Nanobody engineering for SARS-CoV-2 neutralization and detection.

In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "nanobodies." Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants at high potency, with IC50 values as low as 50 pM. Despite simultaneous binding to distinct epitopes, Beta and Omicron variants were more resistant to neutralization by the multimodular nanobodies, which highlights the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present an assay based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious diseases. IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.

Drug repurposing platform for deciphering the druggable SARS-CoV-2 interactome.

The coronavirus disease 2019 (COVID-19) pandemic has heavily challenged the global healthcare system. Despite the vaccination programs, the new virus variants are circulating. Further research is required for understanding of the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and for discovery of therapeutic agents against the virus. Here, we took advantage of drug repurposing to identify if existing drugs could inhibit SARS-CoV-2 infection. We established an open high throughput platform for in vitro screening of drugs against SARS-CoV-2 infection. We screened ∼1000 drugs for their ability to inhibit SARS-CoV-2-induced cell death in the African green monkey kidney cell line (Vero-E6), analyzed how the hit compounds affect the viral N (nucleocapsid) protein expression in human cell lines using high-content microscopic imaging and analysis, determined the hit drug targets in silico, and assessed their ability to cause phospholipidosis, which can interfere with the viral replication. Duvelisib was found by in silico interaction assay as a potential drug targeting virus-host protein interactions. The predicted interaction between PARP1 and S protein, affected by Duvelisib, was further validated by immunoprecipitation. Our results represent a rapidly applicable platform for drug repurposing and evaluation of the new emerging viruses' responses to the drugs. Further in silico studies help us to discover the druggable host pathways involved in the infectious cycle of SARS-CoV-2.

Challenges for the Applications of Human Pluripotent Stem Cell-Derived Liver Organoids.

The current organoid culture systems allow pluripotent and adult stem cells to self-organize to form three-dimensional (3D) structures that provide a faithful recapitulation of the architecture and function of in vivo organs. In particular, human pluripotent stem cell-derived liver organoids (PSC-LOs) can be used in regenerative medicine and preclinical applications, such as disease modeling and drug discovery. New bioengineering tools, such as microfluidics, biomaterial scaffolds, and 3D bioprinting, are combined with organoid technologies to increase the efficiency of hepatic differentiation and enhance the functional maturity of human PSC-LOs by precise control of cellular microenvironment. Long-term stabilization of hepatocellular functions of in vitro liver organoids requires the combination of hepatic endodermal, endothelial, and mesenchymal cells. To improve the biological function and scalability of human PSC-LOs, bioengineering methods have been used to identify diverse and zonal hepatocyte populations in liver organoids for capturing heterogeneous pathologies. Therefore, constructing engineered liver organoids generated from human PSCs will be an extremely versatile tool in in vitro disease models and regenerative medicine in future. In this review, we aim to discuss the recent advances in bioengineering technologies in liver organoid culture systems that provide a timely and necessary study to model disease pathology and support drug discovery in vitro and to generate cell therapy products for transplantation.

SARS-CoV-2-host proteome interactions for antiviral drug discovery.

Treatment options for COVID-19, caused by SARS-CoV-2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS-CoV-2-host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS-CoV-2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP-MS) and the complementary proximity-based labeling MS method (BioID-MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS-CoV-2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image-based drug screen with infectious SARS-CoV-2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein-protein interactions.

Differentiation of Human Pluripotent Stem Cells Into Definitive Endoderm Cells in Various Flexible Three-Dimensional Cell Culture Systems: Possibilities and Limitations.

The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell-matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.

Thyroid Hormone Effect on the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells.

Human induced pluripotent stem cells (hiPSCs) hold great potential as an unlimited source for obtaining hepatocyte-like cells (HLCs) for drug research. However, current applications of HLCs have been severely limited by the inability to produce mature hepatocytes from hiPSCs in vitro. Thyroid hormones are one of the hormones that surge during the perinatal period when liver maturation takes place. Here we assessed the influence of thyroid hormone on hepatic progenitor differentiation to HLCs. We analyzed gene and protein expression of early and late hepatic markers and demonstrated the selective activity of thyroid hormone on different genes. Particularly, we demonstrated thyroid hormone-dependent inhibition of the fetal hepatic marker AFP. Our study sheds light on the role of thyroid hormone during liver differentiation and maturation.

RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells.

Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.

Differences in definitive endoderm induction approaches using growth factors and small molecules.

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.

Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells.