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Background & Aims Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking. Methods We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. Results We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. Conclusions In conclusion, we established and validated an in vitro damage–repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518., Graphical abstract

Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%–13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L “Padua” FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile. Keywords: AAV, AAV5, AMT-060, AMT-061, adeno-associated virus, factor IX, gene therapy, hemophilia B, non-human primate

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1-deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy of a low dose (1x10(11) gc/kg) but not of a higher dose (3x10(11) gc/kg) scAAV8-LP1-UT1A1. Administration of poly[i] just before the vector significantly increases the correction of serum bilirubin in female rats. In these, the effect of poly[i] is seen by both doses but is more pronounced in the females receiving the low vector, where it also results in a significant increase of bilirubin glucuronides in bile. In conclusion, this study shows that SR-A mediates the endocytosis of AAV8 in vitro and in vivo and that blocking this receptor can improve the efficacy of AAV-mediated liver-directed gene therapy.

The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4(-/-)) is a relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in increased bile toxicity. Expression of insulin like growth factor binding protein 5 (Igfbp5) increases in time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a role in the progression of liver pathology. The aim of this study was therefore to investigate the effect of IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy. IGFBP5 and, as a control, green fluorescent protein were overexpressed in the hepatocytes of Abcb4(-/-) mice, using an adeno-associated viral vector (AAV). Progression of liver fibrosis was studied 3, 6, and 12 weeks after vector injection by analyzing serum parameters, collagen deposition, expression of pro-fibrotic genes, inflammation and oxidative stress. A single administration of the AAV vectors provided prolonged expression of IGFBP5 and GFP in the livers of Abcb4(-/-) mice. Compared to GFP control, fractional liver weight, extracellular matrix deposition and amount of activated hepatic stellate cells significantly decreased in IGFBP5 oyerexpressing mice even 12 weeks after treatment. This effect was not due to a change in bile composition, but driven by reduced inflammation, oxidative stress, and proliferation. Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this model for chronic cholangiopathy. (C) 2012 Elsevier B.V. All rights reserved

Adeno-associated virus (AAV) liver-directed gene therapy seems a feasible treatment for Crigler-Najjar syndrome type I, an inherited liver disorder characterized by severe unconjugated hyperbilirubinemia. Transient immunosuppression coupled with vector administration seems needed to overcome host immune responses that prevent long-term expression in patients. The immunosuppressive mycophenolate mofetil (MMF), which inhibits de novo synthesis of purines, is a promising candidate. To investigate the potential use of MMF in patients with Crigler-Najjar syndrome, we studied its effect on single-stranded AAV (ssAAV)-mediated correction of hyperbilirubinemia in the relevant preclinical model, the Gunn rat. Although MMF was well tolerated and effective it also impaired the efficacy of ssAAV. Subsequent in vitro studies showed that this effect is not specific for UGT1A deficiency. In fact, clinical relevant concentrations of mycophenolic acid (MPA), the active compound of MMF, also impair the transduction of HEK-293T cells by ssAAV. Because this effect was reversed by guanosine addition, it seems that intracellular levels of this nucleotide become limited, suggesting that MPA impairs second-strand DNA synthesis. This is corroborated by observations that MPA did not impair transduction of 293T cells by a self-complementary AAV (scAAV) vector and that MMF did not reduce the scAAV efficacy in the Gunn rat. In conclusion, MMF impairs ssAAV-mediated liver-directed gene therapy, which is relevant for the use of this immunosuppressive agent with single-stranded vectors. Furthermore, because this effect is due to impaired second-strand synthesis, the use of MMF with scAAV seems warranted.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

Adenoviral (AdV) and Adenovirus-associated viral (AAV) vectors both are used for in vivo gene therapy of inherited liver disorders, such as Crigler-Najjar syndrome type 1. In a relevant animal model, the Gunn rat, both vectors efficiently correct the severe hyperbilirubinemia characteristic of this liver disorder. Although the clinical use of AAV is more advanced, as demonstrated by the successful phase 1 trial in hemophilia B patients, because of its large cloning capacity AdV remains an attractive option. A direct comparison of the efficacy of these two vectors in the liver in a relevant disease model has not been reported. Aim of this study was to compare the efficiency of clinically applicable doses of both vectors in the Gunn rat. AdV or scAAV (self-complimentary AAV) ferrying identical liver-specific expression cassettes of the therapeutic gene, UGT1A1, were injected into the tail vein. As the titration methods of these two vectors are very different, a comparison based on vector titers is not valid. Therefore, their efficacy was compared by determining the amount of vector genomes delivered to the liver required for therapeutic correction of serum bilirubin. Like AAV, the liver-specific first-generation AdV also provided sustained correction in this relevant disease model. UGT1A1 mRNA expression provided per genome was comparable for both vectors. Flanking the expression cassette in AdV with AAV-ITRs (inverted terminal repeats), increased UGT1A1 mRNA expression eightfold which resulted in a significant improvement of efficacy. Compared with AAV, less AdV genomes were needed for complete correction of hyperbilirubinemia.

As recently demonstrated in patients with factor IX deficiency, adeno-associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and MV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity. (C) 2012 Elsevier B.V. All rights reserved.

Crigler-Najjar (CN) syndrome is a recessive inherited disorder caused by deficiency of uridine diphospho-glucuronosyl transferase 1A1. This hepatic enzyme catalyzes the glucuronidation of bilirubin, an essential step in excretion into bile of this neurotoxic compound. As a result, CN patients suffer from severe unconjugated hyperbilirubinemia and are at risk of bilirubin encephalopathy. Over the last decades ex vivo and in vivo gene therapy using viral and non-viral vectors has been used to correct hyperbilirubinemia in the relevant animal model for CN syndrome, the Gunn rat. Several of these approaches did result in long-term correction of serum bilirubin levels in this animal model. However, none have been translated into a clinical trial. In this review we will recapitulate the strategies used and discuss their suitability for clinical application in the near future. We will also address specific safety measures in the gene therapy protocol needed to prevent adverse effects such as bilirubin toxicity. Since CN seems an ideal model for other monogenetic inherited metabolic liver disorders, development of liver-directed gene-therapy has relevance beyond this rare disease.

Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species.

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1–deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy o...