Protective role of RIPK1 scaffolding against HDV-induced hepatocyte cell death and the significance of cytokines in mice.

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/ßR 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.

NatB Catalytic Subunit Depletion Disrupts DNA Replication Initiation Leading to Senescence in MEFs.

Alpha-aminoterminal acetyltransferase B (NatB) is a critical enzyme responsible for acetylating the aminoterminal end of proteins, thereby modifying approximately 21% of the proteome. This post-translational modification impacts protein folding, structure, stability, and interactions between proteins which, in turn, play a crucial role in modulating several biological functions. NatB has been widely studied for its role in cytoskeleton function and cell cycle regulation in different organisms, from yeast to human tumor cells. In this study, we aimed to understand the biological importance of this modification by inactivating the catalytic subunit of the NatB enzymatic complex, Naa20, in non-transformed mammal cells. Our findings demonstrate that depletion of NAA20 results in decreased cell cycle progression and DNA replication initiation, ultimately leading to the senescence program. Furthermore, we have identified NatB substrates that play a role in cell cycle progression, and their stability is compromised when NatB is inactivated. These results underscore the significance of N-terminal acetylation by NatB in regulating cell cycle progression and DNA replication.

Gene Therapy in Combination with Nitrogen Scavenger Pretreatment Corrects Biochemical and Behavioral Abnormalities of Infant Citrullinemia Type 1 Mice.

Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.

Use of an adeno-associated virus serotype Anc80 to provide durable cure of phenylketonuria in a mouse model.

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pahenu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pahenu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 1012 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

N-terminal acetylation mutants affect alpha-synuclein stability, protein levels and neuronal toxicity.

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Transient Expression of Transgenic IL-12 in Mouse Liver Triggers Unremitting Inflammation Mimicking Human Autoimmune Hepatitis.

The etiopathogenesis of autoimmune hepatitis (AIH) remains poorly understood. In this study, we sought to develop an animal model of human AIH to gain insight into the immunological mechanisms driving this condition. C57BL/6 mice were i.v. injected with adeno-associated viral vectors encoding murine IL-12 or luciferase under the control of a liver-specific promoter. Organ histology, response to immunosuppressive therapy, and biochemical and immunological parameters, including Ag-specific humoral and cellular response, were analyzed. Mechanistic studies were carried out using genetically modified mice and depletion of lymphocyte subpopulations. Adeno-associated virus IL-12-treated mice developed histological, biochemical, and immunological changes resembling type 1 AIH, including marked and persistent liver mononuclear cell infiltration, hepatic fibrosis, hypergammaglobulinemia, anti-nuclear and anti-smooth muscle actin Abs, and disease remission with immunosuppressive drugs. Interestingly, transgenic IL-12 was short-lived, but endogenous IL-12 expression was induced, and both IL-12 and IFN-γ remained elevated during the entire study period. IFN-γ was identified as an essential mediator of liver damage, and CD4 and CD8 T cells but not NK, NKT, or B cells were essential executors of hepatic injury. Furthermore, both MHC class I and MHC class II expression was upregulated at the hepatocellular membrane, and induction of autoreactive liver-specific T cells was detected. Remarkably, although immunoregulatory mechanisms were activated, they only partially mitigated liver damage. Thus, low and transient expression of transgenic IL-12 in hepatocytes causes loss of tolerance to hepatocellular Ags, leading to chronic hepatitis resembling human AIH type 1. This model provides a practical tool to explore AIH pathogenesis and novel therapies.

A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-ß induction.

BACKGROUND & AIMS: Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented. METHODS: HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated. RESULTS: AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice. CONCLUSION: The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Dysregulation of interferon regulatory factors impairs the expression of immunostimulatory molecules in hepatitis C virus genotype 1-infected hepatocytes.

BACKGROUND: IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of CD8 T cells. METHODS: We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. RESULTS: Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. CONCLUSIONS: HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.

Characterization of the CD40L/Oncostatin M/Oncostatin M receptor axis as an antiviral and immunostimulatory system disrupted in chronic HCV infection.

BACKGROUND & AIMS: Oncostatin M (OSM) is an inflammatory cytokine which interacts with a heterodimeric receptor formed by gp130 and either OSMRß or LIFR. Here we have analysed OSM and its receptors in livers with chronic hepatitis C (CHC) and studied the factors that regulate this system. METHODS: OSM, OSM receptors and OSM-target molecules were studied by immunohistochemistry and/or qPCR analysis in livers from CHC patients and controls. We determined the production of OSM by CD40L-stimulated antigen presenting cells (APC) and its biological effects on HuH7 cells containing HCV replicon (HuH7 Core-3'). RESULTS: OSM was upregulated in livers with CHC and its production was mapped to CD11c+ cells. OSM levels correlated directly with inflammatory activity and CD40L expression. In vitro studies showed that OSM is released by APC upon interaction with activated CD4+ T cells in a CD40L-dependent manner. Culture of HuH7 Core-3' cells with supernatant from CD40L-stimulated APC repressed HCV replication and induced IL-7 and IL-15Rα. These effects were dampened by antibodies blocking OSM or gp130 and by silencing OSMRß. In CHC livers OSMRß and LIFR were significantly downregulated and their values correlated with those of OSM-induced molecules. Experiments in HuH7 cells showed that impaired STAT3 signaling and exposure to TGFß1, two findings in CHC, are factors involved in repressing OSMRß and LIFR, respectively. CONCLUSIONS: OSM is a cytokine possessing vigorous antiviral and immunostimulatory properties which is released by APC upon interaction with CD40L present on activated CD4+ T cells. In livers with CHC, OSM is overexpressed but its biological activity appears to be hampered because of downregulation of its receptor subunits.

The infectivity of AAV9 is influenced by the specific location and extent of chemically modified capsid residues.

BACKGROUND: Several treatments for genetic diseases utilizing recombinant adeno-associated viruses (AAVs) have recently gained approval. However, the development of a greater number of therapeutic AAVs is constrained by certain limitations. While extensive efforts have concentrated on screening AAV genetic libraries, an alternative strategy involves modifying the AAV capsid by attaching various moieties. The capsid of AAV plays a pivotal role in transducing target cells and evading immune responses, making modifications a key avenue for engineering improved variants. RESULTS: In our study, we replaced specific AAV9 capsid residues with an unnatural amino acid bearing a bioorthogonal group, identifying four positions with no adverse impact on production. Utilizing click chemistry, we attached varying proportions of Cy5.5 to these positions, allowing us to assess the impact of these modifications on AAV9 infectivity in cultured cells. Our findings reveal that both the position and degree of capsid modification significantly affect AAV transduction. While higher amounts of attached molecules lead to an increased number of AAV genomes within cells, this does not positively impact transgene expression. Conversely, a negative impact on transgene expression is observed when the AAV capsid is highly modified, with the degree of this effect associated with the modified residue. CONCLUSION: Careful control of both the degree and specific position of capsid modifications is crucial for optimizing transduction efficiency and minimizing undesired effects on transgene expression. These results underscore the importance of precision in AAV capsid modification to achieve optimal transduction efficiency while mitigating potential drawbacks on transgene expression.

Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

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.

Evaluating possible maternal effect lethality and genetic background effects in Naa10 knockout mice.

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting approximately 80% of all human proteins. The human essential X-linked gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. There is extensive genetic variation in humans with missense, splice-site, and C-terminal frameshift variants in NAA10. In mice, Naa10 is not an essential gene, as there exists a paralogous gene, Naa12, that substantially rescues Naa10 knockout mice from embryonic lethality, whereas double knockouts (Naa10-/Y Naa12-/-) are embryonic lethal. However, the phenotypic variability in the mice is nonetheless quite extensive, including piebaldism, skeletal defects, small size, hydrocephaly, hydronephrosis, and neonatal lethality. Here we replicate these phenotypes with new genetic alleles in mice, but we demonstrate their modulation by genetic background and environmental effects. We cannot replicate a prior report of "maternal effect lethality" for heterozygous Naa10-/X female mice, but we do observe a small amount of embryonic lethality in the Naa10-/Y male mice on the inbred genetic background in this different animal facility.

Evaluating possible maternal effect lethality and genetic background effects in Naa10 knockout mice.

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting approximately 80% of all human proteins. The human essential X-linked gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. There is extensive genetic variation in humans with missense, splice-site, and C-terminal frameshift variants in NAA10. In mice, Naa10 is not an essential gene, as there exists a paralogous gene, Naa12, that substantially rescues Naa10 knockout mice from embryonic lethality, whereas double knockouts (Naa10-/Y Naa12-/-) are embryonic lethal. However, the phenotypic variability in the mice is nonetheless quite extensive, including piebaldism, skeletal defects, small size, hydrocephaly, hydronephrosis, and neonatal lethality. Here we replicate these phenotypes with new genetic alleles in mice, but we demonstrate their modulation by genetic background and environmental effects. We cannot replicate a prior report of "maternal effect lethality" for heterozygous Naa10-/X female mice, but we do observe a small amount of embryonic lethality in the Naa10-/y male mice on the inbred genetic background in this different animal facility.

Hepatitis C virus induces the expression of CCL17 and CCL22 chemokines that attract regulatory T cells to the site of infection.

BACKGROUND & AIMS: The mechanisms by which Foxp3+ T regulatory cells (Treg) accumulate in HCV infected livers are not known. Here, we studied the role of chemokines CCL17 and CCL22 in this process. METHODS: Chemokine mRNA levels were determined by qPCR in liver biopsies from 26 HCV chronically infected patients (CHC), 11 patients with treatment-induced sustained virological response (SVR), 16 patients with other liver diseases unrelated to HCV, and 24 normal livers. Double-immunofluorescence Foxp3/CD3 or CD11c/CCL22 was performed in liver sections. Chemokine production by monocyte-derived dendritic cells (MDDC) co-cultured with uninfected or HCV-JFH1 infected Huh7 cells was measured by qPCR and ELISA. Chemotactic activity of culture supernatants was also tested. RESULTS: Foxp3+ Treg were increased in CHC livers as compared to controls. Patients with CHC showed elevated intrahepatic levels of CCL17 mRNA compared to normal livers or livers from subjects with SVR or other forms of liver disease. Intrahepatic CCL22 expression was also higher in CHC than in healthy subjects or SVR patients but similar to that observed in other liver diseases. Dendritic cells producing CCL22 could be found inside the hepatic lobule in CHC patients. Contact between MDDC and HCV-JFH1-infected Huh7 cells induced the expression of CCL17 and CCL22 in a process partially dependent on ICAM-1. Transwell experiments showed that upregulation of these chemokines enhanced Treg migration. CONCLUSIONS: Contact of HCV-infected cells with dendritic cells induces the production of Treg-attracting chemokines, an effect which may favour liver accumulation of Treg in CHC. Our findings contribute to explain the mechanism by which HCV escapes the immune response and thus reveals novel therapeutic targets.

Consequences of Mammalian Target of Rapamycin Inhibition on Adeno-Associated Virus Hepatic Transduction Efficacy.

The efficiency of recombinant adeno-associated virus (AAV) vectors transducing host cells is very low, limiting their therapeutic potential in patients. There are several cellular pathways interacting and interfering with the journey of the AAV from the cell surface to the nucleus, opening the possibility to enhance AAV transduction by modifying these interactions. In this study, we explored the results of AAV hepatic transduction when different mammalian target of rapamycin (mTOR) inhibitors, rapamycin, MLN0128, RapaLink-1, were used in preconditioned juvenile and adult mice. We confirmed rapamycin as an AAV hepatic transduction enhancer in juvenile and adult mice; however, RapaLink-1, a stronger mTOR inhibitor and a clear hepatic autophagy inducer, had no positive effect. Moreover, MLN0128 reduced AAV hepatic transduction. Therefore, our results show a complex interaction between the mTOR pathway and AAV-mediated hepatic transduction and indicate that mTOR inhibition is not a straightforward strategy for improving AAV transduction. More studies are necessary to elucidate the molecular mechanisms involved in the positive and negative effects of mTOR inhibitors on AAV transduction efficiency.

Novel vectors and approaches for gene therapy in liver diseases.

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.

Oligonucleotide-Based Therapies for Renal Diseases.

The global burden of chronic kidney disease (CKD) is increasing every year and represents a great cost for public healthcare systems, as the majority of these diseases are progressive. Therefore, there is an urgent need to develop new therapies. Oligonucleotide-based drugs are emerging as novel and promising alternatives to traditional drugs. Their expansion corresponds with new knowledge regarding the molecular basis underlying CKD, and they are already showing encouraging preclinical results, with two candidates being evaluated in clinical trials. However, despite recent technological advances, efficient kidney delivery remains challenging, and the presence of off-targets and side-effects precludes development and translation to the clinic. In this review, we provide an overview of the various oligotherapeutic strategies used preclinically, emphasizing the most recent findings in the field, together with the different strategies employed to achieve proper kidney delivery. The use of different nanotechnological platforms, including nanocarriers, nanoparticles, viral vectors or aptamers, and their potential for the development of more specific and effective treatments is also outlined.

AAV-HDV: An Attractive Platform for the In Vivo Study of HDV Biology and the Mechanism of Disease Pathogenesis.

Hepatitis delta virus (HDV) infection causes the most severe form of viral hepatitis, but little is known about the molecular mechanisms involved. We have recently developed an HDV mouse model based on the delivery of HDV replication-competent genomes using adeno-associated vectors (AAV), which developed a liver pathology very similar to the human disease and allowed us to perform mechanistic studies. We have generated different AAV-HDV mutants to eliminate the expression of HDV antigens (HDAgs), and we have characterized them both in vitro and in vivo. We confirmed that S-HDAg is essential for HDV replication and cannot be replaced by L-HDAg or host cellular proteins, and that L-HDAg is essential to produce the HDV infectious particle and inhibits its replication. We have also found that lack of L-HDAg resulted in the increase of S-HDAg expression levels and the exacerbation of liver damage, which was associated with an increment in liver inflammation but did not require T cells. Interestingly, early expression of L-HDAg significantly ameliorated the liver damage induced by the mutant expressing only S-HDAg. In summary, the use of AAV-HDV represents a very attractive platform to interrogate in vivo the role of viral components in the HDV life cycle and to better understand the mechanism of HDV-induced liver pathology.