Gloria González-Aseguinolaza

Insulin-like growth factor I gene transfer to cirrhotic liver induces fibrolysis and reduces fibrogenesis leading to cirrhosis reversion in rats.

We investigated whether gene transfer of insulin‐like growth factor I (IGF‐I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF‐I (SVIGF‐I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF‐I was synthesized by hepatocytes, IGF‐I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF‐I showed increased hepatic levels of IGF‐I, improved liver function tests, and reduced fibrosis in association with diminished α‐smooth muscle actin expression, up‐regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM‐1 and TIM‐2. SVIGF‐I therapy induced down‐regulation of the profibrogenic molecules transforming growth factor beta (TGFβ), amphiregulin, platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF‐I‐treated animals showed decreased expression of Wilms tumor‐1 (WT‐1; a nuclear factor involved in hepatocyte dedifferentiation) and up‐regulation of hepatocyte nuclear factor 4 alpha (HNF4α) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF‐I was also tested in rats with thioacetamide‐induced liver cirrhosis. Also in this model, SVIGF‐I improved liver function and reduced liver fibrosis in association with up‐regulation of HGF and MMPs and down‐regulation of tissue inhibitor of metalloproteinase 1 (TIMP‐1). Conclusion: IGF‐I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF‐I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation. (HEPATOLOGY 2010;51:912–921.)

Insulin-like growth factor 2 reverses memory and synaptic deficits in APP transgenic mice.

Insulin‐like growth factor 2 (IGF2) was recently found to play a critical role in memory consolidation in rats and mice, and hippocampal or systemic administration of recombinant IGF2 enhances memory. Here, using a gene therapy‐based approach with adeno‐associated virus (AAV), we show that IGF2 overexpression in the hippocampus of aged wild‐type mice enhances memory and promotes dendritic spine formation. Furthermore, we report that IGF2 expression decreases in the hippocampus of patients with Alzheimers disease, and this leads us to hypothesize that increased IGF2 levels may be beneficial for treating the disease. Thus, we used the AAV system to deliver IGF2 or IGF1 into the hippocampus of the APP mouse model Tg2576 and demonstrate that IGF2 and insulin‐like growth factor 1 (IGF1) rescue behavioural deficits, promote dendritic spine formation and restore normal hippocampal excitatory synaptic transmission. The brains of Tg2576 mice that overexpress IGF2 but not IGF1 also show a significant reduction in amyloid levels. This reduction probably occurs through an interaction with the IGF2 receptor (IGF2R). Hence, IGF2 and, to a lesser extent, IGF1 may be effective treatments for Alzheimers disease.

Gene Therapy: A Pharmacokinetic/Pharmacodynamic Modelling Overview

ABSTRACTSince gene therapy started over 20xa0years ago, more than one-thousand clinical trials have been carried out. Nonviral vectors present interesting properties for their clinical application, but their efficiency in vivo is relatively low, and further improvements in these vectors are needed. Elucidating how nonviral vectors behave at the intracellular level is enlightening for vector improvement and optimization. Model-based approach is a powerful tool to understand and describe the different processes that gene transfer systems should overcome inside the body. Model-based approach allows for proposing and predicting the effect of parameter changes on the overall gene therapy response, as well as the known application of the pharmacokinetic/pharmacodynamic modelling in conventional therapies. The objective of this paper is to critically review the works in which the time-course of naked or formulated DNA have been quantitatively studied or modelled.

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8+ T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods

High‐capacity adenoviruses (HC‐Ad) hold great promise for the treatment of many diseases. The major drawbacks for the clinical application of this vector concern difficulties with respect to large‐scale production, and the absence of standardized methods for production and titration. In the present study, we compare the different methods found in the literature for characterizing HC‐Ad production.

Semi-mechanistic pharmacodynamic modelling of gene expression and silencing processes

BACKGROUNDnSuppressed expression of transgene is a major obstacle in gene therapy. Understanding of the mechanisms involved in expression and silencing of exogenous genes is required to overcome gene therapy hurdles.nnnPURPOSEnTo develop a semi-mechanistic model describing the effects of transgenes over the activity of an expression cassette.nnnMETHODSnTwelve Balb/c mice received 40microg of plasmid DNA. Animals were assigned to one of the following treatments: (I) 20microg of the plasmid expressing luciferase (pEF-Luc) and 20microg of empty plasmid; (II) pEF-Luc (20microg) and 20microg of plasmid expressing murine interferon alpha (IFNalpha); and (III) pEF-Luc (20microg), and 20microg of plasmid expressing beta-galactosidase (pCMVbeta). The expression of luciferase over time, quantified by a noninvasive method, was used as a measured of pEF-Luc activity and modelled using NONMEM.nnnRESULTSnThe selected model suggests the co-existence of two forms of active DNA differing in their transcription efficiencies. The core model was expanded to describe reversible and irreversible silencing processes, induced by the coexpression of IFNalpha or beta-galactosidase, respectively.nnnCONCLUSIONnCoupling noninvasive in vivo imaging and mathematical modelling allows quantitative description of gene transfer, providing a tool to select the best regulatory elements to construct a therapeutic expression cassette.

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

BACKGROUND & AIMSnStudying 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.nnnMETHODSnHDV 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.nnnRESULTSnAAV-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.nnnCONCLUSIONnThe 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.

Complementary Effects of Interleukin-15 and Alpha Interferon Induce Immunity in Hepatitis B Virus Transgenic Mice

ABSTRACT In chronic hepatitis B (CHB), failure to control hepatitis B virus (HBV) is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T cell unresponsiveness, and thus represent an appropriate model in which to test novel therapeutic strategies. To date, the tolerant state of CD8+ T cells in these animals could be altered only by strong immunogens or by immunization with HBV antigen-pulsed dendritic cells; however, the effectors induced were unable to suppress viral gene expression or replication. Because of the known stimulatory properties of alpha interferon (IFN-α) and interleukin-15 (IL-15), this study explored the therapeutic potential of liver-directed gene transfer of these cytokines in a murine model of CHB using adeno-associated virus (AAV) delivery. This combination not only resulted in a reduction in the viral load in the liver and the induction of an antibody response but also gave rise to functional and specific CD8+ immunity. Furthermore, when splenic and intrahepatic lymphocytes from IFN-α- and IL-15-treated animals were transferred to new HBV carriers, partial antiviral immunity was achieved. In contrast to previous observations made using either cytokine alone, markedly attenuated PD-L1 induction in hepatic tissue was observed upon coadministration. An initial study with CHB patient samples also gave promising results. Hence, we demonstrated synergy between two stimulating cytokines, IL-15 and IFN-α, which, given together, constitute a potent approach to significantly enhance the CD8+ T cell response in a state of immune hyporesponsiveness. Such an approach may be useful for treating chronic viral infections and neoplastic conditions. IMPORTANCE With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly and not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.

Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

ABSTRACT Scavenger receptor class B type I (SR-B1) binds pathogen-associated molecular patterns participating in the regulation of the inflammatory reaction but there is no information regarding potential interactions between SR-B1 and the interferon system. Herein, we report that SR-B1 ligands strongly regulate the transcriptional response to interferon α (IFNα) and enhance its antiviral and antitumor activity. This effect was mediated by the activation of TLR2 and TLR4 as it was annulled by the addition of anti-TLR2 or anti-TLR4 blocking antibodies. In vivo, we maximized the antitumor activity of IFNα co-expressing in the liver a SR-B1 ligand and IFNα by adeno-associated viruses. This gene therapy strategy eradicated liver metastases from colon cancer with reduced toxicity. On the other hand, genetic and pharmacological inhibition of SR-B1 blocks the clathrin-dependent interferon receptor recycling pathway with a concomitant reduction in IFNα signaling and bioactivity. This effect can be applied to enhance cancer immunotherapy with oncolytic viruses. Indeed, SR-B1 antagonists facilitate replication of oncolytic viruses amplifying their tumoricidal potential. In conclusion, SR-B1 agonists behave as IFNα enhancers while SR-B1 inhibitors dampen IFNα activity. These results demonstrate that SR-B1 is a suitable pharmacology target to enhance cancer immunotherapy based on IFNα and oncolytic viruses.