Innate Immune Sensing of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8+ T cell activation. Alternatively, pDCs may also promote CD8+ T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response.

Optimizing Liver Health Before and After Gene Therapy for Hemophilia A.

Gene therapy for severe hemophilia A employs an adeno-associated virus (AAV) vector and liver-specific promoters that depend on healthy hepatocyte function to achieve safe and long-lasting increases in FVIII activity. Thus, hepatocyte health is an essential aspect of safe and successful gene therapy. Many people living with hemophilia A have current or past chronic hepatitis C virus infection, metabolic dysfunction-associated steatosis or steatohepatitis, or other conditions that may compromise the efficacy and safety of AAV-mediated gene therapy. In addition, gene therapy may induce an immune response to transduced hepatocytes, leading to liver inflammation and reduced FVIII activity. The immune response can be treated with immunosuppression, but close monitoring of liver function tests and factor levels is necessary. The long-term risk of hepatocellular carcinoma associated with gene therapy is unknown. Routine screening by imaging for hepatocellular carcinoma, preferable every 6 months, is essential in patients at high risk and recommended in all recipients of hemophilia A gene therapy. This paper describes our current understanding of the biologic underpinnings of how liver health affects hemophilia A gene therapy, and provides practical clinical guidance for assessing, monitoring, and managing liver health both before and after gene therapy.

Increased Protein Kinase A Activity Induces Fibrolamellar Hepatocellular Carcinoma Features Independent of DNAJB1.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by a fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug-response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype.

Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription.

Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.

Monitoring for liver cancer post-gene therapy-How much and how often?

Hepatocellular carcinoma (HCC) has long been recognized as a complication in people with chronic liver disease, particularly those with cirrhosis. Two gene therapies for haemophilia A and B recently approved in Europe and the US utilize adeno-associated virus (AAV) vectors designed to target hepatocytes. A number of other AAV gene therapies are undergoing clinical investigation for both liver and extrahepatic diseases, many of which likely transduce hepatocytes as well. Although AAV vectors predominantly persist in episomal forms, concerns about insertional mutagenesis have arisen due to findings in pre-clinical models and in a small subset of human HCC cases featuring wild-type AAV integrations in proximity to potential oncogenes. Despite the absence of any causative link between AAV vector therapy and HCC in approved extrahepatic gene therapies or haemophilia gene therapy trials, the package inserts for the recently approved haemophilia gene therapies advise HCC screening in subsets of individuals with additional risk factors. In this review, we discuss HCC risk factors, compare various screening modalities, discuss optimal screening intervals, and consider when to initiate and possibly discontinue screening. At this early point in the evolution of gene therapy, we lack sufficient data to make evidence-based recommendations on HCC screening. While AAV vectors may eventually be shown to be unassociated with risk of HCC, we presently favour a cautious approach that entails regular surveillance until such time as it is hopefully proven to be unnecessary.

TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling.

Upon viral infection of the liver, CD8+ T cell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ T cells. Surprisingly, we find local initiation of a CD8+ T cell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1ß contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ T cells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral T cell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

WNTinib is a multi-kinase inhibitor with specificity against ß-catenin mutant hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. ß-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant ß-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index.

Systematic metareview of prediction studies demonstrates stable trends in bias and low PROBAST inter-rater agreement.

OBJECTIVES: To (1) explore trends of risk of bias (ROB) in prediction research over time following key methodological publications, using the Prediction model Risk Of Bias ASsessment Tool (PROBAST) and (2) assess the inter-rater agreement of the PROBAST. STUDY DESIGN AND SETTING: PubMed and Web of Science were searched for reviews with extractable PROBAST scores on domain and signaling question (SQ) level. ROB trends were visually correlated with yearly citations of key publications. Inter-rater agreement was assessed using Cohen's Kappa. RESULTS: One hundred and thirty nine systematic reviews were included, of which 85 reviews (containing 2,477 single studies) on domain level and 54 reviews (containing 2,458 single studies) on SQ level. High ROB was prevalent, especially in the Analysis domain, and overall trends of ROB remained relatively stable over time. The inter-rater agreement was low, both on domain (Kappa 0.04-0.26) and SQ level (Kappa -0.14 to 0.49). CONCLUSION: Prediction model studies are at high ROB and time trends in ROB as assessed with the PROBAST remain relatively stable. These results might be explained by key publications having no influence on ROB or recency of key publications. Moreover, the trend may suffer from the low inter-rater agreement and ceiling effect of the PROBAST. The inter-rater agreement could potentially be improved by altering the PROBAST or providing training on how to apply the PROBAST.

An RNA-based system to study hepatitis B virus replication and evaluate antivirals.

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.

Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion.

Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.

Cardiorenal Outcomes Among Patients With Atrial Fibrillation Treated With Oral Anticoagulants.

RATIONALE & OBJECTIVE: Direct oral anticoagulants (DOACs) have progressively replaced vitamin K antagonists (VKAs) for stroke prevention in patients with nonvalvular atrial fibrillation (AF). DOACs cause fewer bleeding complications, but their other advantages, particularly related to kidney outcomes, remain inconclusive. We studied the risks of chronic kidney disease (CKD) progression and acute kidney injury (AKI) after DOAC and VKA administration for nonvalvular AF. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Cohort study of Swedish patients enrolled in the Stockholm Creatinine Measurements (SCREAM) project with a diagnosis of nonvalvular AF during 2011-2018. EXPOSURE: Initiation of DOAC or VKA treatment. OUTCOME: Primary outcomes were CKD progression (composite of >30% estimated glomerular filtration rate [eGFR] decline and kidney failure) and AKI (by diagnosis or KDIGO-defined transient creatinine elevations). Secondary outcomes were death, major bleeding, and the composite of stroke and systemic embolism. ANALYTICAL APPROACH: Propensity score weighted Cox regression was used to balance 50 baseline confounders. Sensitivity analyses included falsification end points, subgroups, and estimation of per-protocol effects. RESULTS: We included 32,699 patients (56% initiated DOAC) who were observed for a median of 3.8 years. Their median age was 75 years, 45% were women, and 27% had an eGFR <60mL/min/1.73m2. The adjusted HRs for DOAC versus VKA were 0.87 (95% CI, 0.78-0.98) for the risk of CKD progression and 0.88 (95% CI, 0.80-0.97) for AKI. HRs were 0.77 (95% CI, 0.67-0.89) for major bleeding, 0.93 (95% CI, 0.78-1.11) for the composite of stroke and systemic embolism, and 1.04 (95% CI, 0.95-1.14) for death. The results were similar across subgroups of age, sex, and baseline eGFR when restricting to patients at high risk for thromboembolic events and when censoring follow up at treatment discontinuation or change in type of anticoagulation. LIMITATIONS: Missing information on time in therapeutic range and treatment dosages. CONCLUSIONS: Among patients with nonvalvular AF treated in routine clinical practice compared with VKA use, DOAC use was associated with a lower risk of CKD progression, AKI, and major bleeding but a similar risk of the composite of stroke, systemic embolism, or death.

Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma.

Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65 weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.

Human hepatocyte PNPLA3-148M exacerbates rapid non-alcoholic fatty liver disease development in chimeric mice.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.

Targeting BCL-XL in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal liver cancer with no proven effective systemic therapy. Inhibition of the antiapoptotic protein BCL-XL was found to synergize with a variety of systemic therapies in vitro using cells dissociated from patient-derived xenografts (PDX) of FLC or cells dissociated directly from surgical patient resections. As BCL-XL is physiologically expressed in platelets, prior efforts to leverage this vulnerability in other cancers have been hampered by severe thrombocytopenia. To overcome this toxicity, we treated FLC models with DT2216, a proteolysis targeting chimera (PROTAC) that directs BCL-XL for degradation via the von Hippel-Lindau (VHL) E3 ligase, which is minimally expressed in platelets. The combination of irinotecan and DT2216 in vitro on cells directly acquired from patients or in vivo using several xenografts derived from patients with FLC demonstrated remarkable synergy and at clinically achievable doses not associated with significant thrombocytopenia.

IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice.

Hepatic adeno-associated viral (AAV) gene transfer has the potential to cure the X-linked bleeding disorder hemophilia A. However, declining therapeutic coagulation factor VIII (FVIII) expression has plagued clinical trials. To assess the mechanistic underpinnings of this loss of FVIII expression, we developed a hemophilia A mouse model that shares key features observed in clinical trials. Following liver-directed AAV8 gene transfer in the presence of rapamycin, initial FVIII protein expression declines over time in the absence of antibody formation. Surprisingly, loss of FVIII protein production occurs despite persistence of transgene and mRNA, suggesting a translational shutdown rather than a loss of transduced hepatocytes. Some of the animals develop ER stress, which may be linked to hepatic inflammatory cytokine expression. FVIII protein expression is preserved by interleukin-15/interleukin-15 receptor blockade, which suppresses CD8+ T and natural killer cell responses. Interestingly, mice with initial FVIII levels >100% of normal had diminishing expression while still under immune suppression. Taken together, our findings of interanimal variability of the response, and the ability of the immune system to shut down transgene expression without utilizing cytolytic or antibody-mediated mechanisms, illustrate the challenges associated with FVIII gene transfer. Our protocols based upon cytokine blockade should help to maintain efficient FVIII expression.

Comprehensive comparison of stroke risk score performance: a systematic review and meta-analysis among 6 267 728 patients with atrial fibrillation.

AIMS: Multiple risk scores to predict ischaemic stroke (IS) in patients with atrial fibrillation (AF) have been developed. This study aims to systematically review these scores, their validations and updates, assess their methodological quality, and calculate pooled estimates of the predictive performance. METHODS AND RESULTS: We searched PubMed and Web of Science for studies developing, validating, or updating risk scores for IS in AF patients. Methodological quality was assessed using the Prediction model Risk Of Bias ASsessment Tool (PROBAST). To assess discrimination, pooled c-statistics were calculated using random-effects meta-analysis. We identified 19 scores, which were validated and updated once or more in 70 and 40 studies, respectively, including 329 validations and 76 updates-nearly all on the CHA2DS2-VASc and CHADS2. Pooled c-statistics were calculated among 6 267 728 patients and 359 373 events of IS. For the CHA2DS2-VASc and CHADS2, pooled c-statistics were 0.644 [95% confidence interval (CI) 0.635-0.653] and 0.658 (0.644-0.672), respectively. Better discriminatory abilities were found in the newer risk scores, with the modified-CHADS2 demonstrating the best discrimination [c-statistic 0.715 (0.674-0.754)]. Updates were found for the CHA2DS2-VASc and CHADS2 only, showing improved discrimination. Calibration was reasonable but available for only 17 studies. The PROBAST indicated a risk of methodological bias in all studies. CONCLUSION: Nineteen risk scores and 76 updates are available to predict IS in patients with AF. The guideline-endorsed CHA2DS2-VASc shows inferior discriminative abilities compared with newer scores. Additional external validations and data on calibration are required before considering the newer scores in clinical practice. CLINICAL TRIAL REGISTRATION: ID CRD4202161247 (PROSPERO).

The influence of corn trypsin inhibitor on the contribution of coagulation determinants to the Technoclone Thrombin Generation Assay (TGA) and the Calibrated Automated Thrombogram (CAT).

In thrombin generation (TG) assays, regarded as global coagulation tests, contact activation is considered a major problem which can be eliminated by adding Corn Trypsin Inhibitor (CTI). In previous studies, however, venous thrombosis risk prediction using TG assays did not improve after CTI addition. However, it is unknown whether CTI addition could help to detect subtle but relevant nuances in determinants of TG, making the assay more suitable to detect disturbances in the coagulation system. This study's objective was to assess whether the addition of CTI is associated with a broader contribution of individual coagulation factors to the total amount of thrombin formed in Calibrated Automated Thrombogram (CAT) and Technoclone Thrombin Generation Assay (TGA). Thrombin generation was measured in 326 healthy individuals from THE VTE study at very low tissue factor concentrations, with and without addition of CTI prior to blood sampling. The influence of several coagulation factors on total amount of thrombin formed, i.e. area under the curve (AUC) or endogenous thrombin potential (ETP), was analysed using multiple linear regression with standardisation of all values resulting in Z-scores with 95% confidence intervals (95%CI). Association between coagulation factors and TG changed minimally after addition of CTI. Largest changes after CTI addition were found for following factors: for CAT: free protein S (from 0.00 (95%CI -0.12 to 0.12) to -0.29 (95%CI -0.43 to -0.15)) and protein S (from -0.05 (95%CI -0.18 to 0.08) to -0.21 (95%CI -0.37 to -0.05)); for TGA: antithrombin (from -0.11 (-0.23 to 0.02) to -0.19 (-0.30 to -0.07)), factor VIII (from 0.15 (0.03 to 0.27) to 0.24 (0.13 to 0.36)) and fibrinogen (from 0.12 (-0.01 to 0.26) to 0.19 (0.06 to 0.32)). In conclusion, there is no clear trend towards a broader contribution of coagulation factors in samples handled with CTI compared with those handled without CTI.

Mouse characteristics that affect establishing xenografts from hepatocellular carcinoma patient biopsies in the United States.

BACKGROUND: Hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models hold potential to advance knowledge in HCC biology to help improve systemic therapies. Beside hepatitis B virus-associated tumors, HCC is poorly established in PDX. METHODS: PDX formation from fresh HCC biopsies were obtained and implanted intrahepatically or in subrenal capsule (SRC). Mouse liver injury was induced in immunodeficient Fah-/-  mice through cycling off nitisinone after HCC biopsy implantation, versus continuous nitisinone as non-liver injury controls. Mice with macroscopically detectable PDX showed rising human alpha1-antitrypsin (hAAT) serum levels, and conversely, no PDX was observed in mice with undetectable hAAT. RESULTS: Using rising hAAT as a marker for PDX formation, 20 PDX were established out of 45 HCC biopsy specimens (44%) reflecting the four major HCC etiologies most commonly identified at Memorial SloanKettering similar to many other institutions in the United States. PDX was established only in severely immunodeficient mice lacking lymphocytes and NK cells. Implantation under the renal capsule improved PDX formation two-fold compared to intrahepatic implantation. Two out of 18 biopsies required murine liver injury to establish PDX, one associated with hepatitis C virus and one with alcoholic liver disease. PDX tumors were histologically comparable to biopsy specimens and 75% of PDX lines could be passaged. CONCLUSIONS: Using cycling off nitisinone-induced liver injury, HCC biopsies implanted under the renal capsule of severely immunodeficient mice formed PDX with 57% efficiency as determined by rising hAAT levels. These findings facilitate a more efficient make-up of PDX for research into subset-specific HCC.

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript.

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.