Chemically inducible split protein regulators for mammalian cells.

Chemically inducible systems represent valuable synthetic biology tools that enable the external control of biological processes. However, their translation to therapeutic applications has been limited because of unfavorable ligand characteristics or the immunogenicity of xenogeneic protein domains. To address these issues, we present a strategy for engineering inducible split protein regulators (INSPIRE) in which ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a cognate physiological ligand or clinically approved drug. We show that the INSPIRE platform can be used for dynamic, orthogonal and multiplex control of gene expression in mammalian cells. Furthermore, we demonstrate the functionality of a glucocorticoid-responsive INSPIRE platform in vivo and apply it for perturbing an endogenous regulatory network. INSPIRE presents a generalizable approach toward designing small-molecule responsive systems that can be implemented for the construction of new sensors, regulatory networks and therapeutic applications.

Synergy between 15-lipoxygenase and secreted PLA2 promotes inflammation by formation of TLR4 agonists from extracellular vesicles.

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA2 activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA2 promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA2 enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells.

Protein interactions guide most cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR-dCas9-based transcriptional activators and to amplify the response of light and small molecule-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

The SARS-CoV-2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike-ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor-binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol-reactive compounds are able to inhibit viral entry. N-acetyl cysteine amide, L-ascorbic acid, JTT-705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

Correlation between Phenotype and Genotype in CTNNB1 Syndrome: A Systematic Review of the Literature.

The CTNNB1 Syndrome is a rare neurodevelopmental disorder associated with developmental delay, intellectual disability, and delayed or absent speech. The aim of the present study is to systematically review the available data on the prevalence of clinical manifestations and to evaluate the correlation between phenotype and genotype in published cases of patients with CTNNB1 Syndrome. Studies were identified by systematic searches of four major databases. Information was collected on patients' genetic mutations, prenatal and neonatal problems, head circumference, muscle tone, EEG and MRI results, dysmorphic features, eye abnormalities, early development, language and comprehension, behavioral characteristics, and additional clinical problems. In addition, the mutations were classified into five groups according to the severity of symptoms. The study showed wide genotypic and phenotypic variability in patients with CTNNB1 Syndrome. The most common moderate-severe phenotype manifested in facial dysmorphisms, microcephaly, various motor disabilities, language and cognitive impairments, and behavioral abnormalities (e.g., autistic-like or aggressive behavior). Nonsense and missense mutations occurring in exons 14 and 15 were classified in the normal clinical outcome category/group because they had presented an otherwise normal phenotype, except for eye abnormalities. A milder phenotype was also observed with missense and nonsense mutations in exon 13. The autosomal dominant CTNNB1 Syndrome encompasses a wide spectrum of clinical features, ranging from normal to severe. While mutations cannot be more generally categorized by location, it is generally observed that the C-terminal protein region (exons 13, 14, 15) correlates with a milder phenotype.

Selective inhibition of NLRP3 inflammasome by designed peptide originating from ASC.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is a multiprotein complex which forms within cells in response to various microbial and self-derived triggers. Mutations in the gene encoding NLRP3 cause rare cryopyrin-associated periodic syndromes (CAPS) and growing evidence links NLRP3 inflammasome to common diseases such as Alzheimer´s disease. In order to modulate different stages of NLRP3 inflammasome assembly nine peptides whose sequences correspond to segments of inflammasome components NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) were selected. Five peptides inhibited IL-1ß release, caspase-1 activation and ASC oligomerization in response to soluble and particulate NLRP3 triggers. Modulatory peptides also attenuated IL-1ß maturation induced by constitutive CAPS-associated NLRP3 mutants. Peptide corresponding to H2-H3 segment of ASC pyrin domain selectively inhibited NLRP3 inflammasome by binding to NLRP3 pyrin domain in the micromolar range. The peptide had no effect on AIM2 and NLRC4 inflammasomes as well as NF-κB pathway. The peptide effectively dampened neutrophil infiltration in the silica-induced peritonitis and when equipped with Antennapedia or Angiopep-2 motifs crossed the blood-brain barrier in a mouse model. Our study demonstrates that peptides represent an important tool for targeting multiprotein inflammatory complexes and can serve as the basis for the development of novel anti-inflammatory strategies for neurodegeneration.

Extracellular vesicle-mediated transfer of constitutively active MyD88L265P engages MyD88wt and activates signaling.

The link between inflammation and cancer is particularly strong in Waldenström macroglobulinemia (WM), a diffuse large B-cell lymphoma wherein the majority of patients harbor a constitutively active mutation in the innate immune-signaling adaptor myeloid differentiation primary response 88 (MyD88). MyD88Leu265Pro (MyD88L265P) constitutively triggers the myddosome assembly providing a survival signal for cancer cells. Here, we report detection and a functional role of MyD88 in the extracellular vesicles (EVs) shed from WM cells. MyD88L265P was transferred via EVs into the cytoplasm of the recipient mast cells and macrophages, recruiting the endogenous MyD88 that triggered the activation of proinflammatory signaling in the absence of receptor activation. Additionally, internalization of EVs containing MyD88L265P was observed in mice with an effect on the bone marrow microenvironment. MyD88-loaded EVs were detected in the bone marrow aspirates of WM patients thus establishing the physiological role of EVs for MyD88L265P transmission and shaping of the proinflammatory microenvironment. Results establish the mechanism of transmission of signaling complexes via EVs to propagate inflammation as a new mechanism of intercellular communication.

Calcium Ionophore-Induced Extracellular Vesicles Mediate Cytoprotection against Simulated Ischemia/Reperfusion Injury in Cardiomyocyte-Derived Cell Lines by Inducing Heme Oxygenase 1.

Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner.

A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation.

Inflammation, which is a highly regulated host response against danger signals, may be harmful if it is excessive and deregulated. Ideally, anti-inflammatory therapy should autonomously commence as soon as possible after the onset of inflammation, should be controllable by a physician, and should not systemically block beneficial immune response in the long term. We describe a genetically encoded anti-inflammatory mammalian cell device based on a modular engineered genetic circuit comprising a sensor, an amplifier, a "thresholder" to restrict activation of a positive-feedback loop, a combination of advanced clinically used biopharmaceutical proteins, and orthogonal regulatory elements that linked modules into the functional device. This genetic circuit was autonomously activated by inflammatory signals, including endogenous cecal ligation and puncture (CLP)-induced inflammation in mice and serum from a systemic juvenile idiopathic arthritis (sIJA) patient, and could be reset externally by a chemical signal. The microencapsulated anti-inflammatory device significantly reduced the pathology in dextran sodium sulfate (DSS)-induced acute murine colitis, demonstrating a synthetic immunological approach for autonomous anti-inflammatory therapy.

Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9.

Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4-6 nt from the 5'-end. Activation is augmented by a 5'TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5'-end and the ODN's length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.

In Vivo Bioluminescence and Fluorescence Imaging: Optical Tool for Cancer Research.

In vivo whole-body imaging, using optical tools based on bioluminescence and fluorescence detection, offers tremendous opportunities to specifically determine the spatiotemporal resolution of cancer cells within the tested animals. This enables the study of many aspects of cancer biology, including cell proliferation, trafficking, and invasions. The antitumor therapeutic properties of various tested compounds (e.g., CD19 CAR-T cells, used for cancer immunotherapy) can be monitored within the same animal at different time points, significantly reducing the number of animals used in the study as indicated in this method.

Toll-like receptor 4 signaling activation domains promote CAR T cell function against solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses, there is an exciting prospect of further enhancing CAR functionality. Here, we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-ß (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model, MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors.

Designed allosteric protein logic.

The regulation of protein function by external or internal signals is one of the key features of living organisms. The ability to directly control the function of a selected protein would represent a valuable tool for regulating biological processes. Here, we present a generally applicable regulation of proteins called INSRTR, based on inserting a peptide into a loop of a target protein that retains its function. We demonstrate the versatility and robustness of coiled-coil-mediated regulation, which enables designs for either inactivation or activation of selected protein functions, and implementation of two-input logic functions with rapid response in mammalian cells. The selection of insertion positions in tested proteins was facilitated by using a predictive machine learning model. We showcase the robustness of the INSRTR strategy on proteins with diverse folds and biological functions, including enzymes, signaling mediators, DNA binders, transcriptional regulators, reporters, and antibody domains implemented as chimeric antigen receptors in T cells. Our findings highlight the potential of INSRTR as a powerful tool for precise control of protein function, advancing our understanding of biological processes and developing biotechnological and therapeutic interventions.

TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Mucoadhesive film for oral delivery of vaccines for protection of the respiratory tract.

The delivery of vaccines plays a pivotal role in influencing the strength and longevity of the immune response and controlling reactogenicity. Mucosal immunization, as compared to parenteral vaccination, could offer greater protection against respiratory infections while being less invasive. While oral vaccination has been presumed less effective and believed to target mainly the gastrointestinal tract, trans-buccal delivery using mucoadhesive films (MAF) may allow targeted delivery to the mucosa. Here we present an effective strategy for mucosal delivery of several vaccine platforms incorporated in MAF, including DNA plasmids, viral vectors, and lipid nanoparticles incorporating mRNA (mRNA/LNP). The mRNA/LNP vaccine formulation targeting SARS-CoV-2 as a proof of concept remained stable within MAF consisting of slowly releasing water-soluble polymers and an impermeable backing layer, facilitating enhanced penetration into the oral mucosa. This formulation elicited antibody and cellular responses comparable to the intramuscular injection, but also induced the production of mucosal IgAs, highlighting its efficacy, particularly for use as a booster vaccine and the potential advantage for protection against respiratory infections. The MAF vaccine preparation demonstrates significant advantages, such as efficient delivery, stability, and simple noninvasive administration with the potential to alleviate vaccine hesitancy.

Rehoming and Other Refinements and Replacement in Procedures Using Golden Hamsters in SARS-CoV-2 Vaccine Research.

Effective vaccines are needed to fight the COVID-19 pandemic. Forty golden hamsters were inoculated with two promising vaccine candidates and eighteen animals were used in pilot trials with viral challenge. ELISA assays were performed to determine endpoint serum titres for specific antibodies and virus neutralisation tests were used to evaluate the efficacy of antibodies. All tests with serum from vaccinated hamsters were negative even after booster vaccinations and changes in vaccination protocol. We concluded that antibodies did not have sufficient neutralising properties. Refinements were observed at all steps, and the in vitro method (virus neutralisation test) presented a replacement measure and ultimately lead to a reduction in the total number of animals used in the project. The institutional animal welfare officer and institutional designated veterinarian approved the reuse or rehoming of the surplus animals. Simple socialization procedures were performed and ultimately 19 animals were rehomed, and feedback was collected. Recently, FELASA published recommendations for rehoming of animals used for scientific and educational purposes, with species-specific guidelines, including mice, rats, and rabbits. Based on our positive experience and feedback from adopters, we concluded that the rehoming of rodents, including hamsters, is not only possible, but highly recommended.

Regulation of CD19 CAR-T cell activation based on an engineered downstream transcription factor.

CAR-T cells present a highly effective therapeutic option for several malignant diseases, based on their ability to recognize the selected tumor surface marker in an MHC-independent manner. This triggers cell activation and cytokine production, resulting in the killing of the cancerous cell presenting markers recognized by the chimeric antigen receptor. CAR-T cells are highly potent serial killers that may cause serious side effects, so their activity needs to be carefully controlled. Here we designed a system to control the proliferation and activation state of CARs based on downstream NFAT transcription factors, whose activity can be regulated via chemically induced heterodimerization systems. Chemical regulators were used to either transiently trigger engineered T cell proliferation or suppress CAR-mediated activation when desired or to enhance activation of CAR-T cells upon engagement of cancer cells, shown also in vivo. Additionally, an efficient sensor to monitor activated CD19 CAR-T cells in vivo was introduced. This implementation in CAR-T cell regulation offers an efficient way for on-demand external control of CAR-T cell activity to improve their safety.

Engineered combinatorial cell device for wound healing and bone regeneration.

Growth factors are the key regulators that promote tissue regeneration and healing processes. While the effects of individual growth factors are well documented, a combination of multiple secreted growth factors underlies stem cell-mediated regeneration. To avoid the potential dangers and labor-intensive individual approach of stem cell therapy while maintaining their regeneration-promoting effects based on multiple secreted growth factors, we engineered a "mix-and-match" combinatorial platform based on a library of cell lines producing growth factors. Treatment with a combination of growth factors secreted by engineered mammalian cells was more efficient than with individual growth factors or even stem cell-conditioned medium in a gap closure assay. Furthermore, we implemented in a mouse model a device for allogenic cell therapy for an in situ production of growth factors, where it improved cutaneous wound healing. Augmented bone regeneration was achieved on calvarial bone defects in rats treated with a cell device secreting IGF, FGF, PDGF, TGF-ß, and VEGF. In both in vivo models, the systemic concentration of secreted factors was negligible, demonstrating the local effect of the regeneration device. Finally, we introduced a genetic switch that enables temporal control over combinations of trophic factors released at different stages of regeneration mimicking the maturation of natural wound healing to improve therapy and prevent scar formation.