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Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/βR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

Wilson’s disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.

BackgroundThe main objective was to evaluate the efficacy of intranasal photodynamic therapy (PDT) in SARS-CoV-2 mildly symptomatic carriers on decreasing the infectivity period. SARS-CoV-2-specific immune-stimulating effects and safety were also analysed.MethodsWe performed a randomized, placebo-controlled, clinical trial in a tertiary hospital (NCT05184205). Patients with a positive SARS-CoV-2 PCR in the last 48 hours were recruited and aleatorily assigned to PDT or placebo. Patients with pneumonia were excluded. Participants and investigators were masked to group assignment. The primary outcome was the reduction in in vitro infectivity of nasopharyngeal samples at days 3 and 7. Additional outcomes included safety assessment and quantification of humoral and T-cell immune-responses.FindingsPatients were recruited between December 2021 and February 2022. Most were previously healthy adults vaccinated against COVID-19 and most carried Omicron variant. 38 patients were assigned to placebo and 37 to PDT. Intranasal PDT reduced infectivity at day 3 post-treatment when compared to placebo with a β-coefficient of -812.2 (CI95%= -478660 – -1.3, p34) at day 7 was higher on the PDT-group, with an OR of 0.15 (CI95%=0.04-0.58). There was a decay in anti-Spike titre and specific SARS-CoV-2 T cell immunity in the placebo group 10 and 20 weeks after infection, but not in the PDT-group. No serious adverse events were reported.InterpretationIntranasal-PDT is safe in pauci-symptomatic COVID-19 patients, it reduces SARS-CoV-2 infectivity and decelerates the decline SARS-CoV-2 specific immune-responses.

Dravet syndrome is a genetic encephalopathy characterized by severe epilepsy combined with motor, cognitive, and behavioral abnormalities. Current antiepileptic drugs achieve only partial control of seizures and provide little benefit on the patient’s neurological development. In >80% of cases, the disease is caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of the Nav1.1 voltage-gated sodium channel. Novel therapies aim to restore SCN1A expression in order to address all disease manifestations. We provide evidence that a high-capacity adenoviral vector harboring the 6-kb SCN1A cDNA is feasible and able to express functional Nav1.1 in neurons. In vivo, the best biodistribution was observed after intracerebral injection in basal ganglia, cerebellum, and prefrontal cortex. SCN1A A1783V knockin mice received the vector at 5 weeks of age, when most neurological alterations were present. Animals were protected from sudden death, and the epileptic phenotype was attenuated. Improvement of motor performance and interaction with the environment was observed. In contrast, hyperactivity persisted, and the impact on cognitive tests was variable (success in novel object recognition and failure in Morris water maze tests). These results provide proof of concept for gene supplementation in Dravet syndrome and indicate new directions for improvement.

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27-hydroxylase. Disruption of the bile acid biosynthesis pathway and accumulation of toxic precursors such as cholestanol cause chronic diarrhea, bilateral juvenile cataracts, tissue deposition of cholestanol and cholesterol (xanthomas), and progressive motor/neuropsychiatric alterations. We have evaluated the therapeutic potential of adeno-associated virus (AAV) vectors expressing CYP27A1 in a CTX mouse model. We found that a vector equipped with a strong liver-specific promoter (albumin enhancer fused with the α1 anti-trypsin promoter) is well tolerated and shows therapeutic effect at relatively low doses (1.5 × 1012 viral genomes [vg]/kg), when less than 20% of hepatocytes overexpress the transgene. This vector restored bile acid metabolism and normalized the concentration of most bile acids in plasma. By contrast, standard treatment (oral chenodeoxycholic acid [CDCA]), while reducing cholestanol, did not normalize bile acid composition in plasma and resulted in supra-physiological levels of CDCA and its derivatives. At the transcriptional level, only the vector was able to avoid the induction of xenobiotic-induced pathways in mouse liver. In conclusion, the overexpression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in Cyp27a1−/− mice. These features make gene therapy a feasible option for the etiological treatment of CTX patients.

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

Summary: Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10–15% of the body’s total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Background & Aims: HDV infection induces the most severe form of human viral hepatitis. However, the specific reasons for the severity of the disease remain unknown. Recently, we developed an HDV replication mouse model in which, for the first time, liver damage was detected. Methods: HDV and HBV replication-competent genomes and HDV antigens were delivered to mouse hepatocytes using adeno-associated vectors (AAVs). Aminotransferase elevation, liver histopathology, and hepatocyte death were evaluated and the immune infiltrate was characterized. Liver transcriptomic analysis was performed. Mice deficient for different cellular and molecular components of the immune system, as well as depletion and inhibition studies, were employed to elucidate the causes of HDV-mediated liver damage. Results: AAV-mediated HBV/HDV coinfection caused hepatocyte necrosis and apoptosis. Activated T lymphocytes, natural killer cells, and proinflammatory macrophages accounted for the majority of the inflammatory infiltrate. However, depletion studies and the use of different knockout mice indicated that neither T cells, natural killer cells nor macrophages were necessary for HDV-induced liver damage. Transcriptomic analysis revealed a strong activation of type I and II interferon (IFN) and tumor necrosis factor (TNF)-α pathways in HBV/HDV-coinfected mice. While the absence of IFN signaling had no effect, the use of a TNF-α antagonist resulted in a significant reduction of HDV-associated liver injury. Furthermore, hepatic expression of HDAg resulted in the induction of severe liver damage, which was T cell- and TNF-α-independent. Conclusions: Both host (TNF-α) and viral (HDV antigens) factors play a relevant role in HDV-induced liver damage. Importantly, pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage. Lay summary: Chronic hepatitis delta constitutes the most severe form of viral hepatitis. There is limited data on the mechanism involved in hepatitis delta virus (HDV)-induced liver pathology. Our data indicate that a cytokine (TNF-α) and HDV antigens play a relevant role in HDV-induced liver damage.

Substrate reduction therapies (SRT) are a promising therapeutic approach for monogenic inherited metabolic diseases. Here the authors evaluate the therapeutic potential of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I and demonstrate its safety and efficacy.

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-fat-diet-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. This article has an associated First Person interview with the first author of the paper.

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.

CD8⁺ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8⁺ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H(2b)-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20₃₁₈ and PbTRAP₁₃₀ respectively, as targets of CD8⁺ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20₃₁₈ and PbTRAP₁₃₀ elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP₁₃₀-specific CD8⁺ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP₁₃₀-specific, but not PbS20₃₁₈-specific, CD8⁺ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8⁺ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8⁺ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8⁺ T cells will allow interrogation of the development of immune responses against malaria liver stages.

Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents.

Abstract Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14‐3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14‐3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14‐3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

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 deficiency.

Background & Aims: Gene therapy using recombinant adeno-associated virus (rAAV) vector carrying multidrug resistance protein 3 (MDR3) coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PFIC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy because therapeutic efficacy is expected to wane as rAAV genomes are lost owing to hepatocyte division, and the formation of AAV-specific neutralising antibodies precludes re-administration. Here, we tested a strategy of vector re-administration in infant PFIC3 mice with careful evaluation of its oncogenicity – a particular concern surrounding rAAV treatment. Methods: AAV8-MDR3 was re-administered to infant Abcb4-/- mice 2 weeks after a first dose co-administered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at 2 weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment. Results: Co-administration with ImmTOR mitigated the formation of rAAV-specific neutralising antibodies and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function, as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to developing hepatocellular carcinoma. Conclusions: These outcomes provide strong evidence for rAAV redosing through co-administration with ImmTOR, as it resulted in a long-term therapeutic effect in a paediatric liver metabolic disorder, including the prevention of oncogenesis. Impact and implications: Redosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in paediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration.

Abstract Background Bile acid (BA) homeostasis is mainly regulated by bile salt excretory pump (BSEP), a hepatocyte transporter that transfers BAs to the bile. BSEP expression is regulated by BA levels through activation of farnesoid X receptor transcription factor, which binds to the inverted repeat (IR-1) element in the BSEP promoter. Gene therapy of cholestatic diseases could benefit from using vectors carrying endogenous promoters physiologically regulated by BAs, however their large size limits this approach, especially when using adeno-associated viral vector (AAV) vectors. Results We evaluated the functionality and BA-mediated regulation of minimal versions of human and mouse BSEP promoters containing IR-1 using AAV vectors expressing luciferase. Unexpectedly, a minimal mouse BSEP promoter (imPr) showed higher BA-mediated expression and inducibility than a minimal human promoter (ihPr) or than full-length BSEP promoters in human hepatic cells. In addition, in mice receiving an AAV8 vector carrying imPr promoter-driven luciferase expression was efficiently regulated by administration of a BA-enriched diet. Interestingly, this vector also expressed significantly higher luciferase levels in Abcb4 −/− mice, which have high levels of BAs, compared to wild type mice, or to mice receiving a vector containing the luciferase gene downstream of the constitutive alpha-1 antitrypsin promoter. In contrast, the AAV vector containing ihPr showed very low luciferase expression with no inducibility. Finally, we optimized imPr by adding three IR-1 repeats at its 5′ end. This new promoter provided higher levels of luciferase than imPr both in vitro and in vivo. Conclusions The imPr could represent a useful tool for gene therapy approaches in which physiological BA regulation is desired.

Gene therapy is a promising strategy to treat and cure most inherited metabolic liver disorders. Viral vectors such as those based on adeno-associated viruses (AAVs) and lentiviruses (LVs) are used as vehicles to deliver functional genes to affected hepatocytes. Adverse events associated with the use of high vector doses have motivated the use of small molecules as adjuvants to reduce the dose. In this study, we showed that a one-hour treatment with topoisomerase inhibitors (camptothecin and etoposide) prior to viral transduction is enough to increase AAV and LV reporter expression in non-dividing hepatic cells in culture. Topoisomerase inhibitors increased both integration-competent (ICLV) and integration-deficient (IDLV) LV-derived expression, with a much stronger increase in the IDLV transduction system. In agreement with that, topoisomerase inhibitors increased viral genome integration in both strains, with a greater impact on the IDLV strain, supporting the idea that topoisomerase inhibitors increased episomal DNA integration, especially when viral integrase activity is abolished. These effects correlated with an increase in the DNA damage response produced by the treatments. Our study highlights the need to monitor DNA damage and undesired integration of viral episomal DNAs into the host genome when studying chemical compounds that increase viral transduction.

Oxidative stress-induced myocardial apoptosis and necrosis are critically involved in ischemic infarction, and several sources of extracellular vesicles appear to be enriched in therapeutic activities. The central objective was to identify and validate the differential exosome miRNA repertoire in human cardiac progenitor cells (CPC). CPC exosomes were first analyzed by LC-MS/MS and compared by RNAseq with exomes of human mesenchymal stromal cells and human fibroblasts to define their differential exosome miRNA repertoire (exo-miRSEL). Proteomics demonstrated a highly significant representation of cardiovascular development functions and angiogenesis in CPC exosomes, and RNAseq analysis yielded about 350 different miRNAs; among the exo-miRSEL population, miR-935 was confirmed as the miRNA most significantly up-regulated; interestingly, miR-935 was also found to be preferentially expressed in mouse primary cardiac Bmi1+high CPC, a population highly enriched in progenitors. Furthermore, it was found that transfection of an miR-935 antagomiR combined with oxidative stress treatment provoked a significant increment both in apoptotic and necrotic populations, whereas transfection of a miR-935 mimic did not modify the response. Conclusion. miR-935 is a highly differentially expressed miRNA in exo-miRSEL, and its expression reduction promotes oxidative stress-associated apoptosis. MiR-935, together with other exosomal miRNA members, could counteract oxidative stress-related apoptosis, at least in CPC surroundings.

SARS-CoV-2 is responsible for the COVID-19 pandemic, which has caused almost 570 million infections and over six million deaths worldwide. To help curb its spread, solutions using ultraviolet light (UV) for quick virus inactivation inside buildings without human intervention could be very useful to reduce chances of contagion. The UV dose must be sufficient to inactivate the virus considering the different materials in the room, but it should not be too high, not to degrade the environment. In the present study, we have analyzed the ability of a 254 nm wavelength UV-C lamp to inactivate dried samples of SARS-CoV-2 exposed at a distance of two meters, simulating a full-scale scenario. Our results showed that virus inactivation was extremely efficient in most tested materials, which included plastic, metal, wood, and textile, with a UV-C exposure of only 42 s (equivalent to 10 mJ/cm2). However, porous materials like medium density fibreboard, were hard to decontaminate, indicating that they should be avoided in hospital rooms and public places.

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund’s adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104–3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80–95% inhibition capacity; p

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.