GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice.

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

In vivo generation of CAR T cells in the presence of human myeloid cells.

Pre-clinical humanized mouse models are a powerful tool to evaluate immunotherapies. NSG-SGM3 mice reconstituted with human stem cells (huSGM3) develop pronounced human myeloid cells due to transgenic expression of stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 (IL-3) compared with the widely used humanized NSG (huNSG) model. We assessed in vivo generation of CD19-CAR T cells in huSGM3 mice upon single intravenous injection of the T cell-specific lentiviral vectors (LVs) CD4-LV and CD8-LV. While in vivo CAR T cell generation was clearly detectable in individual mice, generation appeared less efficient than previously observed for huNSG mice. Especially for the CD4-LV group, this correlated with increased IL-15 and decreased GM-CSF levels, indicating activation of monocytes and macrophages. Co-culture assays identified macrophages as a potential barrier for gene transfer. Refining CD4-LV and CD8-LV with a less immunogenic surface by using modified packaging cells substantially improved the transduction of lymphocytes in vitro in the presence of macrophages, as well in vivo in huSGM3 mice. Notably, two mice that developed less CAR T cells showed high interferon-α or -ß levels before vector injection. Our data emphasize the relevance of innate immune responses for in vivo generation of CAR T cells, which can be overcome by vector surface engineering.

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates.

Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application.

Robotic surgery vs laparoscopic surgery in patients with diagnosis of endometriosis: a systematic review and meta-analysis.

Endometriosis is one of the most common medical conditions affecting the women. The study aimed to evaluate the safety and efficacy of robotic-assisted laparoscopic surgery (RAS) versus conventional laparoscopic surgery (LPS) in the treatment of endometriosis. PubMed, Embase, Cochrane and CINAHL databases were searched from January 1995 to March 2019. According to meta-analysis criteria, five comparative studies were selected. A total of 1527 patients were identified. In the meta-analysis, there were no significant differences in blood loss, complication, and hospital stay between RAS and LPS surgeries in the treatment of patients with endometriosis. However, RAS surgery required a higher weighted mean operating time than LPS surgery, 0.54 (95% confidence interval; 0.37 to 0.70; p < 0.00001) min. This meta-analysis confirmed that the robotic surgery is safe and feasible in patients affected by endometriosis. We could suggest that RAS is a valid option and might be considered an alternative to LPS especially in advanced cases.

Risk factors for abnormally invasive placenta: a systematic review and meta-analysis.

Purpose of the article. To explore the strength of association between different maternal and pregnancy characteristics and the occurrence of abnormally invasive placenta (AIP).Materials and methods: Pubmed, Embase, CINAHL databases were searched. The risk factors for AIP explored were: obesity, age >35 years, smoking before or during pregnancy, placenta previa, prior cesarean section (CS), placenta previa and prior CS, prior uterine surgery, abortion and uterine curettage, in vitro fertilization (IVF) pregnancy and interval between a previous CS, and a subsequent pregnancy. Random-effect head-to-head meta-analyses were used to analyze the data.Results: Forty-six were included in the systematic review. Maternal obesity (Odd ratio, OR: 1.4, 95% CI 1.0-1.8), advanced maternal age (OR: 3.1, 95% CI 1.4-7.0) and parity (OR: 2.5, 95% CI 1.7-3.6), but not smoking were associated with a higher risk of AIP. The presence of placenta previa in women with at least a prior CS was associated with a higher risk of AIP compared to controls, with an OR of 12.0, 95% CI 1.6-88.0. Furthermore, the risk of AIP increased with the number of prior CS (OR of 2.6, 95% CI 1.6-4.4 and 5.4, 95% CI 1.7-17.4 for two and three prior CS respectively). Finally, IVF pregnancies were associated with a high risk of AIP, with an OR of 2.8 (95% CI 1.2-6.8).Conclusion: A prior CS and placenta previa are among the strongest risk factors for the occurrence of AIP.

Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.

Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.

Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy.

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy.

A closer look at prion strains: characterization and important implications.

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrP (Sc) ), a pathogenic isoform of the host-encoded cellular prion protein (PrP (C) ). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrP (Sc) conformational and aggregation states. Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrP (Sc) biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages. This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves.