Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

STUDY QUESTION: Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? SUMMARY ANSWER: Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. WHAT IS KNOWN ALREADY: We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. STUDY DESIGN SIZE DURATION: Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 µl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. PARTICIPANTS/MATERIALS SETTING METHODS: EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). LARGE SCALE DATA: Raw data have been uploaded to GEO (accession number GSE234338). LIMITATIONS REASONS FOR CAUTION: The findings of the study will require validation utilizing a second cohort of EV samples. WIDER IMPLICATIONS OF THE FINDINGS: The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Marie Sklodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

Dendritic cell-targeted therapy expands CD8 T cell responses to bona-fide neoantigens in lung tumors.

Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage.

Stool Microbiome Signature Associated with Response to Neoadjuvant Pembrolizumab in Patients with Muscle-invasive Bladder Cancer.

Neoadjuvant pembrolizumab has been shown to be a valid treatment for patients affected by muscle-invasive bladder cancer (MIBC), as demonstrated in the PURE-01 clinical trial (NCT02736266). Among the tumor-extrinsic factors influencing immunotherapy efficacy, extensive data highlighted that the microbiome is a central player in immune-mediated anticancer activity. This report aimed to investigate the composition and role of stool microbiome in patients enrolled in the PURE-01 clinical trial. An orthotopic animal model of bladder cancer (MB49-Luc) was used to support some of the findings from human data. An analysis of stool microbiome before pembrolizumab was conducted for 42 patients, of whom 23 showed a pathologic response. The information in the preclinical model of orthotopic bladder cancer treated with anti-PD-1 antibody or control isotype was validated. Linear discriminant analysis effect size and linear models were used to identify the bacterial taxa enriched in either responders or nonresponders. The identified taxa were also tested for their association with event-free survival (EFS). Survival at 31 d after tumor instillation was used as the study endpoint in the preclinical model. Responders and nonresponders emerged to differ in terms of enrichment for 16 bacterial taxa. Of these, the genus Sutterella was enriched in responders, while the species Ruminococcus bromii was enriched in nonresponders. The negative impact of R. bromii on anti-PD-1 antibody activity was also observed in the preclinical model. EFS and survival of the preclinical model showed a negative role of R. bromii. We found different stool bacterial taxa associated with the response or lack of response to neoadjuvant pembrolizumab. Moreover, we provided experimental data about the negative role of R. bromii on immunotherapy response. Further studies are needed to externally validate our findings and provide mechanistic insights about the host-pathogen interactions in MIBC. PATIENT SUMMARY: Using prepembrolizumab stool samples collected from patients enrolled in the PURE-01 clinical trials, we identified some bacterial taxa that were enriched in patients who either responded or did not respond to immunotherapy. Using an animal model of bladder cancer, we gathered further evidence of the negative impact of the Ruminococcus bromii on immunotherapy efficacy. Further studies are needed to confirm the current findings and test the utility of these bacteria as predictive markers of immunotherapy response.

PML modulates epigenetic composition of chromatin to regulate expression of pro-metastatic genes in triple-negative breast cancer.

The promyelocytic leukemia (PML) protein organizes nuclear aggregates known as PML nuclear bodies (PML-NBs), where many transcription factors localize to be regulated. In addition, associations of PML and PML-NBs with chromatin are described in various cell types, further implicating PML in transcriptional regulation. However, a complete understanding of the functional consequences of PML association to DNA in cellular contexts where it promotes relevant phenotypes is still lacking. We examined PML chromatin association in triple-negative breast cancer (TNBC) cell lines, where it exerts important oncogenic functions. We find that PML associates discontinuously with large heterochromatic PML-associated domains (PADs) that contain discrete gene-rich euchromatic sub-domains locally depleted of PML. PML promotes heterochromatic organization in PADs and expression of pro-metastatic genes embedded in these sub-domains. Importantly, this occurs outside PML-NBs, suggesting that nucleoplasmic PML exerts a relevant gene regulatory function. We also find that PML plays indirect regulatory roles in TNBC cells by promoting the expression of pro-metastatic genes outside PADs. Our findings suggest that PML is an important transcriptional regulator of pro-oncogenic metagenes in TNBC cells, via transcriptional regulation and epigenetic organization of heterochromatin domains that embed regions of local transcriptional activity.

Pro-tumor Tfh2 cells induce detrimental IgG4 production and PGE2-dependent IgE inhibition in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and it is characterized by predominant pro-tumor Th2-type inflammation. T follicular helper (Tfh) cells are relevant immunoregulators in cancer, and often correlate with better survival. How the Th2-skewed microenvironment in PDAC modulates the differentiation of Tfh cells and their immunoregulatory function is unknown. METHODS: We carried out high-dimensional flow cytometry and T-cell receptor- and RNA-sequencing, as well as bioinformatics, immunohistochemistry and in vitro mechanistic studies. FINDINGS: We identified Tfh1-, Tfh2-, and Tfh17-like cell clusters in the blood, tumors and tumor-draining lymph-nodes (TDLNs) of chemo-naïve PDAC patients and showed that high percentages of Tfh2 cells within the tumor tissue and TDLNs correlated with reduced patient survival. Moreover, only Tfh2 cells were highly activated and were reduced in frequency in patients who responded to neoadjuvant chemotherapy. RNA-sequencing analysis of immunoglobulin expression showed that tumor and TDLN samples expressed all immunoglobulin (IGH) isotypes apart from IGHE. Consistent with these findings, Tfh2 cells differentiated in vitro by tumor microenvironment-conditioned dendritic cells promoted the production of anti-inflammatory IgG4 antibodies by co-cultured B cells, dependent on IL-13. Moreover, unexpectedly, Tfh2 cells inhibited the secretion of pro-inflammatory IgE, dependent on prostaglandin E2. INTERPRETATION: Our results indicate that in PDAC, highly activated pro-tumor Tfh2 favor anti-inflammatory IgG4 production, while inhibit pro-inflammatory IgE. Thus, targeting the circuits that drive Tfh2 cells, in combination with chemotherapy, may re-establish beneficial anti-tumor Tfh-B cell interactions and facilitate more effective treatment. FUNDING: Research grants from the Italian Association for Cancer Research (AIRC) IG-19119 to MPP and the AIRC Special Program in Metastatic disease: the key unmet need in oncology, 5 per Mille no. 22737 to CB, MF, CD, MR and MPP; the ERA-NET EuroNanoMed III (a collaborative european grant financed by the Italian Ministry of Health, Italy) project PANIPAC (JTC2018/041) to MPP; the Fondazione Valsecchi to SC.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Enhancing clinical potential of liquid biopsy through a multi-omic approach: A systematic review.

In the last years, liquid biopsy gained increasing clinical relevance for detecting and monitoring several cancer types, being minimally invasive, highly informative and replicable over time. This revolutionary approach can be complementary and may, in the future, replace tissue biopsy, which is still considered the gold standard for cancer diagnosis. "Classical" tissue biopsy is invasive, often cannot provide sufficient bioptic material for advanced screening, and can provide isolated information about disease evolution and heterogeneity. Recent literature highlighted how liquid biopsy is informative of proteomic, genomic, epigenetic, and metabolic alterations. These biomarkers can be detected and investigated using single-omic and, recently, in combination through multi-omic approaches. This review will provide an overview of the most suitable techniques to thoroughly characterize tumor biomarkers and their potential clinical applications, highlighting the importance of an integrated multi-omic, multi-analyte approach. Personalized medical investigations will soon allow patients to receive predictable prognostic evaluations, early disease diagnosis, and subsequent ad hoc treatments.

Analyzing genomic and epigenetic profiles in single cells by hybrid transposase (scGET-seq).

scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022)1 and de Pretis and Cittaro (2022).2.

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

RNA sequencing of blood from sex- and age-matched discordant siblings supports immune and transcriptional dysregulation in autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with onset in early childhood, still diagnosed only through clinical observation due to the lack of laboratory biomarkers. Early detection strategies would be especially useful in screening high-risk newborn siblings of children already diagnosed with ASD. We performed RNA sequencing on peripheral blood, comparing 27 pairs of ASD children vs their sex- and age-matched unaffected siblings. Differential gene expression profiling, performed applying an unpaired model found two immune genes, EGR1 and IGKV3D-15, significantly upregulated in ASD patients (both p adj = 0.037). Weighted gene correlation network analysis identified 18 co-expressed modules. One of these modules was downregulated among autistic individuals (p = 0.035) and a ROC curve using its eigengene values yielded an AUC of 0.62. Genes in this module are primarily involved in transcriptional control and its hub gene, RACK1, encodes for a signaling protein critical for neurodevelopment and innate immunity, whose expression is influenced by various hormones and known "endocrine disruptors". These results indicate that transcriptomic biomarkers can contribute to the sensitivity of an intra-familial multimarker panel for ASD and provide further evidence that neurodevelopment, innate immunity and transcriptional regulation are key to ASD pathogenesis.

Mutations in MYO9B are associated with Charcot-Marie-Tooth disease type 2 neuropathies and isolated optic atrophy.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders caused by mutations in at least 100 genes. However, approximately 60% of cases with axonal neuropathies (CMT2) still remain without a genetic diagnosis. We aimed at identifying novel disease genes responsible for CMT2. METHODS: We performed whole exome sequencing and targeted next generation sequencing panel analyses on a cohort of CMT2 families with evidence for autosomal recessive inheritance. We also performed functional studies to explore the pathogenetic role of selected variants. RESULTS: We identified rare, recessive variants in the MYO9B (myosin IX) gene in two families with CMT2. MYO9B has not yet been associated with a human disease. MYO9B is an unconventional single-headed processive myosin motor protein with signaling properties, and, consistent with this, our results indicate that a variant occurring in the MYO9B motor domain impairs protein expression level and motor activity. Interestingly, a Myo9b-null mouse has degenerating axons in sciatic nerves and optic nerves, indicating that MYO9B plays an essential role in both peripheral nervous system and central nervous system axons, respectively. The degeneration observed in the optic nerve prompted us to screen for MYO9B mutations in a cohort of patients with optic atrophy (OA). Consistent with this, we found compound heterozygous variants in one case with isolated OA. CONCLUSIONS: Novel or very rare variants in MYO9B are associated with CMT2 and isolated OA.

A Whole-Genome Sequencing Study Implicates GRAMD1B in Multiple Sclerosis Susceptibility.

While the role of common genetic variants in multiple sclerosis (MS) has been elucidated in large genome-wide association studies, the contribution of rare variants to the disease remains unclear. Herein, a whole-genome sequencing study in four affected and four healthy relatives of a consanguineous Italian family identified a novel missense c.1801T > C (p.S601P) variant in the GRAMD1B gene that is shared within MS cases and resides under a linkage peak (LOD: 2.194). Sequencing GRAMD1B in 91 familial MS cases revealed two additional rare missense and two splice-site variants, two of which (rs755488531 and rs769527838) were not found in 1000 Italian healthy controls. Functional studies demonstrated that GRAMD1B, a gene with unknown function in the central nervous system (CNS), is expressed by several cell types, including astrocytes, microglia and neurons as well as by peripheral monocytes and macrophages. Notably, GRAMD1B was downregulated in vessel-associated astrocytes of active MS lesions in autopsied brains and by inflammatory stimuli in peripheral monocytes, suggesting a possible role in the modulation of inflammatory response and disease pathophysiology.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Longitudinal analysis of T cell receptor repertoires reveals shared patterns of antigen-specific response to SARS-CoV-2 infection.

T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

High-Throughput Monoclonal Antibody Discovery from Phage Libraries: Challenging the Current Preclinical Pipeline to Keep the Pace with the Increasing mAb Demand.

Monoclonal antibodies are among the most powerful therapeutics in modern medicine. Since the approval of the first therapeutic antibody in 1986, monoclonal antibodies keep holding great expectations for application in a range of clinical indications, highlighting the need to provide timely and sustainable access to powerful screening options. However, their application in the past has been limited by time-consuming and expensive steps of discovery and production. The screening of antibody repertoires is a laborious step; however, the implementation of next-generation sequencing-guided screening of single-chain antibody fragments has now largely overcome this issue. This review provides a detailed overview of the current strategies for the identification of monoclonal antibodies from phage display-based libraries. We also discuss the challenges and the possible solutions to improve the limiting selection and screening steps, in order to keep pace with the increasing demand for monoclonal antibodies.

Radiomic and gEnomic approaches for the enhanced DIagnosis of clear cell REnal Cancer (REDIRECt): a translational pilot methodological study.

Background: The combination of radiomic and transcriptomic approaches for patients diagnosed with small clear-cell renal cell carcinoma (ccRCC) might improve decision making. In this pilot and methodological study, we investigate whether imaging features obtained from computed tomography (CT) may correlate with gene expression patterns in ccRCC patients. Methods: Samples from 6 patients who underwent partial nephrectomy for unilateral non-metastatic ccRCC were included in this pilot cohort. Transcriptomic analysis was conducted through RNA-sequencing on tumor samples, while radiologic features were obtained from pre-operative 4-phase contrast-enhanced CT. To evaluate the heterogeneity of the transcriptome, after a 1,000 re-sampling via bootstrapping, a first Principal Component Analyses (PCA) were fitted with all transcripts and a second ones with transcripts deriving from a list of 369 genes known to be associated with ccRCC from The Cancer Genome Atlas (TCGA). Significant pathways in each Principal Components for the 50 genes with the highest loadings absolute values were assessed with pathways enrichment analysis. In addition, Pearson's correlation coefficients among radiomic features themselves and between radiomic features and transcripts expression values were computed. Results: The transcriptomes of the analysed samples showed a high grade of heterogeneity. However, we found four radiogenomic patterns, in which the correlation between radiomic features and transcripts were statistically significant. Conclusions: We showed that radiogenomic approach is feasible, however its clinical meaning should be further investigated.

Rapid SARS-CoV-2 Intra-Host and Within-Household Emergence of Novel Haplotypes.

In February 2020, the municipality of Vo', a small town near Padua (Italy) was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. To investigate the viral prevalence and clinical features, the entire population was swab tested in two sequential surveys. Here we report the analysis of 87 viral genomes, which revealed that the unique ancestor haplotype introduced in Vo' belongs to lineage B, carrying the mutations G11083T and G26144T. The viral sequences allowed us to investigate the viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo'. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptom severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype that was carried by an index case. The lockdown extinguished both the viral spread and the emergence of new variants.

Time-controlled and muscle-specific CRISPR/Cas9-mediated deletion of CTG-repeat expansion in the DMPK gene.

CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3' untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo.

Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.

The frequency of somatic mutations in cancer predicts the phenotypic relevance of germline mutations.

Genomic sequence mutations can be pathogenic in both germline and somatic cells. Several authors have observed that often the same genes are involved in cancer when mutated in somatic cells and in genetic diseases when mutated in the germline. Recent advances in high-throughput sequencing techniques have provided us with large databases of both types of mutations, allowing us to investigate this issue in a systematic way. Hence, we applied a machine learning based framework to this problem, comparing multiple models. The models achieved significant predictive power as shown by both cross-validation and their application to recently discovered gene/phenotype associations not used for training. We found that genes characterized by high frequency of somatic mutations in the most common cancers and ancient evolutionary age are most likely to be involved in abnormal phenotypes and diseases. These results suggest that the combination of tolerance for mutations at the cell viability level (measured by the frequency of somatic mutations in cancer) and functional relevance (demonstrated by evolutionary conservation) are the main predictors of disease genes. Our results thus confirm the deep relationship between pathogenic mutations in somatic and germline cells, provide new insight into the common origin of cancer and genetic diseases, and can be used to improve the identification of new disease genes.