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Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death,but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis,or of its underlying transcriptome network. Therefore,the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes,whereas MeHg altered fewer transcripts. To attenuate batch effects,analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (backslashtextless20 % overlap). Moreover,within one test system,little overlap between the PS changed by the two compounds has been observed. However,using TFBS enrichment,a relatively large ‘common response' to VPA and MeHg could be distinguished from ‘compound-specific' responses. In conclusion,the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles. View Publication

The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However,the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S,and the leukemia cell lines K562,HL60,U937,KG1,and NB4. In contrast,normal CD34(+) cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition,dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases,protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed,shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore,apoptin-mediated cell death proceeded through the upregulation of PKCβ,activation of caspase-9/3,cleavage of the PKCδ catalytic domain,and downregulation of the MERTK and AKT kinases. Collectively,these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further,they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma. View Publication

Asthma is a complex and heterogeneous respiratory disorder characterized by chronic airway inflammation. It has generally been associated with allergic mechanisms related to type 2 airway inflammation. Nevertheless,between 10 and 33{\%} of asthmatic individuals have nonallergic asthma (NA). Several targeted treatments are in clinical development for patients with Th2 immune response,but few biomarkers are been defined for low or non-Th2-mediated inflammation asthma. We have recently defined by gene expression a set of genes as potential biomarkers of NA,mainly associated with disease severity: IL10,MSR1,PHLDA1,SERPINB2,CHI3L1,IL8,and PI3. Here,we analyzed their protein expression and specificity using sera and isolated peripheral blood mononuclear cells (PBMCs). First,protein quantification was carried out using ELISA (in sera) or Western blot (proteins extracted from PBMCs by Trizol procedure),depending on the biomarker in 30 healthy controls (C) subjects and 30 NA patients. A receiver operating characteristic curve analysis was performed by using the R program to study the specificity and sensitivity of the candidate biomarkers at a gene- and protein expression level. Four kinds of comparisons were performed: total NA group vs C group,severe NA patients vs C,moderate-mild NA patients vs C,and severe NA patients vs moderate-mild NA patients. We found that all the single genes showed good sensitivity vs specificity for some phenotypic discrimination,with CHI3L1 and PI3 exhibiting the best results for C vs NA: CHI3L1 area under the curve (AUC) (CI 95{\%}): 0.95 (0.84-1.00) and PI3 AUC: 0.99 (0.98-1.00); C vs severe NA: PI3 AUC: 1 (0.99-1.00); and C vs moderate-mild NA: CHI3L1 AUC: 1 (0.99-1.00) and PI3 AUC: 0.99 (0.96-1.00). However,the results for discriminating asthma disease and severity with protein expression were better when two or three biomarkers were combined. In conclusion,individual genes and combinations of proteins have been evaluated as reliable biomarkers for classifying NA subjects and their severity. These new panels could be good diagnostic tests. View Publication

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study,we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively,while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs,multiple uniquely shared amino acid configurations were identified,warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes,which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation. View Publication

BackgroundPrimary cilia mediate vertebrate development and growth factor signalling. Defects in primary cilia cause inherited developmental conditions termed ciliopathies. Ciliopathies often present with cystic kidney disease,a major cause of early renal failure. Currently,only one drug,Tolvaptan,is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions are needed.MethodsWe screened clinical development compounds to identify those that reversed cilia loss due to siRNA knockdown. In parallel,we undertook a whole genome siRNA-based reverse genetics phenotypic screen to identify positive modulators of cilia formation.ResultsUsing a clinical development compound screen,we identify fasudil hydrochloride. Fasudil is a generic,off-patent drug that is a potent,broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In parallel,the siRNA screen identifies ROCK2 and we demonstrate that ROCK2 is a key mediator of cilium formation and function through its possible effects on actin cytoskeleton remodelling.ConclusionsOur results indicate that specific ROCK2 inhibitors (e.g. belumosudil) could be repurposed for cystic kidney disease treatment. We propose that ROCK2 inhibition represents a novel,disease-modifying therapeutic approach for heterogeneous ciliopathies. Plain language summaryPrimary cilia are antennae-like structures on cells that are important for early development and healthy cell function. Defects in primary cilia can cause inherited diseases called ciliopathies. Ciliopathies often cause fluid-filled sacs,called cysts,that are a major cause of kidney disease and failure. There is currently one drug licensed to slow kidney disease progression,but it is poorly tolerated in patients. Therefore,new drugs are needed. In this study,we used screening assays to identify potential drugs and their targets that are effective in promoting the formation of primary cilia. Our results identified ROCK2 (Rho-associated coiled-coil-containing protein kinase 2),an inhibitor of protein signalling,as a key mediator of cilium function. These findings suggest that drugs that specifically target ROCK2 could be a potential treatment option for cystic kidney disease. Smith et al. use clinical development screen and whole genome siRNA-reverse genetics phenotypic screen to identify ROCK2,as a modulator of cilia formation and function via its effects on actin cytoskeleton remodelling. Repurposing ROCK2 is a viable treatment for ciliopathies,for which a limited therapeutic option is available. View Publication

AbstractBackgroundThe pro-inflammatory cytokine,interleukin-18 (IL-18),plays an instrumental role in bolstering anti-tumor immunity. However,the therapeutic application of IL-18 has been limited due to its susceptibility to neutralization by IL-18 binding protein (IL-18BP),short in vivo half-life,and unfavorable physicochemical properties.MethodsIn order to overcome the poor drug-like properties of IL-18,we installed an artificial disulfide bond,removed the native,unpaired cysteines,and fused the stabilized cytokine to an IgG Fc domain. The stability,potency,pharmacokinetic and pharmacodynamic properties as well as efficacy of disulfide-stabilized IL-18 Fc-fusion (dsIL-18-Fc) were assessed via in vitro and in vivo studies.ResultsThe stability and mammalian host cell production yields of dsIL-18-Fc were improved,compared to the wild-type (WT) cytokine,while maintaining its biological potency and interactions with IL-18 receptor α (IL-18Rα) and IL-18BP. Recombinant fusion of the cytokine to an IgG Fc domain provided extended half-life. Notably,despite maintaining sensitivity to IL-18BP,dsIL-18-Fc was effective at activating both T and natural killer (NK) cells,and elicited a strong anti-tumor response,either as a single agent,or in conjunction with anti-programmed cell death-ligand 1 (anti-PD-L1) therapy.ConclusionsWe engineered IL-18 for reinforced stability,extended half-life,and improved manufacturability. The therapeutic benefit of dsIL-18-Fc,coupled with a more favorable manufacturability profile and enhanced drug-like properties,underscores the potential utility of this engineered cytokine in cancer immunotherapy. View Publication

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets,bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody,which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification,Elisa assay for antigen binding,cytotoxicity assays,T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially,BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover,PHE1 domain was further shortened with D14G and R41S mutations,named PHE-S,and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo,bringing more possibilities for the development and optimization of different bsAbs. To sum up,PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy. View Publication

Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR),with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here,we introduce enGager,an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types,showing up to 6-fold higher efficiency compared to unfused Cas9,especially for large transgenes in primary cells. Notably,enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells,enhancing anti-tumor functionality. This ‘tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer,more efficient alternative to viral vectors for therapeutic gene modification. Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination. Here the authors present the TESOGENASE system which enhances CRISPR-based gene integration by tethering circular single-stranded DNA to Cas9. View Publication

Telomeres as the protective ends of linear chromosomes,are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes,telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study,we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently,the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect,highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies. The online version contains supplementary material available at 10.1007/s00018-024-05239-7. View Publication

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise,with a negligible affinity for mismatched substrates,but its low cellular targeting efficiency limits therapeutic use. Here,we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency,knock-in rates,and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally,we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor,highlighting its therapeutic utility. Subject terms: CRISPR-Cas9 genome editing,Molecular medicine,Genetic engineering,CRISPR-Cas9 genome editing View Publication