High-throughput functional assay in cystic fibrosis patient-derived organoids allows drug repurposing.

Background: Cystic fibrosis (CF) is a rare hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent therapies enable effective restoration of CFTR function of the most common F508del CFTR mutation. This shifts the unmet clinical need towards people with rare CFTR mutations such as nonsense mutations, of which G542X and W1282X are most prevalent. CFTR function measurements in patient-derived cell-based assays played a critical role in preclinical drug development for CF and may play an important role to identify new drugs for people with rare CFTR mutations. Methods: Here, we miniaturised the previously described forskolin-induced swelling (FIS) assay in intestinal organoids from a 96-well to a 384-well plate screening format. Using this novel assay, we tested CFTR increasing potential of a 1400-compound Food and Drug Administration (FDA)-approved drug library in organoids from donors with W1282X/W1282X CFTR nonsense mutations. Results: The 384-well FIS assay demonstrated uniformity and robustness based on coefficient of variation and Z'-factor calculations. In the primary screen, CFTR induction was limited overall, yet interestingly, the top five compound combinations that increased CFTR function all contained at least one statin. In the secondary screen, we indeed verified that four out of the five statins (mevastatin, lovastatin, simvastatin and fluvastatin) increased CFTR function when combined with CFTR modulators. Statin-induced CFTR rescue was concentration-dependent and W1282X-specific. Conclusions: Future studies should focus on elucidating genotype specificity and mode-of-action of statins in more detail. This study exemplifies proof of principle of large-scale compound screening in a functional assay using patient-derived organoids.

Analysis of urinary oligosaccharide excretion patterns by UHPLC/HRAM mass spectrometry for screening of lysosomal storage disorders.

Oligosaccharidoses, sphingolipidoses and mucolipidoses are lysosomal storage disorders (LSDs) in which defective breakdown of glycan-side chains of glycosylated proteins and glycolipids leads to the accumulation of incompletely degraded oligosaccharides within lysosomes. In metabolic laboratories, these disorders are commonly diagnosed by thin-layer chromatography (TLC) but more recently also mass spectrometry-based approaches have been published. To expand the possibilities to screen for these diseases, we developed an ultra-high-performance liquid chromatography (UHPLC) with a high-resolution accurate mass (HRAM) mass spectrometry (MS) screening platform, together with an open-source iterative bioinformatics pipeline. This pipeline generates comprehensive biomarker profiles and allows for extensive quality control (QC) monitoring. Using this platform, we were able to identify α-mannosidosis, ß-mannosidosis, α-N-acetylgalactosaminidase deficiency, sialidosis, galactosialidosis, fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, GM2 gangliosidosis (M. Sandhoff) and mucolipidosis II/III in patient samples. Aberrant urinary oligosaccharide excretions were also detected for other disorders, including NGLY1 congenital disorder of deglycosylation, sialic acid storage disease, MPS type IV B and GSD II (Pompe disease). For the latter disorder, we identified heptahexose (Hex7), as a potential urinary biomarker, in addition to glucose tetrasaccharide (Glc4), for the diagnosis and monitoring of young onset cases of Pompe disease. Occasionally, so-called "neonate" biomarker profiles were observed in young patients, which were probably due to nutrition. Our UHPLC/HRAM-MS screening platform can easily be adopted in biochemical laboratories and allows for simple and robust screening and straightforward interpretation of the screening results to detect disorders in which aberrant oligosaccharides accumulate.

Targeted Locus Amplification and Haplotyping.

Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids.

Forskolin-induced organoid swelling is associated with long-term cystic fibrosis disease progression.

RATIONALE: Cystic fibrosis (CF) is a monogenic life-shortening disease associated with highly variable individual disease progression which is difficult to predict. Here we assessed the association of forskolin-induced swelling (FIS) of patient-derived organoids with long-term CF disease progression in multiple organs and compared FIS with the golden standard biomarker sweat chloride concentration (SCC). METHODS: We retrieved 9-year longitudinal clinical data from the Dutch CF Registry of 173 people with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individual CFTR function was defined by FIS, measured as the relative size increase of intestinal organoids after stimulation with 0.8 µM forskolin, quantified as area under the curve (AUC). We used linear mixed-effect models and multivariable logistic regression to estimate the association of FIS with long-term forced expiratory volume in 1 s % predicted (FEV1pp) decline and development of pancreatic insufficiency, CF-related liver disease and diabetes. Within these models, FIS was compared with SCC. RESULTS: FIS was strongly associated with longitudinal changes of lung function, with an estimated difference in annual FEV1pp decline of 0.32% (95% CI 0.11-0.54%; p=0.004) per 1000-point change in AUC. Moreover, increasing FIS levels were associated with lower odds of developing pancreatic insufficiency (adjusted OR 0.18, 95% CI 0.07-0.46; p<0.001), CF-related liver disease (adjusted OR 0.18, 95% CI 0.06-0.54; p=0.002) and diabetes (adjusted OR 0.34, 95% CI 0.12-0.97; p=0.044). These associations were absent for SCC. CONCLUSION: This study exemplifies the prognostic value of a patient-derived organoid-based biomarker within a clinical setting, which is especially important for people carrying rare CFTR mutations with unclear clinical consequences.

Abnormal VLCADD newborn screening resembling MADD in four neonates with decreased riboflavin levels and VLCAD activity.

Early detection of congenital disorders by newborn screening (NBS) programs is essential to prevent or limit disease manifestation in affected neonates. These programs balance between the detection of the highest number of true cases and the lowest number of false-positives. In this case report, we describe four unrelated cases with a false-positive NBS result for very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Three neonates presented with decreased but not deficient VLCAD enzyme activity and two of them carried a single heterozygous ACADVL c.1844G>A mutation. Initial biochemical investigations after positive NBS referral in these infants revealed acylcarnitine and organic acid profiles resembling those seen in multiple acyl-CoA dehydrogenase deficiency (MADD). Genetic analysis did not reveal any pathogenic mutations in the genes encoding the electron transfer flavoprotein (ETF alpha and beta subunits) nor in ETF dehydrogenase. Subsequent further diagnostics revealed decreased levels of riboflavin in the newborns and oral riboflavin administration normalized the MADD-like biochemical profiles. During pregnancy, the mothers followed a vegan, vegetarian or lactose-free diet which probably caused alimentary riboflavin deficiency in the neonates. This report demonstrates that a secondary (alimentary) maternal riboflavin deficiency in combination with reduced VLCAD activity in the newborns can result in an abnormal VLCADD/MADD acylcarnitine profile and can cause false-positive NBS. We hypothesize that maternal riboflavin deficiency contributed to the false-positive VLCADD neonatal screening results.

An open-source high-content analysis workflow for CFTR function measurements using the forskolin-induced swelling assay.

MOTIVATION: The forskolin-induced swelling (FIS) assay has become the preferential assay to predict the efficacy of approved and investigational CFTR-modulating drugs for individuals with cystic fibrosis (CF). Currently, no standardized quantification method of FIS data exists thereby hampering inter-laboratory reproducibility. RESULTS: We developed a complete open-source workflow for standardized high-content analysis of CFTR function measurements in intestinal organoids using raw microscopy images as input. The workflow includes tools for (i) file and metadata handling; (ii) image quantification and (iii) statistical analysis. Our workflow reproduced results generated by published proprietary analysis protocols and enables standardized CFTR function measurements in CF organoids. AVAILABILITY AND IMPLEMENTATION: All workflow components are open-source and freely available: the htmrenamer R package for file handling https://github.com/hmbotelho/htmrenamer; CellProfiler and ImageJ analysis scripts/pipelines https://github.com/hmbotelho/FIS_image_analysis; the Organoid Analyst application for statistical analysis https://github.com/hmbotelho/organoid_analyst; detailed usage instructions and a demonstration dataset https://github.com/hmbotelho/FIS_analysis. Distributed under GPL v3.0. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

R117H-CFTR function and response to VX-770 correlate with mRNA and protein expression in intestinal organoids.

INTRODUCTION: Variability in disease severity and CFTR modulator responses exists between patients with identical CFTR genotypes. Here, we characterized transcription, translation and function of R117H-CFTR using intestinal organoids and correlated them with in vitro responses to ivacaftor (VX-770). METHODS: Organoids were generated from individuals possessing at least one R117H-CFTR allele. The forskolin-induced swelling (FIS) assay was used to measure CFTR function and response to VX-770 treatment. R117H-CFTR protein and mRNA expression levels were determined in parallel and Pearson's correlation coefficients were assessed. RESULTS: Variability in R117H-CFTR FIS responses was observed and correlated significantly with mRNA and protein expression. Response to VX-770 treatment in organoids correlated with mRNA and protein expression as well. CONCLUSIONS: Our results indicate that gene expression, protein expression and CFTR function are strongly correlated in organoids from people with CFTR-R117H-7T/9T, which may suggest that CFTR gene expression may have consequences for CF diagnosis, prognosis and therapeutic benefit.

Comparison of ex vivo and in vitro intestinal cystic fibrosis models to measure CFTR-dependent ion channel activity.

BACKGROUND: New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis. METHODS: CFTR-mediated ion transport was measured in rectal organoid-derived monolayers grown from subjects with various CFTR mutations and compared to donor-matched intestinal current measurements (ICM) in rectal biopsies and forskolin-induced swelling of rectal organoids. RESULTS: Rectal organoid-derived monolayers were generated within four days. Ion transport measurements of CFTR function using these monolayers correlated with ICM and organoid swelling (r = 0.73 and 0.79 respectively). Culturing the monolayers under differentiation conditions enhanced the detection of mucus-secreting cells and was accompanied by reduced CFTR function. CONCLUSIONS: CFTR-dependent intestinal epithelial ion transport properties can be measured in rectal organoid-derived monolayers of subjects and correlate with donor-matched ICM and rectal organoid swelling.

Translational research to enable personalized treatment of cystic fibrosis.

Translational research efforts in cystic fibrosis (CF) aim to develop therapies for all subjects with CF. To reach this goal new therapies need to be developed that target multiple aspects of the disease. To enable individuals to benefit maximally from these treatments will require improved methods to tailor these therapies specifically to individuals who suffer from CF. This report highlights current examples of translational CF research efforts to reach this goal. The use of intestinal organoids and genetics to better understand individual assessment of CFTR modulator treatment effects to ultimately enable a better personalized treatment for CF subjects will be discussed. In addition, development of viral vectors and non-viral synthetic nanoparticles for delivery of mRNA, sgRNA and DNA will be highlighted. New approaches to restore function of CFTR with early premature termination codons using nanoparticle delivery of suppressor tRNAs and new insights into mechanisms of airway epithelial repair will be reviewed as well. The state-of-the-art approaches that are discussed in this review demonstrate significant progress towards the development of optimal individual therapies for CF patients, but also reveal that remaining challenges still lie ahead.

Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

A field-proven yeast two-hybrid protocol used to identify coronavirus-host protein-protein interactions.

Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein-protein interaction networks. In the field of virology, it has proven instrumental to identify virus-host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libraries, and only requires a minimum of equipment. We also discuss reasons that motivated our technical choices and compromises that had to be made. This protocol has been used to screen most non-structural proteins of murine hepatitis virus (MHV), a member of betacoronavirus genus, against a mouse brain cDNA library. Typical results were obtained and are presented in this report.

Studying the dynamics of coronavirus replicative structures.

Coronaviruses (CoVs) generate specialized membrane compartments, which consist of double membrane vesicles connected to convoluted membranes, the so-called replicative structures, where viral RNA synthesis takes place. These sites harbor the CoV replication-transcription complexes (RTCs): multi-protein complexes consisting of 16 nonstructural proteins (nsps), the CoV nucleocapsid protein (N) and presumably host proteins. To successfully establish functional membrane-bound RTCs all of the viral and host constituents need to be correctly spatiotemporally organized during viral infection. Few studies, however, have investigated the dynamic processes involved in the formation and functioning of the (subunits of) CoV RTCs and the replicative structures in living cells. In this chapter we describe several protocols to perform time-lapse imaging of CoV-infected cells and to study the kinetics of (subunits of) the CoV replicative structures. The approaches described are not limited to CoV-infected cells; they can also be applied to other virus-infected or non-infected cells.

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.

Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells.

Biogenesis and dynamics of the coronavirus replicative structures.

Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins.

Visualizing coronavirus RNA synthesis in time by using click chemistry.

Coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (DMVs) and convoluted membranes, where viral RNA synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. Double-stranded RNA (dsRNA), the presumed intermediate in RNA synthesis, is localized to the DMV interior. However, as pores connecting the DMV interior with the cytoplasm have not been detected, it is unclear whether RNA synthesis occurs at these same sites. Here, we studied coronavirus RNA synthesis by feeding cells with a uridine analogue, after which nascent RNAs were detected using click chemistry. Early in infection, nascent viral RNA and nsp's colocalized with or occurred adjacent to dsRNA foci. Late in infection, the correlation between dsRNA dots, then found dispersed throughout the cytoplasm, and nsp's and nascent RNAs was less obvious. However, foci of nascent RNAs were always found to colocalize with the nsp12-encoded RNA-dependent RNA polymerase. These results demonstrate the feasibility of detecting viral RNA synthesis by using click chemistry and indicate that dsRNA dots do not necessarily correspond with sites of active viral RNA synthesis. Rather, late in infection many DMVs may harbor dsRNA molecules that are no longer functioning as intermediates in RNA synthesis.

Mobility and interactions of coronavirus nonstructural protein 4.

Green fluorescent protein (GFP)-tagged mouse hepatitis coronavirus nonstructural protein 4 (nsp4) was shown to localize to the endoplasmic reticulum (ER) and to be recruited to the coronavirus replicative structures. Fluorescence loss in photobleaching and fluorescence recovery after photobleaching experiments demonstrated that while the membranes of the ER are continuous with those harboring the replicative structures, the mobility of nsp4 at the latter structures is relatively restricted. In agreement with that observation, nsp4 was shown to be engaged in homotypic and heterotypic interactions, the latter with nsp3 and nsp6. In addition, the coexpression of nsp4 with nsp3 affected the subcellular localization of the two proteins.

The coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes.

The coronavirus nucleocapsid (N) protein is a virion structural protein. It also functions, however, in an unknown way in viral replication and localizes to the viral replication-transcription complexes (RTCs). Here we investigated, using recombinant murine coronaviruses expressing green fluorescent protein (GFP)-tagged versions of the N protein, the dynamics of its interactions with the RTCs and the domain(s) involved. Using fluorescent recovery after photobleaching, we showed that the N protein, unlike the nonstructural protein 2, is dynamically associated with the RTCs. Recruitment of the N protein to the RTCs requires the C-terminal N2b domain, which interacts with other N proteins in an RNA-independent manner.

Dynamics of coronavirus replication-transcription complexes.

Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.

Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning.

Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps-nsp3, nsp4, and nsp6-multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.