An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation.

BACKGROUND AND AIMS: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. APPROACH AND RESULTS: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. CONCLUSIONS: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.

Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting.

Genome-wide computational analysis reveals cardiomyocyte-specific transcriptional Cis-regulatory motifs that enable efficient cardiac gene therapy.

Gene therapy is a promising emerging therapeutic modality for the treatment of cardiovascular diseases and hereditary diseases that afflict the heart. Hence, there is a need to develop robust cardiac-specific expression modules that allow for stable expression of the gene of interest in cardiomyocytes. We therefore explored a new approach based on a genome-wide bioinformatics strategy that revealed novel cardiac-specific cis-acting regulatory modules (CS-CRMs). These transcriptional modules contained evolutionary-conserved clusters of putative transcription factor binding sites that correspond to a "molecular signature" associated with robust gene expression in the heart. We then validated these CS-CRMs in vivo using an adeno-associated viral vector serotype 9 that drives a reporter gene from a quintessential cardiac-specific α-myosin heavy chain promoter. Most de novo designed CS-CRMs resulted in a >10-fold increase in cardiac gene expression. The most robust CRMs enhanced cardiac-specific transcription 70- to 100-fold. Expression was sustained and restricted to cardiomyocytes. We then combined the most potent CS-CRM4 with a synthetic heart and muscle-specific promoter (SPc5-12) and obtained a significant 20-fold increase in cardiac gene expression compared to the cytomegalovirus promoter. This study underscores the potential of rational vector design to improve the robustness of cardiac gene therapy.

Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates.

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk.

UNLABELLED: Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity. CONCLUSION: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis.

Novel hyperactive transposons for genetic modification of induced pluripotent and adult stem cells: a nonviral paradigm for coaxed differentiation.

Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust nonviral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm. We now demonstrate that these hyperactive transposases resulted in superior gene transfer efficiencies and expression in mesenchymal and muscle stem/progenitor cells, consistent with higher expression levels of therapeutically relevant proteins including coagulation factor IX. Their differentiation potential and karyotype was not affected. Moreover, stable transposition could also be achieved in iPS, which retained their ability to differentiate along neuronal, cardiac, and hepatic lineages without causing cytogenetic abnormalities. Most importantly, transposon-mediated delivery of the myogenic PAX3 transcription factor into iPS coaxed their differentiation into MYOD(+) myogenic progenitors and multinucleated myofibers, suggesting that PAX3 may serve as a myogenic "molecular switch" in iPS. Hence, this hyperactive transposon system represents an attractive nonviral gene transfer platform with broad implications for regenerative medicine, cell and gene therapy.

Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.

The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50% stable gene transfer in human CD34(+) cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34(+) cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.

IL-10 dampens TNF/inducible nitric oxide synthase-producing dendritic cell-mediated pathogenicity during parasitic infection.

Antiparasite responses are associated with the recruitment of monocytes that differentiate to macrophages and dendritic cells at the site of infection. Although classically activated monocytic cells are assumed to be the major source of TNF and NO during Trypanosoma brucei brucei infection, their cellular origin remains unclear. In this study, we show that bone marrow-derived monocytes accumulate and differentiate to TNF/inducible NO synthase-producing dendritic cells (TIP-DCs) in the spleen, liver, and lymph nodes of T. brucei brucei-infected mice. Although TIP-DCs have been shown to play a beneficial role in the elimination of several intracellular pathogens, we report that TIP-DCs, as a major source of TNF and NO in inflamed organs, could contribute actively to tissue damage during the chronic stage of T. brucei brucei infection. In addition, the absence of IL-10 leads to enhanced differentiation of monocytes to TIP-DCs, resulting in exacerbated pathogenicity and early death of the host. Finally, we demonstrate that sustained production of IL-10 following IL-10 gene delivery treatment with an adeno-associated viral vector to chronically infected mice limits the differentiation of monocytes to TIP-DCs and protects the host from tissue damage.