Anti-platelet therapy in the prevention of hepatitis B virus-associated hepatocellular carcinoma

n/

Variations in Biodistribution and Acute Response of Differently Shaped Titania Nanoparticles in Healthy Rodents

Biodistribution; Nanotoxicity; Physico-chemical propertiesBiodistribución; Nanotoxicidad; Propiedades fisicoquímicasBiodistribució; Nanotoxicitat; Propietats fisicoquímiquesTitanium dioxide nanoparticles (TiO2 NPs) are one of the main sources of the nanoparticulate matter exposure to humans. Although several studies have demonstrated their potential toxic effects, the real nature of the correlation between NP properties and their interaction with biological targets is still far from being fully elucidated. Here, engineered TiO2 NPs with various geometries (bipyramids, plates, and rods) have been prepared, characterized and intravenously administered in healthy mice. Parameters such as biodistribution, accumulation, and toxicity have been assessed in the lungs and liver. Our data show that the organ accumulation of TiO2 NPs, measured by ICP-MS, is quite low, and this is only partially and transiently affected by the NP geometries. The long-lasting permanence is exclusively restricted to the lungs. Here, bipyramids and plates show a higher accumulation, and interestingly, rod-shaped NPs are the most toxic, leading to histopathological pulmonary alterations. In addition, they are also able to induce a transient increase in serum markers related to hepatocellular injury. These results indicate that rods, more than bipyramidal and spherical geometries, lead to a stronger and more severe biological effect. Overall, small physico-chemical differences can dramatically modify both accumulation and safety.This research was funded by National Key Research and Development Program of China (No. 2017FYA0205301), Projects of International Cooperation and Exchanges NSFC (No. 82020108017), National Natural Science Foundation of China (No. 82272821); MCIN/AEI (PID2019-111218RB-I00 and RYC-2017-23457) and Centro Singular De Investigación de Galicia Accreditation 2019−2022, and ED431G 2019/03. Moreover, parts of this work were funded by the Cluster of Excellence ‘Advanced Imaging of Matter’ of the Deutsche Forschungsgemeinschaft (DFG)-EXC 2056-project ID 390715994; by Deutscher Akademischer Austauschdienst (DAAD); and by an Alexander von Humboldt fellowship. G.S. is supported by the Italian Association for Cancer Research (AIRC), grants 22820 and 22737. NGB and VP receive financial support from the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU) (RTI2018-099965-B-I00, AEI/FEDER, UE) proyectos de I + D + i de programación conjunta internacional MCIN/AEI (CONCORD, PCI2019-103436), co-funded by the European Union. P.B. is supported by the CONCORD project (EuroNanoMed III)

42 correction of scid x1 by targeted genome editing of hematopoietic stem progenitor cells hspc in the mouse model

Targeted genome editing by engineered nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy trials with integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of leukemias due to insertional mutagenesis and unregulated transgene expression. To model SCID-X1 gene correction in preclinical studies, we developed a mouse model carrying the IL2RG human gene harboring a common disease-causing mutation in place of the murine Il2rg, allowing to use of the same reagents developed for gene correction of human cells. These mice have impaired lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute in part the T and B cell compartments. We then tested gene correction of the murine Lin- HSPC by the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol yielded high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR but also high cytotoxicity (65% cell loss) under the conditions we used. The surviving cells remained capable of expansion in culture and maintained their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over the un-corrected SCID cells. These data indicate functional correction of the defective IL2RG gene by targeted editing. Furthermore, upon challenging the mice with a murine pathogen we observed viral-specific γIFN production by CD8+ gene corrected cells, proving their in vivo functionality. Yet, measuring the percentage of edited cells (either by NHEJ or HDR) within the HSC compartment long-term, we found that it was nearly undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells persisted in the mice up to 7 months post transplantation within all the hematopoietic organs, indicating successful editing of at least 1% progenitors able to sustain long-term lymphopoiesis and partially correct the disease phenotype. We then developed a new protocol exploiting CRISPR/Cas9 technology that enabled to achieve substantial levels of targeted DNA repair by NHEJ (up to 70%) and HDR (up to 25%) with minimal cytotoxicity and provided stable engraftment of the edited cells in transplanted mice. By this strategy we are now assessing the impact of HSC vs. progenitor targeted editing and conditioning regimen for the extent and stability of disease correction. These studies will help establish the key factors underlying safe and effective rescue of the disease by HSPC gene editing and assist in the design of the protocol for its first clinical testing

481. Targeted Genome Editing in Mouse Hematopoietic Stem/Progenitor Cells (HSPC) To Model Gene Correction of SCID-X1

Targeted genome editing by artificial nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy with early generation integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of adverse events due to insertional mutagenesis and unregulated transgene expression. We recently reported a strategy that enabled targeted integration of a corrective cDNA into the IL2RG gene in 6% of human HSPC with high specificity. Gene corrected HSPC generated polyclonal lymphoid cells that express the IL2RG protein and have a selective growth advantage over those carrying disruptive IL2RG mutations (Genovese, Nature, 2014). Here, to model SCID-X1 disease correction, we developed a mouse model carrying the IL2RG human gene including a common disease-causing mutation in place of the murine Il2rg gene, allowing to use the same reagents utilized for gene correction of human cells. These mice have impairment in lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we first performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute at least in part the T and B cell compartments. We then developed a protocol to obtain gene correction in murine Lin- HSPC based on the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol was associated with high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR, but also with high levels of acute cytotoxicity (65% cell loss). The surviving cells remained capable of expansion in culture and preserved their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over un-corrected cells. These data indicate functional correction of the IL2RG gene by our strategy. Yet, measuring percentage of correction within myeloid cells at long-term we found that it was almost undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells stably persisted in the mice up to 7 months post transplantation within all the hematopoietic organs. Furthermore, upon challenging the transplanted mice with a murine pathogen (LCMV Arm.) we observed viral-specific γIFN production by CD8+ gene corrected cells at a similar extent as for WT mice, proving in vivo the functionality of corrected T cells. These results suggest that our protocol achieves biologically relevant levels of gene correction in progenitors that sustain long-term lymphopoiesis but is limited in multipotent HSC. Ongoing studies aim to improve murine HSC gene targeting and to compare safety and efficacy of gene correction vs gene replacement in our disease model

Nkx2-5+Islet1+ Mesenchymal Precursors Generate Distinct Spleen Stromal Cell Subsets and Participate in Restoring Stromal Network Integrity

SummarySecondary lymphoid organ stromal cells comprise different subsets whose origins remain unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originate from embryonic mesenchymal progenitors of the Nkx2-5+Islet1+ lineage. This lineage include embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable also of supporting ectopic lymphoid-like structures and a subset of resident spleen stromal cells that proliferate and regenerate the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity

Variations in Biodistribution and Acute Response of Differently Shaped Titania Nanoparticles in Healthy Rodents

Titanium dioxide nanoparticles (TiO NPs) are one of the main sources of the nanoparticulate matter exposure to humans. Although several studies have demonstrated their potential toxic effects, the real nature of the correlation between NP properties and their interaction with biological targets is still far from being fully elucidated. Here, engineered TiO NPs with various geometries (bipyramids, plates, and rods) have been prepared, characterized and intravenously administered in healthy mice. Parameters such as biodistribution, accumulation, and toxicity have been assessed in the lungs and liver. Our data show that the organ accumulation of TiO NPs, measured by ICP-MS, is quite low, and this is only partially and transiently affected by the NP geometries. The long-lasting permanence is exclusively restricted to the lungs. Here, bipyramids and plates show a higher accumulation, and interestingly, rod-shaped NPs are the most toxic, leading to histopathological pulmonary alterations. In addition, they are also able to induce a transient increase in serum markers related to hepatocellular injury. These results indicate that rods, more than bipyramidal and spherical geometries, lead to a stronger and more severe biological effect. Overall, small physico-chemical differences can dramatically modify both accumulation and safety

Kupffer Cells Hasten Resolution of Liver Immunopathology in Mouse Models of Viral Hepatitis

Kupffer cells (KCs) are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV)-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1) protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology

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

AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans