Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production.

Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level.

Activation of cell membrane-localized Toll-like receptor 3 by siRNA.

Small interfering RNA molecules (siRNA) are short dsRNAs that are used for different therapeutic applications. On the other hand, dsRNAs can bind to and activate cell RNA sensors and consequently trigger inflammatory response. Here we show that siRNA activates primary human endothelial cells and human lymphatic endothelial cells and that this response is inhibited by antibodies against TLR3. In contrast, the activation of human lymphatic endothelial cells by poly(I:C) was inhibited by bafilomycin but not by anti-TLR3 antibodies. Bafilomycin also inhibited poly(I:C) but not siRNA cell stimulation in TLR3-transfected HEK293. The response to siRNA required the expression of UNC93B1, which directs TLR3 to the surface of HEK293 cells. We propose that the engaged signaling pathway of TLR3 depends on the receptor localization and on the length of the dsRNA, where the activation of cell membrane TLR3 by short dsRNA leads to a predominantly proinflammatory response, whereas TLR3 activation in endosomal compartments by long dsRNA is characterized by the production of type I IFN. A molecular model suggests that the siRNA can bind to the binding sites of the TLR3 ectodomain and trigger receptor dimerization. These results contribute to understanding of the mechanism of side effects seen in the therapeutic application of naked, unmodified siRNA as a result of the activation of TLR3 localized at the plasma membrane.

Toll-like receptor 4 senses oxidative stress mediated by the oxidation of phospholipids in extracellular vesicles.

Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4) in a manner dependent on its co-receptor MD-2. EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation gained the ability to stimulate TLR4-expressing cells, whereas prolonged oxidation abrogated this property. Furthermore, we found that 15-lipoxygenase generated hydro(pero)xylated phospholipids that stimulated TLR4-expressing cells. Molecular modeling suggested that the mechanism of activation of TLR4 by oxidized phospholipids in EVs was structurally similar to that of the TLR4 ligand lipopolysaccharide (LPS). This was supported by experiments showing that EV-mediated stimulation of cells required MD-2, that mutations that block LPS binding to TLR4 abrogated the stimulatory effect of EVs, and that EVs induced TLR4 dimerization. On the other hand, analysis of gene expression profiles showed that genes encoding factors that resolve inflammation were more abundantly expressed in responses to EVs than in response to LPS. Together, these data suggest that EVs act as an oxidative stress-induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases.

The ectodomain of TLR3 receptor is required for its plasma membrane translocation.

Toll-like receptor 3 (TLR3) is a dsRNA sensing receptor that is localized in the cellular compartments but also at the plasma membrane. Overexpression of UNC93B1 promoted localization of TLR3, but not other nucleic acid sensing TLRs, to the plasma membrane. Here we show that UNC93B1 itself is localized at the plasma membrane. We investigated the role of different domains of TLR3 on cell signaling by preparing chimeric receptors between TLR3 and TLR9 where each of the transmembrane segments or cytosolic domains has been exchanged. While the ectodomain completely governs ligand specificity and the cytosolic TIR domain determines the engagement of the signaling adapters as well as the potentiation of receptor activation by UNC93B1, the ectodomain but not transmembrane segment or cytosolic domain determines plasma membrane localization of TLR3. Nevertheless, TLR3 receptor and ligand endocytosis as well as endosomal acidification are important for the robust signaling of TLR3.

Delivery system for the enhanced efficiency of immunostimulatory nucleic acids.

Toll-like receptors (TLRs) play a key role in the recognition of pathogen-associated molecular patterns, including immunostimulatory nucleic acids (INAs). INAs are recognized by TLRs in endosomes, leading to the activation of signalling pathways that activate the innate immune response. This feature makes INAs and their synthetic analogues useful as adjuvants in vaccines and in cancer treatment. We tested a delivery system for the improvement of the therapeutic effect of INAs which consists of a conjugate between transferrin (Tf) and poly-L-lysine (PLL). Tf is a ligand of the transferrin receptor (TfR) and is internalized via receptor-mediated endocytosis, while PLL binds negatively charged INAs. The TfPLL conjugate protected TLR3 ligand polyinosinic:polycytidylic acid [poly(I:C)] from RNase degradation and enhanced the uptake of poly(I:C) in HeLa cells. Co-localization between TfPLL-bound poly(I:C) and lysosomes demonstrated delivery into the endosomal pathway. Time dependence of the production of IL-6 in the primary cell line showed that TfPLL conjugate enabled a gradual release of poly(I:C) and stronger activation of TLR3 receptor in comparison with poly(I:C) alone. Only 3 h of stimulation by poly(I:C) + TfPLL complexes initiated a strong immune response in contrast to poly(I:C) alone. The poly(I:C) + TfPLL complexes have potential use for development of advanced vaccine adjuvants and targeted cancer immune therapy in cells that express higher levels of TfR.

The role of UNC93B1 protein in surface localization of TLR3 receptor and in cell priming to nucleic acid agonists.

Translocation of nucleic acid-sensing (NAS) Toll-like receptors (TLRs) to endosomes is essential for response to microbial nucleic acids as well as for prevention of the autoimmune response. The accessory protein UNC93B1 is indispensable for activation of NAS TLRs because it regulates their response through trafficking to endosomes. We observed that poly(I:C) up-regulates transcription of UNC93B1 and promotes trafficking of TLR3 to the plasma membrane in human epithelial cell line. Up-regulation of UNC93B1 is triggered through TLR3 activation by poly(I:C). Further studies revealed that expression of UNC93B1 promotes trafficking of differentially glycosylated TLR3, but not other NAS TLRs, to the plasma membrane. UNC93B1 promoter region contains binding sites for poly(I:C)- and type I interferon-inducible regulatory elements. UNC93B1 also increases the protein lifetime of TLR3 and TLR9 and augments signaling of all NAS TLRs. Furthermore, we discovered that poly(I:C) pretreatment primes B-cells to the activation by ssDNA via up-regulation of UNC93B1. Our findings identified TLR3 as the important regulator of UNC93B1 that in turn governs the responsiveness of all NAS TLRs.

A second binding site for double-stranded RNA in TLR3 and consequences for interferon activation.

We show that substrate specificity of Toll-like receptor 3 (TLR3) is due to the presence of two binding sites in the ectodomain, separated by 50 A. This corresponds to two turns of a double-stranded RNA duplex, allowing differentiation between nucleic acids in the A- or B-type conformation. We propose that there are different arrangements of TLR3 ectodomains along the double-stranded RNA that could modulate the strength of the interferon response.