Protective Effects of Human and Mouse Soluble Scavenger-Like CD6 Lymphocyte Receptor in a Lethal Model of Polymicrobial Sepsis

Sepsis still constitutes an unmet clinical need, which could benefit from novel adjunctive strategies to conventional antibiotic therapy. The soluble form of the scavenger-like human CD6 lymphocyte receptor (shCD6) binds to key pathogenic components from Gram-positive and -negative bacteria and shows time- and dose-dependent efficacy in mouse models of monobacterial sepsis. The objective of the present work was to demonstrate the effectiveness of infusing mouse and human sCD6 by different systemic routes, either alone or as adjunctive therapy to gold standard antibiotics, in a lethal model of polymicrobial sepsis. To this end, C57BL/6 mice undergoing high-grade septic shock induced by cecal ligation and puncture (CLP; ≥90% lethality) were infused via the intraperitoneal (i.p.) or intravenous (i.v.) route with shCD6 at different doses and time points, either alone or in combination with imipenem/cilastatin (I/C) at a dose of 33 mg/kg of body weight every 8 h. Significantly reduced mortality and proinflammatory cytokine levels were observed by i.p. infusion of a single shCD6 dose (1.25 mg/kg) 1 h pre- or post-CLP. When using the i.v. route, mice survival was significantly extended by starting shCD6 infusion at later time points post-CLP (up to 6 h after CLP). Significant adjunctive effects on mouse survival were observed by i.p. or i.v. infusion of shCD6 in combination with i.p. I/C post-CLP. Similar results were obtained in mice expressing high sustained levels (5 to 10 μg/ml) of mouse sCD6 in serum by means of transduction with hepatotropic adeno-associated virus (AAV). Taken together, the data support the conserved antibacterial effects of human and mouse sCD6 and their use as adjunctive therapy in experimental models of complex and severe polymicrobial sepsis.Ministerio de Economía y Competitividad (España) SAF2013-46151-R PCIN-2015-070Instituto de Salud Carlos III RD12/0015/0018European Development Regional Fund RD12/0015/0018Fundació La Marató TV3 201319-30-3

Intratumoral injection of dendritic cells transduced by an SV40-based vector expressing interleukin-15 induces curative immunity mediated by CD8+ T lymphocytes and NK cells

Cancer immunotherapy has been extensively attempted by gene transfer of cytokines with viral vectors. In this work, we compared the therapeutic effects of interleukin 12 and 15 (IL-12 and IL-15) genes transferred to tumor cells or to dendritic cells (DCs), which were subsequently injected into established tumors. For this purpose, we used viral vectors based on simian virus 40 (rSV40). Importantly, we observed that nonmatured DCs infected with rSV40 vectors remained phenotypically immature. Infection of CT-26 tumor cells with rSV40 expressing IL-12 (rSVIL-12) or IL-15 (rSVIL-15) failed to inhibit tumor development. In contrast, the intratumoral administration of syngeneic DCs transduced with rSVIL-12 or rSVIL-15 was associated with a strong antitumor response; up to 40% tumor remissions were achieved with DCs transduced by rSVIL-12 and 73% with DCs expressing IL-15. This antitumor effect correlated with the in vivo priming of tumor-specific CD8+ T lymphocytes. Depletion studies showed that rSVIL-15-mediated antitumor efficacy was mediated mainly by CD8+ T lymphocytes and NK cells. We conclude that (i) SV40-derived vectors are an advantageous alternative to transduce genes into DCs and (ii) DCs transferred with IL-15 have an enhanced capability to induce curative antitumor immunity when injected into malignant lesions

Danio Rerio as Model Organism for Adenoviral Vector Evaluation

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectorsThis research was funded by Xunta de Galicia, Local Government, grant number ED431C 2018/28, and Gobierno de Navarra, Local Government, grant numbers 0011-1383-2018-000011 PT038 and 0011-1383-2019-000006 PT013 (XANTOGEN)S

Animal models of chronic hepatitis delta virus infection host–virus immunologic interactions

Hepatitis delta virus (HDV) is a defective RNA virus that has an absolute requirement for a virus belonging to the hepadnaviridae family like hepatitis B virus (HBV) for its replication and formation of new virions. HDV infection is usually associated with a worsening of HBV-induced liver pathogenesis, which leads to more frequent cirrhosis, increased risk of hepatocellular carcinoma (HCC), and fulminant hepatitis. Importantly, no selective therapies are available for HDV infection. The mainstay of treatment for HDV infection is pegylated interferon alpha; however, response rates to this therapy are poor. A better knowledge of HDV–host cell interaction will help with the identification of novel therapeutic targets, which are urgently needed. Animal models like hepadnavirus-infected chimpanzees or the eastern woodchuck have been of great value for the characterization of HDV chronic infection. Recently, more practical animal models in which to perform a deeper study of host virus interactions and to evaluate new therapeutic strategies have been developed. Therefore, the main focus of this review is to discuss the current knowledge about HDV host interactions obtained from cell culture and animal models

Intrahepatic injection of recombinant adeno-associated virus serotype 2 overcomes gender-related differences in liver transduction

The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was associated with the fastest and strongest onset of luciferase expression. Moreover, no gender differences in liver transduction were observed and luciferase expression was confined to the site of injection. Thus, direct intrahepatic injection of rAAV offers specific advantages, which support the potential of this route of administration for future clinical applications

Gene therapy for progressive familial intrahepatic cholestasis type 3 in a clinically relevant mouse model

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare monogenic disease caused by mutations in the ABCB4 gene, resulting in a reduction in biliary phosphatidylcholine. Reduced biliary phosphatidylcholine cannot counteract the detergent effects of bile salts, leading to cholestasis, cholangitis, cirrhosis and ultimately liver failure. Here, we report results from treating two- or five-week-old Abcb4-/- mice with an AAV vector expressing human ABCB4, resulting in significant decreases of PFIC3 disease biomarkers. All male mice achieved a sustained therapeutic effect up through 12 weeks, but the effect was achieved in only 50% of females. However, two-week-old females receiving a second inoculation three weeks later maintained the therapeutic effect. Upon sacrifice, markers of PFIC3 disease such as, hepatosplenomegaly, biliary phosphatidylcholine and liver histology were significantly improved. Thus, AAV-mediated gene therapy successfully prevented PFIC3 symptoms in a clinically relevant mouse model, representing a step forward in improving potential therapy options for PFIC3 patients

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver

The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficienc

Cardiac Progenitor Cell Exosomal miR-935 Protects against Oxidative Stress

This work has been funded by grants from the Spanish Ministry of Science and Innovation RTI2018-097604-B-I00 (funded by MCIN/AEI/10.13039/501100011033/ and FEDER) and PID2021- 128698OB-I00 (MCIN/AEI/10.13039/501100011033); by the Regional Government of Madrid (S2017/ BMD-3692, Avancell), and by the Instituto de Salud Carlos III (RD16/0011/0037) to AB and by the Instituto Salud Carlos III (ISCIII), co-funded by European Regional Development Fund-FEDER (PI19/00501 and PI22/00029), and Gobierno de Navarra (s/n) to BP. JV has been funded by MCIN grant PID2021-122348NB-I00 and Regional Government of Madrid grant P2022/BMD-7333.S

Liver-specific insulin receptor isoform A expression enhances hepatic glucose uptake and ameliorates liver steatosis in a mouse model of diet-induced obesity

Among the main complications associated with obesity are insulin resistance and altered glucose and lipid metabolism within the liver. It has previously been described that insulin receptor isoform A (IRA) favors glucose uptake and glycogen storage in hepatocytes compared with isoform B (IRB), improving glucose homeostasis in mice lacking liver insulin receptor. Thus, we hypothesized that IRA could also improve glucose and lipid metabolism in a mouse model of high-fatdiet-induced obesity. We addressed the role of insulin receptor isoforms in glucose and lipid metabolism in vivo. We expressed IRA or IRB specifically in the liver by using adeno-associated viruses (AAVs) in a mouse model of diet-induced insulin resistance and obesity. IRA, but not IRB, expression induced increased glucose uptake in the liver and muscle, improving insulin tolerance. Regarding lipid metabolism, we found that AAV-mediated IRA expression also ameliorated hepatic steatosis by decreasing the expression of Fasn, Pgc1a, Acaca and Dgat2 and increasing Scd-1 expression. Taken together, our results further unravel the role of insulin receptor isoforms in hepatic glucose and lipid metabolism in an insulin-resistant scenario. Our data strongly suggest that IRA is more efficient than IRB at favoring hepatic glucose uptake, improving insulin tolerance and ameliorating hepatic steatosis. Therefore, we conclude that a gene therapy approach for hepatic IRA expression could be a safe and promising tool for the regulation of hepatic glucose consumption and lipid metabolism, two key processes in the development of non-alcoholic fatty liver disease associated with obesity