hour equilibration step in PBS. Explants were incubated in PBS, SynLubricin, or bovine synovial fluid for 15-20 minutes prior to loading onto the tribometer in 1 mL baths of the respective fluids. Explants were compressed against a glass counterface to approximately 30% strain and allowed to decompress over the course of one hour. After the equilibrium normal load was achieved, the counterface was linearly reciprocated at a speed of 0.3 mm / s for three cycles. Simultaneously, biaxial load recording measured the normal and shear loads. Friction coefficients were calculated as the average shear force over the sliding time divided by the equilibrium normal load for both forward and reverse directions and at each speed.

[0686] Statistical Analysis

[0687] Statistical significance was determined using one-way ANOVA or Student’s t-test (two-tailed) as appropriate using Prism (GraphPad). For lubrication data, one-way ANOVA and Turkey’s post-hoc tests were performed to compare the mean friction coefficients of all lubricants. All figures were generated in Prism (GraphPad, La Jolla, CA).

[0688] Table 1 of Part III: Amino acid composition of tandem repeats in human PRG4 isoform A and SynLubricin.

[0689]

[0690] The following sequences are human, canine, and equine SynLubricin. Italicized letters indicate the secretion signal. Bold nucleotides lead up to the GS between the SynLubricin sequence.

[0691] DNA:

[0692]

[0693]

[0694] Amino acid:

[0695]

[0696]

[0697] Canine SynLubricin

[0698] Italics = canine secreted peptide sequence

[0699] DNA:

[0700]

[0701]

[0702] Amino acid:

[0703]

[0704] (1) Equine SynLubricin

[0705] Red = equine secreted peptide sequence

[0706] DNA:

[0707]

[0708]

[0709] Amino acid:

[0710]

[0711]

[0712] References

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[0754] Part IV

[0755] Among other things, this Part IV provides a description of the physical principles of membrane shape regulation by the glycocalyx.

[0756] In connection with this Part IV, it is well known that cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape regulation occurs is not fully resolved. This Part IV describes a broad synergy between the shape generation process inside the cell and the external organization and composition of the glycocalyx on the cell surface. The mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the protrusion of spherical and finger-like extensions from the cell surface. The polymer brush model of the glycocalyx successfully predicts the influence of polymer size and cell surface density on membrane morphology. Specific glycocalyx compositions can also induce plasma membrane instability to generate more exotic wavy and beaded membrane structures and drive extracellular vesicle secretion. In summary, the results presented in this Part IV indicate a fundamental role of the glycocalyx in regulating the features of curved membranes that play a role in different modes of communication between cells and with the extracellular matrix.

[0757] Introduction to Part IV

[0758] Tubular and spherical extensions of the plasma membrane play key roles in human development and daily cellular functions. While it has long been recognized that curved membrane protrusions increase the cell surface area for secretion, absorption, and receptor-mediated communication, modern studies have provided compelling examples of more diverse and complex functions (Marshall, 2012). For example, T cells of the adaptive immune system produce high-density tubular microvilli to engage antigen-presenting cells, and such structures can be equally important for engineered immune cell therapies to recognize tumor cells (D’Aloia et al., 2018; Jung et al., 2016). Membrane protrusions also enable intercellular communication at precise three-dimensional locations within long ranges and tissues. During development, long and thin membrane protrusions called cytonemes precisely deliver morphogens from ‘sender’ cells to specific ‘receiver’ cells up to 40 micrometers away (Bischoff et al., 2013; Kornberg and Roy, 2014). Stem cells, immune cells, and many other cell types are also known to curve their plasma membranes into spherical microvesicles that directly shed and can deliver macromolecular cargo over long distances (Tricarico et al., 2017). Furthermore, curved membrane features are ubiquitous in physical cellular behaviors, including migration and force transmission. For example, spherical membrane bulges called blebs are produced by primordial germ cells, tumor cells, and other cell types for protruding and frictionally coupling with tissue matrices during migration (Paluch and Raz, 2013).

[0759] Dysregulation of membrane shape generation processes can directly contribute to disease progression. As one notable example, invasive tumor cells frequently extend large numbers of microvilli for adhesion and rolling in blood vessels (Kramer and Nicolson, 1979; Liu et al., 2018). Invasive tumor cells can also protrude blebs for amoeboid migration (Bergert et al., 2015; Friedl and Wolf, 2010). Microvesicles are commonly budded from tumor cell plasma membranes at abnormal high rates (Antonyak et al., 2011; Becker et al., 2016). It is now recognized that the cargo carried by these particles has diverse regulatory roles, including reprogramming of other cell types in the stroma and preparation of distant metastatic niches for colonization (Becker et al., 2016).

[0760] It is postulated that forces arising from cytoskeletal dynamics generate membrane curvature of different spherical and tubular structures on the cell surface. It is envisaged that polymerized cytoskeletal filaments are pushed out along the plasma membrane at discrete points in order to extend microvilli, flagella, filopodia, and other finger-like projections (Footer et al., 2007; Gupton and Gertler, 2007; Peskin et al., 1993). During bleb formation, contraction of the cytoskeleton generates hydrostatic pressure of the spherical expansion of the membrane (Charras et al., 2005). The physical dynamics of bending the plasma membrane subregion into a microvesicle are still not fully understood; however, reports have implicated the actin cytoskeleton in its biosynthesis (Tricarico et al., 2017).

[0761] While the cell surface glycocalyx is not highlighted in the canonical membrane shape regulation model, the correlation between glycocalyx composition and cell surface morphology is high in both normal and disease states. In normal cell physiology, polypeptides and sugar copolymers called mucins are frequently anchored at high density on the surface of epithelial microvilli (Hattrup and Gendler, 2008; Kesavan et al., 2009; Kesimer et al., 2013), flagella (Button et al., 2012), and filopodia (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 chain modifications of sialic acid and hyaluronan coat the highly curved surfaces of neuronal axons (Fowke et al., 2017; van den Pol and Kim, 1993; Zhang et al., 1992). T cells and dendritic cells express cell surface mucins upon activation or maturation, which is often coincident with dramatic changes in membrane tubulation and microvilli production (Agrawal et al., 1998; Cloosen et al., 2004; Jung et al., 2016; Pilon et al., 2009). Invasive tumor cells frequently produce large amounts of mucins and hyaluronan on their cell surface (Kufe, 2009; Turley et al., 2016), and there is anecdotal evidence that expression of these polymers is associated with their unique membrane features, such as extensive microvilli (Polefka et al., 1984). Mucin and hyaluronan polymers also densely arrange on the surface of enterocytes, reactive astrocytes, dendritic cells, and tumor cells 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 morphology, the specific mechanism of action has not been described. The present disclosure helps to understand this mechanism of action.

[0762] Mucins and long-chain glycopolymers are anchored to membranes in a manner such that long polymer chains or loops are expected to extend from the cell surface (Hattrup and Gendler, 2008; Lee et al., 1993). This assembly resembles the well-studied structure known as a brush in polymer physics, where polymers are grafted to a surface at one end (Chen et al., 2017). Polymer brush theory has long recognized that the spatial interactions of densely packed brushes limit the number of molecular conformations each polymer can explore, increasing the free energy of the system through reduced entropy (de Gennes, 1980). Similar to the thermodynamic basis of gas pressure, the entropy penalty associated with molecular crowding can in principle generate a pressure sufficient to deform a flexible surface, such as a membrane (Hiergeist and Lipowsky, 1996; Lipowsky, 1995).

[0763] Results

[0764] Glycocalyx polymers and membrane morphology:

[0765] In this Section IV, we analyzed whether glycocalyx polymers can generate an entropic bending force to promote the formation of specific membrane forms. As a corollary to this, we tested whether the emerging membrane structure can be tuned by rational manipulation of the glycocalyx.

[0766] To test this, we constructed a genetically encoded library of native, semi-synthetic, and rationally designed mucin polymers of varying size, backbone sequence, and membrane anchoring ( Figure 36 A and Figure 38 A). Each construct encodes a mucin polymer domain composed of an unstructured polypeptide backbone with a high density of serine and threonine sites for O-glycosylation. When expressed in cells, the mucin domain is post-translationally modified with O-linked glycoside side chains to form the bottlebrush molecular structure that defines mucins ( Figure 33 A, B).

[0767] The polymer domains in the library include 42 native tandem repeats of Mucin-1 (Muc1-42TR), the serine and threonine-rich polymer domain of podocalyxin (Podxl; S / T-rich), and a new synthetic mucin we rationally designed and constructed by fusing 80 perfect repeats of the consensus sequence PPASTSAPGA based on the mucin O-glycosylation sequence (Rational) Figure 38 A and Figure 33 A). Each polymer domain was fused to either the native Muc1 transmembrane anchor with the cytoplasmic tail deleted (ACT), or a 21 amino acid synthetic transmembrane anchor (TM21), or the native mucin anchor with a membrane proximal green fluorescent protein for imaging (GFP- ACT) Figure 38 A and Figure 33 A).

[0768] When expressed and assembled at high levels on epithelial cell surfaces, each of the mucin polymers in our library triggered dramatic tubulation of the plasma membrane, as observed by scanning electron microscopy (SEM) Figure 38 B, C, and Figure 38 B). Without wishing to be bound by any particular theory, we infer that this tubulation can be a general consequence of polymer anchoring to the plasma membrane and does not require a particular biopolymer sequence or transmembrane anchor. Notably, Muc1-42TRACr, which is identical to native Mucin-1 except for the cytoplasmic tail, indicates that a native glycocalyx component can influence plasma membrane morphology in addition to our rationally designed polymers. Mucin expression had no significant effect on endocytosis, arguing against lipid recycling and membrane tension regulation as the primary mechanism for morphological changes Figure 33 C, D).

[0769] The tubulation phenomenon was relatively insensitive to the length of the mucin polymer domain, provided that the polymer was expressed at a medium to high density on the cell surface. cDNAs for 0, 10, or 42 Muc1 repeats were fused to a GFP-tagged transmembrane anchor to encode cell surface mucins of expected contour lengths of 0, 65, and 270 nm, respectively Figure 38 D, and Figure 33 E). Cell lines expressing the constructs were sorted into populations with similar mucin surface densities using nanobodies that probe cell surface GFP Figure 33 D). Flexible polymer domains were required for efficient membrane tubulation, and despite the size difference between the 10 and 42-TR mucins, they induced comparable levels of membrane tubulation Figure 38 E, and Figure 33 F). We compared cells of similar spreading area to rule out the possibility that changes in membrane surface tension and other effects associated with cell spreading could explain the morphological differences Figure 36 E).

[0770] Similar to mucins, we found that the glycocalyx, which is rich in large linear polysaccharides, can also trigger dramatic changes in plasma membrane morphology. Notably, Hyaluronan synthase 3 (HAS3) expression increased the density of high molecular weight hyaluronan (HA) polymers on the cell surface and caused many finger-like membrane extensions to protrude Figure 33 A-D), consistent with previous observations by others (Koistinen et al., 2015). Together, these results indicate that different glycocalyx polymer types and sizes can influence the cell morphological state.

[0771] We next tested whether the glycocalyx biopolymers could induce spontaneous curvature in model membranes independent of intracellular machinery. When anchored to the surface of giant unilamellar vesicles (GUVs), we found that the S / T-rich polymer domain of Podxl triggered the spontaneous generation of both spherical and tubular membrane structures Figure 37 F and Figure 33 A, B). Small tubes were also observed in very high density of folded proteins (human serum albumin (HSA)), consistent with previous findings that large clusters of folded or intrinsically disordered proteins can induce spontaneous membrane curvature in GUVs (Stachowiak et al., 2010) Figure 37 F and Figure 33 B, C). However, the surface density required for Podxl mucin to induce spontaneous tubulation was significantly lower than for HSA Figure 37 F and Figure 34 B).

[0772] Specialized cells in vivo:

[0773] Motivated by these in vitro observations, we considered whether the glycocalyx polymers could play a role in the morphogenesis of specialized cell types in vivo. We chose to evaluate synoviocytes, as these secretory cells are known to produce large amounts of HA for joint lubrication and are therefore expected to display high densities of HA polymers on their surface. We isolated synovial tissue from the knee joint of a horse Figure 34 A), and found that primary synoviocytes expressing HAS3 were highly tubulated, but treatment with hyaluronidase (HyA) to degrade HA caused rapid destabilization and disappearance of membrane tubes Figure 34 B, C). We also evaluated the synoviocyte morphology in freshly extracted and briefly cultured (<1 hour) tissue in vitro. Synoviocytes in native synovial tissue displayed HA-rich heads that were highly tubulated and projected from the tissue matrix Figure 34 D, E). Brief treatment of the tissue with HyA in vitro caused a dramatic retraction of synoviocyte tubes, suggesting a role for the glycocalyx in maintaining membrane projection in vivo Figure 38 E).

[0774] Polymer brush framework:

[0775] We considered whether the observed membrane shapes and their frequencies could be rationalized by the framework of polymer brush theory. We note that two limiting regimes are classically described for end-grafted polymers in polymer physics: the "mushroom" regime, in which low grafting densities of polymers have limited interactions with one another, and the "brush" regime, in which crowded polymers can spatially and electrostatically interact with one another to exert large pressures on the anchoring surface (Milner, 1991) Figure 38A). For mucins, we expect the transition of the mushroom-to-brush regime to occur at a surface density where the average distance between polymers is about twice their radius of gyration in solution, Figure 41 A).

[0776] To measure the radius of gyration and flexibility of individual mucins, we generated recombinant Muc1-42TR with terminal purification tags instead of a transmembrane anchor Figure 41 A-C). Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) reports a mucin radius of gyration of 32 nm ± 0.4% in physiological buffer. Based on an estimated Muc1-42TR contour length of about 270 nm, and without wishing to be bound by any particular theory, we infer that the mucin has a persistence length of about 7.5 nm and adopts an extended random coil conformation in solution as expected for a semi-flexible polymer.

[0777] We next tested whether the polymer brush theory can capture the physical behavior of mucin assemblies on the cell surface. We tested whether mucins stretch and extend in a predictable manner as they become increasingly crowded, a characteristic physical behavior originally described by Alexander and de Gennes in their seminal theory on polymer brushes (Alexander, 1977; Milner, 1991). We chose to assess mucin extension on actin-containing microvilli-like filaments because the curvature of these structures is highly uniform and essentially independent of mucin surface density Figure 35 D). Thus, we can approximate the surface of the filament as a rigid cylinder of fixed radius for direct comparison with classical theory. We constructed a cDNA for Muc1-42TR with a complementary epitope tag flanking the mucin polymer domain. After cell expression, the coding tag was labeled with a fluorophore-conjugated probe and resolved on microvilli cross-sections using a super-resolution optical technique called expansion microscopy (ExM) Figure 41 B and Figure 35 E). We found that mucin extension has an exponential dependence, or'scaling', on the fluorescence intensity and thus surface density, with an exponent of 0.48 ± 0.10 Figure 35 B). For polyelectrolytes grafted to a rigid cylindrical surface at physiological salt concentrations, this value is comparable to the power law exponents of 0.33 and 0.5 derived from theory (Zhulina and Borisov, 1996).

[0778] We created a polymer brush model to describe the physical behavior of the mucin-rich glycocalyx assembled on the plasma membrane. The entropic pressure contributed by the mucin brush generates a spontaneous membrane curvature that scales strongly with the polymer density and weakly with the polymer chain length (Hiergeist and Lipowsky, 1996) Figure 42C and Figure 33 The weak dependence on polymer length is consistent with the following findings: mucins with 10 and 42 repeats had comparable effects on cell surface morphology, despite a 4-fold difference in size. Figure 38 E and Figure 42 F). For these two mucins, our brush model predicts only about 20% difference in the induction of spontaneous membrane curvature (F). Figure 36 ).

[0779] Preferred membrane shape:

[0780] We tested whether the polymer model could explain the frequency of finger-like and spherical protrusions from the cell surface. We inferred that protrusions of a particular membrane feature are disadvantageous when high intracellular forces are required to extend or maintain the protrusions, and advantageous when these forces are minimized. By minimizing the standard Helfrich free energy function of a membrane with induced spontaneous curvature, we calculated the equilibrium cytoplasmic pressure required to maintain spherical vesicles and the point force required to maintain membrane tubules. Figure 36 D). For experimental comparisons, we evaluated the type, size, and frequency of plasma membrane features as a function of mucin cell surface density. Cells expressing Muc1-42TR GFP were labeled with anti-GFP nanobodies and sorted into populations with different mucin surface levels ( Figure 43 A). The average mucin surface density in each population was estimated by SDS-PAGE using interpolation of nanobody standard curves. Figure 35 The molecular surface density in the sorted population ranged from 180 to approximately 50,000 mucins / μm. 2 Within this range. For reference, based on the measured radius of gyration of recombinant Muc1-42TR in solution, we expect the mushroom-to-brush transition to occur at approximately 250 mucin / μm. 2 .

[0781] Initially, we evaluated membrane vesicles. Using physical parameters measured against the Muc1-42TR, we predicted that the pressure required to maintain a vesicle with a typical radius of 250 nm would be minimal at moderate mucin densities close to the mushroom-brush transition. Figure 35 D). The unexpected model predicts that the required maintenance pressure will rise sharply at higher mucin densities, rapidly exceeding the known limits of cellular contraction mechanisms (Charras et al., 2008). Therefore, theory suggests that bubbling will be inhibited by highly dense glycocalyxes ( Figure 36 D). Our experimental observations showed good qualitative agreement with these predictions. Cells with mucin densities close to the estimated mushroom-brush transition exhibited a considerable number of large vesicle-like forms with an average radius of 260 ± 100 nm. Figure 36 BD; 180 mucins / μm2 ). Upon exchange into the brush regime, the frequency of blebs abruptly decreases, consistent with the model prediction of a secondary rise in the pressure necessary to maintain blebs Figure 35 B, D).

[0782] The glycocalyx polymer model predicts a very different dependence of tubule protrusion on mucin density. The predicted point force necessary to maintain an extended tubule gradually decreases with high mucin density and does not exhibit a sharp transition Figure 36 D). Thus, the frequency of cell surface tubules observed in our sorted cell populations increases continuously with mucin density throughout the mushroom and brush regimes, until the cells are completely saturated with tubules at very high mucin density Figure 36 B-E). Notably, the model predicts that the force necessary to extend a tubule at these high densities is comparable to the polymerization force of a single cellular cytoskeletal microfilament, approximately 1 pN (Footer et al., 2007). Based on experimentally measured mucin density, we estimate the theoretical point force f necessary to maintain a tubule. Notably, the experimentally observed tubule frequency is almost perfectly inversely correlated with the theoretical point force Figure 37 F). The Pearson correlation coefficient describing the relationship between tubule density and 1 / f is 0.97.

[0783] The polymer model also predicts that the spontaneous curvature generated by high mucin surface density exceeds the curvature of the lamellipodia observed on the surface of our cells. We note that the tubular membrane protrusions on our cells typically contain a filamentous actin (F-actin) core and do not contain microtubules Figure 44 A, B, Figure 37 A-D). Disruption of F-actin assembly with the drug latrunculin A (LatA) causes the tubule diameter to decrease by approximately 30 nm Figure 44 C, D and Figure 37 E, F), indicating that the spontaneous curvature induced by mucin exceeds the curvature of an actin-filled protrusion. Note that our measurements on LatA-treated cells likely exclude very thin and fragile membrane tubules, which are difficult to preserve throughout SEM sample preparation. Nonetheless, these results clearly indicate that the spontaneous curvature generated by the glycocalyx can meet or exceed the curvature requirements of thin lamellipodia with characteristic diameters of approximately 100-200 nm, such as microtubules, flagella, filopodia, axons, and cell conduits.

[0784] Membrane instability and extracellular vesicle production:

[0785] We next considered whether other functional membrane shapes could arise through the action of the glycocalyx. We noted that a gradual increase in spontaneous curvature is known to trigger membrane instability and morphological changes in membrane vesicles (Campelo and Hernandez-Machado, 2007; Tsafrir et al., 2001). Thus, we reasoned that if the F-actin core, which physiologically resists the spontaneous curvature of mucins, were disrupted, membrane instability could arise. Indeed, our model indicated that approximately 400 mucins per pm 2 or more would be sufficient to drive membrane instability in tubules. Thus, we observed that LatA treatment triggered the formation of beading and undulating structures characteristic of membrane instability Figure 37 D).

[0786] Deuling, Helfrich, and others have considered theoretically the instability in membrane tubules of volume-to-area ratio λ and found that for certain spontaneous curvatures c0, the bending energy of the membrane vanishes by adopting one of three "Delaunay" shapes: for c0= ½ λ, a cylinder (shape 1); for ½ λ < c0< 2 / 3 λ, a set of smoothly varying undulations (shape 2); and for c0= 2 / 3 λ, a set of "beads" of equal size (shape 3) (Campelo and Hernandez-Machado, 2007; Tsafrir et al., 2001). For spontaneous curvatures beyond 2 / 3 λ, the lowest energy shape satisfying the volume and surface area constraints was found to comprise a set of small beads with a preferred curvature, and one or more large beads required to hold the excess volume (shape 4) and a set of gradient-sized beads (shape 5) (Campelo and Hernandez-Machado, 2007; Tsafrir et al., 2001). We assessed whether the minimum energy surface shapes 1-5 would form on cells expressing moderate to high levels of mucins in the absence of exogenous treatment and found common instances of each of the expected shapes Figure 37 E). The observation of these shapes provides a compelling argument that membrane instability can be driven by the specific composition of the glycocalyx.

[0787] Remarkably, we found that membrane beading is an intermediate step toward the direct secretion of extracellular vesicles from the plasma membrane Figure 5 F). Conditioned media from cells expressing Muc1-42TR contained high concentrations of particles in the size range of approximately 100-nm to 400-nm Figure 37 G), characteristic of microvesicles (Pol et al., 2016). Particle production was further enhanced by LatA treatment to disrupt the supporting F-actin core of surface protrusions and locally destabilize the plasma membrane Figure 37H). Cryogenic transmission electron microscopy (cryo-TEM) confirmed that the secreted particles are indeed membrane vesicles and that they are grafted with a clear glycocalyx ultrastructure on their surface Figure 5 I). Previous reports showed that vesicles are generated from microvilli in enterocytes and other mucin-expressing cells (McConnell et al., 2009), and these observations of the present application are consistent with this. However, and without wishing to be bound by any particular theory, our results currently suggest a possible three-step mechanism of microvesicle production: (1) cytoskeletal microfilaments help extend and stabilize long and thin protrusions from the plasma membrane in a glycocalyx-dependent manner; (2) after cytoskeletal core disassembly, the spontaneous curvature imposed by the glycocalyx induces membrane instability of the tubule; and (3) the membrane bead snaps to release the vesicle Figure 35 E, F).

[0788] Discussion

[0789] The description presented in this Section IV suggests an entropic mechanism by which the glycocalyx can strongly influence the preference of different plasma membrane shapes and protrusions. Morphological changes regulated by the glycocalyx can in principle have a broad impact on membrane processes, from absorption and secretion to cell communication, signaling, and motility (Lange, 2011; Paluch and Raz, 2013; Sauvanet et al., 2015; Schmick and Bastiaens, 2014). Given that glycosylation changes dramatically and in conjunction with cell fate transitions (Buck et al., 1971; Freeze, 2013; Satomaa et al., 2009), and that the pool of monomers used to build the glycocalyx polymers is tightly linked to specific metabolic programs (Dennis et al., 2009; Koistinen et al., 2015; Ying et al., 2012), this Section IV raises the intriguing possibility that the glycocalyx can act as a conduit that links physical morphology to specific cell states.

[0790] Contemporary frameworks for understanding membrane shape regulation lack, to a large extent, a physical description of the glycocalyx. However, it is almost universally found that long-chain biopolymers in the glycocalyx are anchored to the surface of curved membrane features and cell surface organelles (Bennett et al., 2001; Button et al., 2012; Evanko et al., 2007; Fowke et al., 2017; Hattrup and Gendler, 2008; Kesavan et al., 2009; Kesimer et al., 2013; Makabe Sayoko et al., 2006; van den Pol and Kim, 1993; Zhang et al., 1992). The results in this Section IV suggest that principles and theories of polymer physics can be employed to understand (at least a first approximation of) the physical regulation of the glycocalyx on membrane shape generation. The model of end-anchored polymer mushrooms and polymer brushes is a simple physical representation of the glycocalyx. The actual glycocalyx architecture can include additional levels of structure from cross-linking, entanglement, and molecular heterogeneity (Tammi et al., 2002). However, the almost perfect inverse relationship between the force requirement for membrane extension and the frequency of these extensions observed experimentally, as estimated using the relatively simple glycocalyx model, demonstrates that at least some of the physical behavior of the glycocalyx can be captured using a polymer network model. In fact, according to the classic scaling laws developed for polymer brushes by de Gennes and others (Gennes, 1979; Zhulina and Borisov, 1996), we find that glycocalyx polymer extension is related to the cell surface density.

[0791] How the glycocalyx and intracellular shape generation processes are coordinated in space and time to control membrane protrusion is not fully resolved. In particular, Rho family GTPases are master regulators of cytoskeletal dynamics and cell surface morphology (Hall, 1998). The description in this Section IV suggests that the glycocalyx, by regulating the barrier to membrane curvature, prepares the membrane to swell into a particular type of spherical or tubular form subject to Rho GTPase regulation. This integrated view suggests that interference with normal cell surface morphology can be achieved through dysregulation of intracellular shape generation processes, glycocalyx polymer assembly, or both. For example, dysregulation 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 each can contribute to unique cell surface dynamics that contribute to the lethality of metastatic cancer cells.

[0792] Anchoring polymers cause surfaces to bend as a general physical phenomenon. Thus, the regulation of membrane shape by the glycocalyx can be a relevant universal feature in all cell types. Future efforts can elucidate the physical function of the glycocalyx in the biogenesis of specific membrane organelles and signaling structures, including flagella, axons, cell conduits, and microvilli. Nonetheless, the description in this Section IV supports a more holistic model of membrane shape regulation that includes consideration of forces on both the intracellular and extracellular faces of the plasma membrane.

[0793] Methods

[0794] 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). The following lectins were used: 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). Hyaluronan (HA) was probed in blots with fluorescently labeled or biotinylated bovine nasal hyaluronan binding protein (HABP, Millipore). Biotin-HABP was detected with horseradish peroxidase-conjugated streptavidin (HRP-streptavidin; R&D Systems). For HA ELISA, the DuoSet Hyaluronan kit was from R&D Systems. Actin depolymerization was induced by treatment with latrunculin A (LatA; 76343-93-6; Cayman Chemicals).

[0795] For the formation of giant unilamellar vesicles (GUVs), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-((N-(5-amino-1-carboxypentyl) iminodiacetic acid) butanedioyl), nickel-containing salt (DOGS-NTA-Ni) were purchased from Avanti Polar Lipids; 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- pentanoyl)-1-hexadecanoyl-sn-glycero-2-phosphocholine (Bodipy-PC) was purchased from Invitrogen; His-tagged recombinant human podocalyxin (Ser23-Arg427; Accession No. AAB61574.1) was from R&D Systems; His-tagged human serum albumin (Accession No. NP_000468) was from ACROBiosystems.

[0796] GFP-binding proteins (nanobodies) were 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% OsO4for scanning electron microscopy (SEM) were obtained from Electron Microscopy Sciences.

[0797] Cloning and constructs. cDNAs of human Muc1 with cytoplasmic tail deletion of 42 tandem repeats (Muc1-42TRACT), fusion of Muc1-42TR polymerization domain with TM21 synthetic membrane domain (Muc1-42TR TM21), cytoplasmic tail deletion of human glypican (rich in S / T ACT) were generated and cloned into tetracycline-inducible PiggyBac expression vector (pPB TetOn Puro) or mammalian expression vector pcDNA3.1 as previously described (Paszek et al., 2014; Shurer et al.). For preparation of lentiviral vector pLV Hygro TetOn HAS3, cDNA of human HAS3 (accession number NP_005320) was obtained from OriGene and amplified by PCR with forward primer 5'-GGCACCTCGAGGATGCCGGTGCAGCTGACGACA-3' (SEQ ID NO: 88) and reverse primer 5'-GGCAGAATTCTTACACCTCAGCAAAAGCCAAGCT-3' (SEQ ID NO: 89). PCR product was cloned into pJET1.2 (Thermo Fisher) and subcloned into AbsI and EcoRI sites of pLV Hygro Te...

Claims

1. A recombinant lubricin polypeptide comprising a repeating sequence, wherein the polypeptide comprises the sequence SEQ ID NO:

68.

2. The recombinant lubricant polypeptide according to claim 1, further comprising a secretion signal, said secretion signal being the sequence MAWKTLPIYLLLLLSVFVIQQVSS shown in SEQ ID NO:

72.

3. The recombinant lubricant polypeptide according to claim 1, further comprising a secretion signal, said secretion signal being the sequence METDTLLLWVLLLWVPGSTGD shown in SEQ ID NO:

81.

4. The recombinant lubricant polypeptide according to claim 1, further comprising a secretion signal, said secretion signal being the sequence MQWKILPIYLLLLSVFLIQQVSS shown in SEQ ID NO:

82.

5. The recombinant lubricant polypeptide according to claim 1, further comprising a secretion signal, said secretion signal being the sequence MEWKILPIYLLLLLSIFSIQEVSS shown in SEQ ID NO:

74.

6. A composition comprising a recombinant lubricant polypeptide according to any one of claims 1 to 5.

7. One or more mammalian cells comprising the recombinant lubricant polypeptide according to any one of claims 1 to 5.

8. One or more mammalian cells according to claim 7, wherein the cells are in a suspension culture.

9. An isolated polynucleotide and / or expression vector encoding a recombinant lubricin polypeptide, said isolated polynucleotide and / or expression vector comprising the sequence SEQ ID NO:

67.

10. One or more mammalian cells comprising the isolated polynucleotide of claim 9, wherein the one or more mammalian cells are adapted to grow in a suspension culture.

11. A suspension culture comprising mammalian cells expressing a recombinant lubricin polypeptide according to any one of claims 1 to 5.

12. A method for preparing a recombinant lubricant polypeptide according to any one of claims 1 to 5, the method comprising introducing a polynucleotide encoding the recombinant lubricant polypeptide into a mammalian cell, such that the cell expresses the recombinant lubricant polypeptide.

13. The method of claim 12, further comprising isolating the recombinant lubricant polypeptide from the mammalian cells.

14. An inanimate article, wholly or partially coated with a composition comprising any one of claims 1 to 5 of a recombinant lubricant polypeptide.

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