Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy.

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

Severe α1-antitrypsin deficiency associated with lower blood pressure and reduced risk of ischemic heart disease: a cohort study of 91,540 individuals and a meta-analysis.

BACKGROUND: Increased elastase activity in α1-antitrypsin deficiency may affect elasticity of the arterial walls, and thereby blood pressure and susceptibility to cardiovascular disease. We hypothesized that severe α1-antitrypsin deficiency is associated with reduced blood pressure and susceptibility to cardiovascular disease. METHODS: We genotyped 91,353 adults randomly selected from the Danish general population and 187 patients from the Danish α1-Antitrypsin Deficiency Registry and recorded baseline blood pressure, baseline plasma lipids and cardiovascular events during follow-up. 185 participants carried the ZZ genotype, 207 carried the SZ genotype and 91,148 carried the MM genotype. RESULTS: α1-Antitrypsin deficiency was associated with decreases in blood pressure of up to 5 mmHg for systolic blood pressure and up to 2 mmHg for diastolic blood pressure, in ZZ vs SZ vs MM individuals (trend test, P's ≤ 0.01). Plasma triglycerides and remnant cholesterol were reduced in ZZ individuals compared with MM individuals (t-test, P's < 0.001). α1-Antitrypsin deficiency was associated with lower risk of myocardial infarction (trend test P = 0.03), but not with ischemic heart disease, ischemic cerebrovascular disease or hypertension (trend test, P's ≥ 0.59). However, when results for ischemic heart disease were summarized in meta-analysis with results from four previous studies, individuals with versus without α1-antitrypsin deficiency had an odds ratio for ischemic heart disease of 0.66 (95% CI:0.53-0.84). CONCLUSIONS: Individuals with severe α1-antitrypsin deficiency have lower systolic and diastolic blood pressure, lower plasma triglycerides and remnant cholesterol, reduced risk of myocardial infarction, and a 34% reduced risk of ischemic heart disease.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Identification of an alpha-1 antitrypsin variant with enhanced specificity for factor XIa by phage display, bacterial expression, and combinatorial mutagenesis.

Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis.

Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

CHOP and c-JUN up-regulate the mutant Z α1-antitrypsin, exacerbating its aggregation and liver proteotoxicity.

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop-/- mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop-/- mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter-based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.

Post-Transcriptional Regulation of Alpha One Antitrypsin by a Proteasome Inhibitor.

Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.

Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture.

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.

In Vivo Analysis of Alpha-1-Antitrypsin Functions in Autoimmune Disease Models.

Alpha-1-antitrypsin (AAT) is a circulating protein, a serpin, with multiple protective functions. Beside the well-known proteinase inhibitory function, which protects the lungs from chronic obstructive pulmonary disease (COPD), many studies have shown that AAT inhibits pro-inflammatory cytokine gene expression and functions. These anti-inflammatory and immune-regulatory properties have led to studies testing the therapeutic effect of AAT in autoimmune disease models. Initially, a study using recombinant adeno-associated viral (rAAV) vector showed that AAT gene therapy prevented type 1 diabetes (T1D) development in a nonobese diabetic (NOD) mouse model. Consequently, several studies confirmed that AAT therapy prevented and reversed T1D. AAT therapy has also been tested and has demonstrated protective effects in a collagen-induced arthritis model and a systemic lupus erythematosus (SLE) mouse model. This chapter describes methods that evaluate AAT functions in autoimmune mouse models.

Gene Delivery of Alpha-1-Antitrypsin Using Recombinant Adeno-Associated Virus (rAAV).

The challenge for alpha-1-antitrypsin (AAT also known as SERPINA1) gene therapy is to achieve long term and high levels of AAT production. Recombinant adeno-associated virus (rAAV) vector has several advantages for AAT gene delivery including no viral genes in the vector, no requirement of integration for long-term transgene expression, low immunogenicity, and wide tropism. AAV-mediated AAT gene therapy has been developed and tested in animal models for AAT deficiency, type 1 diabetes, rheumatoid arthritis, and osteoporosis. AAV-mediated AAT gene therapy has also been tested in clinical studies and has shown promising results. Here we describe the methods of rAAV-AAT vector construction and production as well as AAT gene delivery through (1) liver-directed, (2) muscle-directed, and (3) mesenchymal stem cell (MSC)-mediated routes. We will also describe methods for the evaluation of AAT expression for each delivery approach.

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

A comparative proteomic analysis of bile for biomarkers of cholangiocarcinoma.

Cholangiocarcinoma is a primary malignant tumor of the bile duct epithelium. Cholangiocarcinoma is usually detected at an advanced stage when successful treatment is no longer possible. As the tumor originates from the bile duct epithelium, bile is an ideal source of tumor biomarkers for cholangiocarcinoma. In this study, we used a quantitative proteomics approach to identify potential tumor-associated proteins in the bile fluid of six cholangiocarcinoma patients. Three different gross-appearance tumor types were used in the analysis: mass-forming type ( n = 2), periductal infiltrating type ( n = 2), and intraductal growth type ( n = 2). Two bile samples from non-cancerous patients were used as controls. Isobaric labeling, coupled with Tandem mass spectrometry, was used to quantify protein levels in the bile of cholangiocarcinoma and control patients. In all, 63 proteins were significantly increased in cholangiocarcinoma bile compared to normal bile. Alpha-1-antitrypsin was one of the overexpressed proteins that increased in cholangiocarcinoma bile samples. Immunohistochemical analysis revealed that alpha-1-antitrypsin was detected in 177 (50%) of 354 cholangiocarcinoma tissues from our Tissue Bank. Immunoblotting of 54 cholangiocarcinoma bile samples showed that alpha-1-antitrypsin was positive in 38 (70%) samples. Fecal enzyme-linked immunosorbent assay showed that alpha-1-antitrypsin level was able to distinguish cholangiocarcinoma patients from normal individuals. In conclusion, alpha-1-antitrypsin is a potential marker for early diagnosis of cholangiocarcinoma.

Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells.

Calreticulin is a lectin chaperone of the endoplasmic reticulum that interacts with newly synthesized glycoproteins by binding to Glc1Man9GlcNAc2 oligosaccharides as well as to the polypeptide chain. In vitro, the latter interaction potently suppresses the aggregation of various non-glycosylated proteins. Although the lectin-oligosaccharide association is well understood, the polypeptide-based interaction is more controversial because the binding site on calreticulin has not been identified, and its significance in the biogenesis of glycoproteins in cells remains unknown. In this study, we identified the polypeptide binding site responsible for the in vitro aggregation suppression function by mutating four candidate hydrophobic surface patches. Mutations in only one patch, P19K/I21E and Y22K/F84E, impaired the ability of calreticulin to suppress the thermally induced aggregation of non-glycosylated firefly luciferase. These mutants also failed to bind several hydrophobic peptides that act as substrate mimetics and compete in the luciferase aggregation suppression assay. To assess the relative contributions of the glycan-dependent and -independent interactions in living cells, we expressed lectin-deficient, polypeptide binding-deficient, and doubly deficient calreticulin constructs in calreticulin-negative cells and monitored the effects on the biogenesis of MHC class I molecules, the solubility of mutant forms of α1-antitrypsin, and interactions with newly synthesized glycoproteins. In all cases, we observed a profound impairment in calreticulin function when its lectin site was inactivated. Remarkably, inactivation of the polypeptide binding site had little impact. These findings indicate that the lectin-based mode of client interaction is the predominant contributor to the chaperone functions of calreticulin within the endoplasmic reticulum.

Acute lung injury induced by whole gastric fluid: hepatic acute phase response contributes to increase lung antiprotease protection.

BACKGROUND: Gastric contents aspiration in humans is a risk factor for severe respiratory failure with elevated mortality. Although aspiration-induced local lung inflammation has been studied in animal models, little is known about extrapulmonary effects of aspiration. We investigated whether a single orotracheal instillation of whole gastric fluid elicits a liver acute phase response and if this response contributes to enrich the alveolar spaces with proteins having antiprotease activity. METHODS: In anesthetized Sprague-Dawley rats receiving whole gastric fluid, we studied at different times after instillation (4 h -7 days): changes in blood cytokines and acute phase proteins (fibrinogen and the antiproteases alpha1-antitrypsin and alpha2-macroglobulin) as well as liver mRNA expression of the two antiproteases. The impact of the systemic changes on lung antiprotease defense was evaluated by measuring levels and bioactivity of antiproteases in broncho-alveolar lavage fluid (BALF). Markers of alveolar-capillary barrier derangement were also studied. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. RESULTS: Severe peribronchiolar injury involving edema, intra-alveolar proteinaceous debris, hemorrhage and PMNn cell infiltration was seen in the first 24 h and later resolved. Despite a large increase in several lung cytokines, only IL-6 was found elevated in blood, preceding increased liver expression and blood concentration of both antiproteases. These changes, with an acute phase response profile, were significantly larger for alpha2-macroglobulin (40-fold increment in expression with 12-fold elevation in blood protein concentration) than for alpha1-antitrypsin (2-3 fold increment in expression with 0.5-fold elevation in blood protein concentration). Both the increment in capillary-alveolar antiprotease concentration gradient due to increased antiprotease liver synthesis and a timely-associated derangement of the alveolar-capillary barrier induced by aspiration, contributed a 58-fold and a 190-fold increase in BALF alpha1-antitrypsin and alpha2-macroglobulin levels respectively (p < 0.001). CONCLUSIONS: Gastric contents-induced acute lung injury elicits a liver acute phase response characterized by increased mRNA expression of antiproteases and elevation of blood antiprotease concentrations. Hepatic changes act in concert with derangement of the alveolar capillary barrier to enrich alveolar spaces with antiproteases. These findings may have significant implications decreasing protease burden, limiting injury in this and other models of acute lung injury and likely, in recurrent aspiration.

Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients.

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients' sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients' group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients' group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients' group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients' groups, to the contrary high glycan branching was observed in HCC patients' group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients' group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.

Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

Recombinant glycoproteins: The impact of cell lines and culture conditions on the generation of protein species.

Glycosylation is the most complex post-translational modification. Thus, it contributes to versatile chemical compositions of proteins, leading to high amounts of protein species. The structural heterogeneity of glycoproteins was also described by the definition of glycoforms. We therefore introduced a new term called "glycoprotein species" to join the two concepts from different fields of biology. In this study, we further determined the theoretical numbers of glycoprotein species of two recombinant glycoproteins - a therapeutical antibody and the human protease inhibitor alpha-1-antitrypsin (A1AT) - based on structural analysis of their N-glycans. Moreover, we showed that variations in the used cell lines and their cultivation conditions strongly influence the number of glycoprotein species in case of recombinant A1AT production. BIOLOGICAL SIGNIFICANCE: Protein glycosylation is a major source for the huge amount of protein species. This study extends the sight of protein species by the following contributions: 1) The new term "glycoprotein species" was defined to introduce the concept of glycoforms into the field. 2) An estimation of the number of potential glycoprotein species of two particular glycoproteins was given. 3) The influence of production conditions for recombinant glycoproteins on glycoprotein species generation was displayed.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies.