Human AdV-20-42-42, a promising novel adenoviral vector for gene therapy and vaccine product development

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies

General practitioners' reasoning when considering the diagnosis heart failure: a think-aloud study

BACKGROUND: Diagnosing chronic heart failure is difficult, especially in mild cases or early in the course of the disease, and guidelines are not easily implemented in everyday practice. The aim of this study was to investigate general practitioners' diagnostic reasoning about patients with suspected chronic heart failure in comparison with recommendations in European guidelines. METHODS: Think-aloud technique was used. Fifteen general practitioners reasoned about six case vignettes, representing authentic patients with suspected chronic heart failure. Information about each case was added successively in five steps. The general practitioners said their thoughts aloud while reasoning about the probability of the patient having chronic heart failure, and tried to decide about the diagnosis. Arguments for and against chronic heart failure were analysed and compared to recommendations in guidelines. RESULTS: Information about ejection fraction was the most frequent diagnostic argument, followed by information about cardiac enlargement or pulmonary congestion on chest X-ray. However, in a third of the judgement situations, no information about echocardiography was utilized in the general practitioners' diagnostic reasoning. Only three of the 15 doctors used information about a normal electrocardiography as an argument against chronic heart failure. Information about other cardio-vascular diseases was frequently used as a diagnostic argument. CONCLUSIONS: The clinical information was not utilized to the extent recommended in guidelines. Some implications of our study are that 1) general practitioners need more information about how to utilize echocardiography when diagnosing chronic heart failure, 2) guidelines ought to give more importance to information about other cardio-vascular diseases in the diagnostic reasoning, and 3) guidelines ought to treat the topic of diastolic heart failure in a clearer way

CUEDC1 is a primary target of ERα essential for the growth of breast cancer cells

Breast cancer is the most prevalent type of malignancy in women with ∼1.7 million new cases diagnosed annually, of which the majority express ERα (ESR1), a ligand-dependent transcription factor. Genome-wide chromatin binding maps suggest that ERα may control the expression of thousands of genes, posing a great challenge in identifying functional targets. Recently, we developed a CRISPR-Cas9 functional genetic screening approach to identify enhancers required for ERα-positive breast cancer cell proliferation. We validated several candidates, including CUTE, a putative ERα-responsive enhancer located in the first intron of CUEDC1 (CUE-domain containing protein). Here, we show that CUTE controls CUEDC1 expression, and that this interaction is essential for ERα-mediated cell proliferation. Moreover, ectopic expression of CUEDC1, but not a CUE-domain mutant, rescues the defects in CUTE activity. Finally, CUEDC1 expression correlates positively with ERα in breast cancer. Thus, CUEDC1 is a functional target gene of ERα and is required for breast cancer cell proliferation

Single cell derived mRNA signals across human kidney tumors.

Funder: Department of HealthTumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer

From Derailed Development to Childhood Cancer : Using organoid models to study Malignant Rhabdoid Tumors

Further improvement in the survival of children with cancer is likely to come from therapies specifically designed to target the unique oncogenic properties of childhood cancer cells. A pediatric malignancy for which effective targeted treatment is currently lacking is malignant rhabdoid tumor (MRT), which has been the primary focus of our research presented in this Thesis. MRT is driven by a single recurrent genetic aberration, bi-allelic loss of SMARCB1 or in rare cases SMARCA4. MRT remain one of the most lethal childhood cancers. There is therefore an urgent need for novel effective treatment options. A subset of childhood malignancies, including MRT, is indicated to initiate in prenatal life. They are therefore commonly regarded as products of aberrant embryonic development. A recurrent characteristic of these so-called embryonal tumors is a block in cellular maturation that retains the cells in an embryonic, proliferative state. This embryonic profile is absent in adult cells and may therefore serve as a specific therapeutic vulnerability. For this reason, our studies aimed to identify the embryonic identity and cellular origin of MRT as well as to define the SMARCB1-dependent differentiation pathways underpinning MRT development, to uncover novel therapeutic options. Tumor transcriptomes may accommodate clues of the differentiation state and origin of human cancer. Therefore, we interrogated the differentiation state of adult and childhood renal cancers by mapping bulk tumor transcriptomes to single-cell references of normal adult and fetal cells. Our findings indicated a consistent fetal signature across childhood renal cancers, absent in adult tumors, which is supporting evidence of their aberrant developmental state. Furthermore, we studied the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues placed MRT in a lineage with neural crest-derived Schwann cells. Single cell mRNA readouts of MRT differentiation, which we examined by SMARCB1 reconstitution in MRT organoids, suggests that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicated that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirmed experimentally. As SMARCB1 is a subunit of the SWI/SNF chromatin remodeler, we hypothesized that the maturation block that underlies MRT development is established by aberrant chromatin remodeling. Therefore, we interrogated changes in chromatin topology by reconstituting SMARCB1 in MRT organoids, which demonstrated that SMARCB1 is essential for enhancer regulation. Furthermore, by assessment of the 3D genome, we identified a SMARCB1-dependent chromatin loop to putatively regulate oncogenic levels of MYC in a patient-specific manner. In addition, we set-up a CRISPR-Cas9 knock-out screen in MRT organoids to identify potential therapeutic targets in an unbiased fashion. Our findings demonstrated that a genetic screening approach using more physiological cancer models (organoids) can provide novel insights into tumor dependencies. In conclusion, our studies define the developmental block of MRT and reveal potential therapies. MRT remains a continuous challenge for pediatric oncologists, and for the sake of MRT patients that still face a dismal prognosis, we strive that our findings may contribute to significant therapeutic improvement

A sampling-based strategy for distributing taxis in a road network for occupancy maximization (GIS Cup)

We present a weighted sampling strategy for distributing a system of taxi agents on a road network. We consider a setting, in which each agent operates independently, following a prescribed strategy based on historical data. Furthermore, customer requests appear dynamically and are assigned to the closest unoccupied taxi agent. We demonstrate that in this setting a simple sampling strategy based on the spatial distribution of historical data performs well in minimizing the average time that agents are unoccupied. The strategy is evaluated on taxi trip data in Manhattan and compared to various, more complex strategies

Maximum physically consistent trajectories

Trajectories are usually collected with physical sensors, which are prone to errors and cause outliers in the data. We aim to identify such outliers via the physical properties of the tracked entity, that is, we consider its physical possibility to visit combinations of measurements. We describe optimal algorithms to compute maximum subsequences of measurements that are consistent with (simplified) physics models. Our results are output-sensitive with respect to the number k of outliers in a trajectory of n measurements. Specifically, we describe an O(n logn log 2k) time algorithm for 2D trajectories using a model with unbounded acceleration but bounded velocity, and an O(nk) time algorithm for any model where consistency is "concatenable": a consistent subsequence that ends where another begins together form a consistent sequence. We also consider acceleration-bounded models which are not concatenable. We show how to compute the maximum subsequence for such models in O(nk 2logk) time, under appropriate realism conditions. Finally, we experimentally explore the performance of our algorithms on several large real-world sets of trajectories. Our experiments show that we are generally able to retain larger fractions of noisy trajectories than previous work and simpler greedy approaches. We also observe that the speed-bounded model may in practice approximate the acceleration-bounded model quite well, though we observed some variation between datasets

Antigen capsid-display on human adenovirus 35 via pIX fusion is a potent vaccine platform.

Durable protection against complex pathogens is likely to require immunity that comprises both humoral and cellular responses. While heterologous prime-boost regimens based on recombinant, replication-incompetent Adenoviral vectors (AdV) and adjuvanted protein have been able to induce high levels of concomitant humoral and cellular responses, complex manufacturing and handling in the field may limit their success. To combine the benefits of genetic and protein-based vaccination within one vaccine construct and to facilitate their use, we generated Human Adenovirus 35 (HAdV35) vectors genetically encoding a model antigen based on the Plasmodium falciparum (P. falciparum) circumsporozoite (CS) protein and displaying a truncated version of the same antigen (CSshort) via protein IX on the capsid, with or without a flexible glycine-linker and/or a 45Å-spacer. The four tested pIX-antigen display variants were efficiently incorporated and presented on the HAdV35 capsid irrespective of whether a transgene was encoded or not. Transgene-expression and producibility of the display-/expression vectors were not impeded by the pIX-display. In mice, the pIX-modified vectors induced strong humoral antigen-specific immunity that increased with the inclusion of the linker-/spacer molecules, exceeded the responses induced by the genetic, transgene-expressing HAdV35 vector, and surpassed recombinant protein in potency. In addition, the pIX- display/expression vectors elicited high antigen-specific cellular immune responses that matched those of the genetic HAdV35 vector expressing CS. pIX-modified display-/expression HAdV vectors may therefore be a valuable technology for the development of vaccines against complex pathogens, especially in resource-limited settings

SMARCB1 loss activates patient-specific distal oncogenic enhancers in malignant rhabdoid tumors

Abstract Malignant rhabdoid tumor (MRT) is a highly malignant and often lethal childhood cancer. MRTs are genetically defined by bi-allelic inactivating mutations in SMARCB1, a member of the BRG1/BRM-associated factors (BAF) chromatin remodeling complex. Mutations in BAF complex members are common in human cancer, yet their contribution to tumorigenesis remains in many cases poorly understood. Here, we study derailed regulatory landscapes as a consequence of SMARCB1 loss in the context of MRT. Our multi-omics approach on patient-derived MRT organoids reveals a dramatic reshaping of the regulatory landscape upon SMARCB1 reconstitution. Chromosome conformation capture experiments subsequently reveal patient-specific looping of distal enhancer regions with the promoter of the MYC oncogene. This intertumoral heterogeneity in MYC enhancer utilization is also present in patient MRT tissues as shown by combined single-cell RNA-seq and ATAC-seq. We show that loss of SMARCB1 activates patient-specific epigenetic reprogramming underlying MRT tumorigenesis