Metabolically Glycoengineered Neural Stem Cells Boost Neural Repair After Cardiac Arrest.

Cardiac arrest (CA)-induced cerebral ischemia remains challenging with high mortality and disability. Neural stem cell (NSC) engrafting is an emerging therapeutic strategy with considerable promise that, unfortunately, is severely compromised by limited cell functionality after in vivo transplantation. This groundbreaking report demonstrates that metabolic glycoengineering (MGE) using the "Ac5ManNTProp (TProp)" monosaccharide analog stimulates the Wnt/ß-catenin pathway, improves cell adhesion, and enhances neuronal differentiation in human NSCs in vitro thereby substantially increasing the therapeutic potential of these cells. For the first time, MGE significantly enhances NSC efficacy for treating ischemic brain injury after asphyxia CA in rats. In particular, neurological deficit scores and neurobehavioral tests experience greater improvements when the therapeutic cells are pretreated with TProp than with "stand-alone" NSC therapy. Notably, the TProp-NSC group exhibits significantly stronger neuroprotective functions including enhanced differentiation, synaptic plasticity, and reduced microglia recruitment; furthermore, Wnt pathway agonists and inhibitors demonstrate a pivotal role for Wnt signaling in the process. These findings help establish MGE as a promising avenue for addressing current limitations associated with NSC transplantation via beneficially influencing neural regeneration and synaptic plasticity, thereby offering enhanced therapeutic options to boost brain recovery following global ischemia.

A dual-acting DNASE1/DNASE1L3 biologic prevents autoimmunity and death in genetic and induced lupus models.

A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and DNASE1L3 deficiency, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association includes an ultra-rare pediatric population with DNASE1L3 deficiency who develop SLE, adult patients with loss of function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies to DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors. Notably, we found that the biologic prevented the development of lupus in Dnase1-/-/Dnase1L3-/- double knockout mice and rescued animals from death in pristane-induced lupus. Finally, we confirmed that the human isoform of the enzyme biologic was not recognized by autoantibodies in SLE and efficiently degrades genomic and mitochondrial cell free DNA, as well as microparticle DNA, in SLE plasma. Our findings suggest that autoimmune diseases characterized by aberrant DNA accumulation, such as SLE, can be effectively treated with a replacement DNASE tailored to bypass pathogenic mechanisms, both genetic and acquired, that restrict DNASE1L3 activity.

Protocol Considerations for In Vitro Metabolic Glycoengineering of Non-Natural Glycans.

Metabolic glycoengineering (MGE) refers to a technique where non-natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell-surface-displayed oligosaccharides, where they can modulate a host of biological activities or be exploited as tags for bioorthogonal and chemoselective ligation reactions. Over the past decade, azido-modified monosaccharides have become the go-to analogs for MGE; at the same time, analogs with novel chemical functionalities continue to be developed. Therefore, one emphasis of this article is to describe a general approach for analog selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell-surface glycans have been successfully remodeled by MGE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. This manuscript concludes by detailing how one of these detection methods-flow cytometry-can be successfully utilized to quantify MGE analog incorporation and set the stage for numerous follow-up applications. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Incubation of cells with sugar analogs Support Protocol: Routine growth and maintenance of Jurkat cells Basic Protocol 2: Cell viability assays Basic Protocol 3: Periodate-resorcinol assay to measure analog uptake and incorporation into metabolic pathways Basic Protocol 4: Quantitation of cell-surface glycoconjugates.

Improving Schwann Cell Differentiation from Human Adipose Stem Cells with Metabolic Glycoengineering.

Schwann cells (SCs) are myelinating cells that promote peripheral nerve regeneration. When nerve lesions form, SCs are destroyed, ultimately hindering nerve repair. The difficulty in treating nerve repair is exacerbated due to SC's limited and slow expansion capacity. Therapeutic use of adipose-derived stem cells (ASCs) is emerging in combating peripheral nerve injury due to these cells' SC differentiation capability and can be harvested easily in large numbers. Despite ASC's therapeutic potential, their transdifferentiation period typically takes more than two weeks. In this study, we demonstrate that metabolic glycoengineering (MGE) technology enhances ASC differentiation into SCs. Specifically, the sugar analog Ac5ManNTProp (TProp), which modulates cell surface sialylation, significantly improved ASC differentiation with upregulated SC protein S100ß and p75NGFR expression and elevated the neurotrophic factors nerve growth factor beta (NGFß) and glial cell-line-derived neurotrophic factor (GDNF). TProp treatment remarkably reduced the SC transdifferentiation period from about two weeks to two days in vitro, which has the potential to improve neuronal regeneration and facilitate future use of ASCs in regenerative medicine.

Anticancer Properties of Hexosamine Analogs Designed to Attenuate Metabolic Flux through the Hexosamine Biosynthetic Pathway.

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP. By screening a panel of analogs in pancreatic cancer and glioblastoma multiform (GBM) cells, we identified Ac4Glc2Bz─a benzyl-modified GlcNAc mimetic─as an antiproliferative cancer drug candidate that down-regulated oncogenic metabolites and reduced GBM cell motility at concentrations non-toxic to non-neoplastic cells. More specifically, the growth inhibitory effects of Ac4Glc2Bz were linked to reduced levels of UDP-GlcNAc and concomitant decreases in protein O-GlcNAc modification in both pancreatic cancer and GBM cells. Targeted metabolomics analysis in GBM cells showed that Ac4Glc2Bz disturbed glucose metabolism, amino acid pools, and nucleotide precursor biosynthesis, consistent with reduced proliferation and other anti-oncogenic properties of this analog. Furthermore, Ac4Glc2Bz reduced the invasion, migration, and stemness of GBM cells. Importantly, normal metabolic functions mediated by UDP-GlcNAc were not disrupted in non-neoplastic cells, including maintenance of endogenous levels of O-GlcNAcylation with no global disruption of N-glycan production. Finally, a pilot in vivo study showed that a potential therapeutic window exists where animals tolerated 5- to 10-fold higher levels of Ac4Glc2Bz than projected for in vivo efficacy. Together, these results establish GlcNAc analogs targeting the HBP through salvage mechanisms as a new therapeutic approach to safely normalize an important facet of aberrant glucose metabolism associated with cancer.

A versatile design platform for glycoengineering therapeutic antibodies.

Manipulation of glycosylation patterns, i.e., glycoengineering, is incorporated in the therapeutic antibody development workflow to ensure clinical safety, and this approach has also been used to modulate the biological activities, functions, or pharmacological properties of antibody drugs. Whereas most existing glycoengineering strategies focus on the canonical glycans found in the constant domain of immunoglobulin G (IgG) antibodies, we report a new strategy to leverage the untapped potential of atypical glycosylation patterns in the variable domains, which naturally occur in 15% to 25% of IgG antibodies. Glycosylation sites were added to the antigen-binding regions of two functionally divergent, interleukin-2-binding monoclonal antibodies. We used computational tools to rationally install various N-glycosylation consensus sequences into the antibody variable domains, creating "glycovariants" of these molecules. Strikingly, almost all the glycovariants were successfully glycosylated at their newly installed N-glycan sites, without reduction of the antibody's native function. Importantly, certain glycovariants exhibited modified activities compared to the parent antibody, showing the potential of our glycoengineering strategy to modulate biological function of antibodies involved in multi-component receptor systems. Finally, when coupled with a high-flux sialic acid precursor, a glycovariant with two installed glycosylation sites demonstrated superior in vivo half-life. Collectively, these findings validate a versatile glycoengineering strategy that introduces atypical glycosylation into therapeutic antibodies in order to improve their efficacy and, in certain instances, modulate their activity early in the drug development process.

Glycoengineering human neural stem cells (hNSCs) for adhesion improvement using a novel thiol-modified N-acetylmannosamine (ManNAc) analog.

This study sets the stage for the therapeutic use of Ac5ManNTProp, an N-acetylmannosamine (ManNAc) analog that installs thiol-modified sialoglycans onto the surfaces of human neural stem cells (hNSC). First, we compared hNSC adhesion to the extracellular matrix (ECM) proteins laminin, fibronectin, and collagen and found preferential adhesion and concomitant changes to cell morphology and cell spreading for Ac5ManNTProp-treated cells to laminin, compared to fibronectin where there was a modest response, and collagen where there was no observable increase. PCR array transcript analysis identified several classes of cell adhesion molecules that responded to combined Ac5ManNTProp treatment and hNSC adhesion to laminin. Of these, we focused on integrin α6ß1 expression, which was most strongly upregulated in analog-treated cells incubated on laminin. We also characterized downstream responses including vinculin display as well as the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). In these experiments, Ac5ManNTProp more strongly induced all tested biological endpoints compared to Ac5ManNTGc, showing that the single methylene unit that structurally separates the two analogs finely tunes biological responses. Together, the concerted modulation of multiple pro-regenerative activities through Ac5ManNTProp treatment, in concert with crosstalk with ECM components, lays a foundation for using our metabolic glycoengineering approach to treat neurological disorders by favorably modulating endpoints that contribute to the viability of transplanted NSCs.

Strategies for Glycoengineering Therapeutic Proteins.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.g., afucosylated Gazyva®, Poteligeo®, Fasenra™, and Uplizna®). In the future, the deliberate modification of therapeutic protein glycosylation will become more prevalent as glycoengineering strategies, including sophisticated computer-aided tools for "building in" glycans sites, acceptance of a broad range of production systems with various glycosylation capabilities, and supplementation methods for introducing non-natural metabolites into glycosylation pathways further develop and become more accessible.

Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs.

This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac5ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac5ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac5ManNTProp and Ac5ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac5ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac5ManNTProp and Ac5ManNTBut suppressed adipogenic differentiation, with Ac5ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac5ManNTProp and Ac5ManNTBut) for manipulating human stem cells.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3 ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.

Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc.

Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells. Interestingly, the glycosites identified by SPEG and HILIC overlapped considerably whereas NGAG identified many glycosites not observed in the other two methods. Further, utilizing enhanced intact glycopeptide identification afforded by the NGAG workflow, we found that the sugar analog 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression.

Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-Bu3ManNAc-treated cells. Only three categories of proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-Bu3ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.

Exploiting metabolic glycoengineering to advance healthcare.

Metabolic glycoengineering (MGE) is a technique for manipulating cellular metabolism to modulate glycosylation. MGE is used to increase the levels of natural glycans and, more importantly, to install non-natural monosaccharides into glycoconjugates. In this Review, we summarize the chemistry underlying MGE that has been developed over the past three decades and highlight several recent advances that have set the stage for clinical translation. In anticipation of near-term application to human healthcare, we describe emerging efforts to deploy MGE in diverse applications, ranging from the glycoengineering of biotherapeutic proteins and the diagnosis and treatment of complex diseases such as cancer to the development of new immunotherapies.

Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production.

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu3 ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu3 ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of ß-1,4- and ß-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation. Supplementation of 1,3,4-O-Bu3 ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild-type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4-O-Bu3 ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild-type CHO cells. Moreover, a detailed mass spectrometric ESI-LC-MS/MS characterization of glycans at each of the three N-glycosylation sites of EPO showed that the 1st N-site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4-O-Bu3 ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu3 ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.

Improving Immunotherapy Through Glycodesign.

Immunotherapy is revolutionizing health care, with the majority of high impact "drugs" approved in the past decade falling into this category of therapy. Despite considerable success, glycosylation-a key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of an immunotherapeutic-has slowed the development of this class of drugs in the past and remains challenging at present. This article describes how optimizing glycosylation through a variety of glycoengineering strategies provides enticing opportunities to not only avoid past pitfalls, but also to substantially improve immunotherapies including antibodies and recombinant proteins, and cell-based therapies. We cover design principles important for early stage pre-clinical development and also discuss how various glycoengineering strategies can augment the biomanufacturing process to ensure the overall effectiveness of immunotherapeutics.

Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes.

In this report we use 'high-flux' tributanoyl-modified N-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and alkyne N-acyl groups (specifically, 1,3,4-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAz, and 1,3,4-O-Bu3ManNAl respectively) to probe intracellular sialic acid metabolism in the near-normal MCF10A human breast cell line in comparison with earlier stage T-47D and more advanced stage MDA-MB-231 breast cancer lines. An integrated view of sialic acid metabolism was gained by measuring intracellular sialic acid production in tandem with transcriptional profiling of genes linked to sialic acid metabolism. The transcriptional profiling showed several differences between the three lines in the absence of ManNAc analog supplementation that helps explain the different sialoglycan profiles naturally associated with cancer. Only minor changes in mRNA transcript levels occurred upon exposure to the compounds confirming that metabolic flux alone can be a key determinant of sialoglycoconjugate display in breast cancer cells; this result complements the well-established role of genetic control (e.g., the transcription of STs) of sialylation abnormalities ubiquitously associated with cancer. A notable result was that the different cell lines produced significantly different levels of sialic acid upon exogenous ManNAc supplementation, indicating that feedback inhibition of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE)-generally regarded as the 'gatekeeper' enzyme for titering flux into sialic acid biosynthesis-is not the only regulatory mechanism that limits production of this sugar. A notable aspect of our metabolic glycoengineering approach is its ability to discriminate cell subtype based on intracellular metabolism by illuminating otherwise hidden cell type-specific features. We believe that this strategy combined with multi-dimensional analysis of sialic acid metabolism will ultimately provide novel insights into breast cancer subtypes and provide a foundation for new methods of diagnosis.

Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings. We demonstrate that these lipid coated ellipsoidal particles maintain advantageous properties of lipid polymer hybrid particles, such as the ability for modular protein conjugation to the particle surface using versatile bioorthogonal ligation reactions. In addition, they exhibit biomimetic membrane fluidity and demonstrate lateral diffusive properties characteristic of natural membrane proteins. These ellipsoidal particles simultaneously provide benefits of non-spherical particles in terms of stability and resistance to non-specific phagocytosis by macrophages as well as enhanced targeted binding. These biomaterials provide a novel and flexible platform for numerous biomedical applications. STATEMENT OF SIGNIFICANCE: The research reported here documents the ability of non-spherical polymeric particles to be coated with lipids to form anisotropic biomimetic particles. In addition, we demonstrate that these lipid-coated biodegradable polymeric particles can be conjugated to a wide variety of biological molecules in a "click-like" fashion. This is of interest due to the multiple types of cellular mimicry enabled by this biomaterial based technology. These features include mimicry of the highly anisotropic shape exhibited by cells, surface presentation of membrane bound protein mimetics, and lateral diffusivity of membrane bound substrates comparable to that of a plasma membrane. This platform is demonstrated to facilitate targeted cell binding while being resistant to non-specific cellular uptake. Such a platform could allow for investigations into how physical parameters of a particle and its surface affect the interface between biomaterials and cells, as well as provide biomimetic technology platforms for drug delivery and cellular engineering.

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3 ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3 ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3 ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3 ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin, or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3 ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3 ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4-O-Bu3 ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3 ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3 ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

Pharmacological, Physiochemical, and Drug-Relevant Biological Properties of Short Chain Fatty Acid Hexosamine Analogues Used in Metabolic Glycoengineering.

In this study, we catalog structure activity relationships (SAR) of several short chain fatty acid (SCFA)-modified hexosamine analogues used in metabolic glycoengineering (MGE) by comparing in silico and experimental measurements of physiochemical properties important in drug design. We then describe the impact of these compounds on selected biological parameters that influence the pharmacological properties and safety of drug candidates by monitoring P-glycoprotein (Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced by length of the SCFAs (e.g., acetate vs n-butyrate), which are added to MGE analogues to increase the efficiency of cellular uptake, the regioisomeric arrangement of the SCFAs on the core sugar, the structure of the core sugar itself, and by the type of N-acyl modification (e.g., N-acetyl vs N-azido). By cataloging the influence of these SAR on pharmacological properties of MGE analogues, this study outlines design considerations for tuning the pharmacological, physiochemical, and the toxicological parameters of this emerging class of small molecule drug candidates.

General and Facile Coating of Single Cells via Mild Reduction.

Cell surface modification has been extensively studied to enhance the efficacy of cell therapy. Still, general accessibility and versatility are remaining challenges to meet the increasing demand for cell-based therapy. Herein, we present a facile and universal cell surface modification method that involves mild reduction of disulfide bonds in cell membrane protein to thiol groups. The reduced cells are successfully coated with biomolecules, polymers, and nanoparticles for an assortment of applications, including rapid cell assembly, in vivo cell monitoring, and localized cell-based drug delivery. No adverse effect on cellular morphology, viability, proliferation, and metabolism is observed. Furthermore, simultaneous coating with polyethylene glycol and dexamethasone-loaded nanoparticles facilitates enhanced cellular activities in mice, overcoming immune rejection.