CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1(-/-) mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.

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.

RIG-I-like receptors (RLRs) are cellular sensor proteins that detect certain RNA species produced during viral infections. RLRs activate a signaling cascade that results in the production of IFN-ß as well as several other cytokines with antiviral and proinflammatory activities. We explored the potential of different constructs based on RLRs to induce the IFN-ß pathway and create an antiviral state in type I IFN-unresponsive models. A chimeric construct composed of RIG-I 2CARD and the first 200 amino acids of MAVS (2CARD-MAVS200) showed an enhanced ability to induce IFN-ß when compared to other stimulatory constructs. Furthermore, this human chimeric construct showed a superior ability to activate IFN-ß expression in cells from various species. This construct was found to overcome the restrictions of blocking IFN-ß induction or signaling by a number of viral IFN-antagonist proteins. Additionally, the antiviral activity of this chimera was demonstrated in influenza virus and HBV infection mouse models using adeno-associated virus (AAV) vectors as a delivery vehicle. We propose that AAV vectors expressing 2CARD-MAVS200 chimeric protein can reconstitute IFN-ß induction and recover a partial antiviral state in different models that do not respond to recombinant IFN-ß treatment.

Interferon-¿ is a potent antiviral agent and a vigorous adjuvant in the induction of T-cell responses but its use is limited by hematologic toxicity. Interferon-¿ alters hematopoietic stem cell dormancy and impairs myelocytic and erythrocytic/megakaryocytic differentiation from hematopoietic progenitors. However, the effect of chronic interferon-¿ exposure on hematopoietic precursors has still not been well characterized. Here, we transduced the liver of mice with an adenoassociated vector encoding interferon-¿ to achieve sustained high serum levels of the cytokine. The bone marrow of these animals showed diminished long-term and short-term hematopoietic stem cells, reduction of multipotent progenitor cells, and marked decrease of B cells, but significant increase in the proportion of CD8(+) and CD4(+)CD8(+) T cells. Upon adoptive transfer to RAG(-/-) mice, bone marrow cells from interferon-¿-treated animals generated CD4(+) and CD8(+) T cells while CD19(+), CD11b(+) and NK1.1(+) lineages failed to develop. These effects are associated with the transcriptional downregulation of transcription factors involved in B-cell differentiation and modulation of key factors for T-cell development. Thus, sustained interferon-¿ exposure causes hematopoietic stem cells exhaustion and drives common lymphoid progenitors towards T-cell generation.

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-¿ 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.