Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells.

BACKGROUND: Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function. METHODS: Human PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses. RESULTS: GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation. CONCLUSIONS: We generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

Poly(ethylene oxide)/Gelatin-Based Biphasic Photocrosslinkable Hydrogels of Tunable Morphology for Hepatic Progenitor Cell Encapsulation.

Macroporous hydrogels have great potential for biomedical applications. Liquid or gel-like pores were created in a photopolymerizable hydrogel by forming water-in-water emulsions upon mixing aqueous solutions of gelatin and a poly(ethylene oxide) (PEO)-based triblock copolymer. The copolymer constituted the continuous matrix, which dominated the mechanical properties of the hydrogel once photopolymerized. The gelatin constituted the dispersed phase, which created macropores in the hydrogel. The microstructures of the porous hydrogel were determined by the volume fraction of the gelatin phase. When volume fractions were close to 50 v%, free-standing hydrogels with interpenetrated morphology can be obtained thanks to the addition of a small amount of xanthan. The hydrogels displayed Young's moduli ranging from 5 to 30 kPa. They have been found to be non-swellable and non-degradable in physiological conditions. Preliminary viability tests with hepatic progenitor cells embedded in monophasic PEO-based hydrogels showed rapid mortality of the cells, whereas encouraging viability was observed in PEO-based triblock copolymer/gelatin macroporous hydrogels. The latter has the potential to be used in cell therapy.

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

BACKGROUND AND AIMS: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. APPROACH AND RESULTS: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and three-dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. CONCLUSIONS: Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells-derived hepatic stem cells.

Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.

Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection.

BACKGROUND: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed. METHODS: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells. RESULTS: We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis. CONCLUSIONS: Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.

Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system.

Despite decades of investigation on the proliferation of adult human primary hepatocytes, their expansion in vitro still remains challenging. To later be able to consider hepatocytes as a cell therapy alternative or bridge to liver transplantation, dramatically impeded by a shortage in liver donors, the first step is having an almost unlimited source of these cells. The banking of transplantable hepatocytes also implies a protocol for their expansion that can be compatible with large-scale production. We show that adult human primary hepatocytes when grown in 3D organoids are easily amplified, providing a substantial source of functional hepatocytes ready for transplantation. Following their plating, differentiated human hepatocytes are amplified during a transient and reversible step as liver progenitors, and can subsequently be converted back to mature differentiated hepatocytes. The protocol we propose is not only compatible with automated and high-throughput cell culture systems, thanks to the expansion of hepatocytes in suspension, but also guarantees the generation of a high number of functional cells from the same patient sample, with a relatively easy set up.

Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene.

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

21st Nantes Actualités Transplantation: "When Stem Cells Meet Immunology".

"When Stem Cells Meet Immunology" has been the topic of the 21st annual "Nantes Actualités en Transplantation" meeting (June 9-10, 2016, Nantes, France). This meeting brought together pioneers and leading experts in the fields of stem cells, biomaterials and immunoregulation. Presentations covered multipotent (mesenchymal and hematopoietic) and pluripotent stem cells (embryonic and induced) for regenerative medicine of incurable diseases, immunotherapy and blood transfusions. An additional focus had been immune rejections and responses of allogeneic or autologous stem cells. Conversely, stem cells are also able to directly modulate the immune response through the production of immunoregulatory molecules. Moreover, stem cells may also provide an unlimited source of immune cells (DCs, NK cells, B cells, and T cells) that can operate as "super" immune cells, for example, through genetic engineering with chimeric antigen receptors.This meeting report puts presentations into an overall context highlighting new potential biomarkers for potency prediction of mesenchymal stem cell-derived and pluripotent stem cell-derived multicellular organoids. Finally, we propose future directions arising from the flourishing encounter of stem cell and immune biology.

Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence.

Wilson's disease (WD) is a rare autosomal recessive disease due to mutations of the gene encoding the copper-transporter ATP7B. The diagnosis is hampered by the variability of symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and the absence of a reliable diagnostic test. We investigated the diagnostic potential of X-ray fluorescence (XRF) that allows quantitative analysis of multiple elements. Studies were performed on animal models using Wistar rats (n = 10) and Long Evans Cinnamon (LEC) rats (n = 11), and on human samples including normal livers (n = 10), alcohol cirrhosis (n = 8), haemochromatosis (n = 10), cholestasis (n = 6) and WD (n = 22). XRF experiments were first performed using synchrotron radiation to address the elemental composition at the cellular level. High-resolution mapping of tissue sections allowed measurement of the intensity and the distribution of copper, iron and zinc while preserving the morphology. Investigations were further conducted using a laboratory X-ray source for irradiating whole pieces of tissue. The sensitivity of XRF was highlighted by the discrimination of LEC rats from wild type even under a regimen using copper deficient food. XRF on whole formalin-fixed paraffin embedded needle biopsies allowed profiling of the elements in a few minutes. The intensity of copper related to iron and zinc significantly discriminated WD from other genetic or chronic liver diseases with 97.6% specificity and 100% sensitivity. This study established a definite diagnosis of Wilson's disease based on XRF. This rapid and versatile method can be easily implemented in a clinical setting.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis.

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles.

RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34(+) and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing.

CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

BACKGROUND: Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. METHODS: Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. RESULTS: shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. CONCLUSION: We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

Human neonatal hepatocyte transplantation induces long-term rescue of unconjugated hyperbilirubinemia in the Gunn rat.

Crigler-Najjar type 1 disease is a rare inherited metabolic disease characterized by high levels of unconjugated bilirubin due to the complete absence of hepatic uridine diphosphoglucuronate-glucuronosyltransferase activity. Hepatocyte transplantation (HT) has been proposed as an alternative treatment for Crigler-Najjar syndrome, but it is still limited by the quality and the low engraftment and repopulation ability of the cells used. Because of their attachment capability and expression of adhesion molecules as well as the higher proportion of hepatic progenitor cells, neonatal hepatocytes may have an advantage over adult cells. Adult or neonatal hepatocytes were transplanted into Gunn rats, a model for Crigler-Najjar disease. Engraftment and repopulation were studied and compared by immunofluorescence (IF). Additionally, the serum bilirubin levels, the presence of bilirubin conjugates in rat serum, and the expression of uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) in rat liver samples were also analyzed. Here we show that neonatal HT results in long-term correction in Gunn rats. In comparison with adult cells, neonatal cells showed better engraftment and repopulation capability 3 days and 6 months after transplantation, respectively. Bilirubinemia decreased in the transplanted animals during the whole experimental follow-up (6 months). Bilirubin conjugates were also present in the serum of the transplanted animals. Western blots and IF confirmed the presence and expression of UGT1A1 in the liver. This work is the first to demonstrate the advantage of using neonatal hepatocytes for the treatment of Crigler-Najjar in vivo.

Hepatic lentiviral gene transfer prevents the long-term onset of hepatic tumours of glycogen storage disease type 1a in mice.

Glycogen storage disease type 1a (GSD1a) is a rare disease due to the deficiency in the glucose-6-phosphatase (G6Pase) catalytic subunit (encoded by G6pc), which is essential for endogenous glucose production. Despite strict diet control to maintain blood glucose, patients with GSD1a develop hepatomegaly, steatosis and then hepatocellular adenomas (HCA), which can undergo malignant transformation. Recently, gene therapy has attracted attention as a potential treatment for GSD1a. In order to maintain long-term transgene expression, we developed an HIV-based vector, which allowed us to specifically express the human G6PC cDNA in the liver. We analysed the efficiency of this lentiviral vector in the prevention of the development of the hepatic disease in an original GSD1a mouse model, which exhibits G6Pase deficiency exclusively in the liver (L-G6pc(-/-) mice). Recombinant lentivirus were injected in B6.G6pc(ex3lox/ex3lox). SA(creERT2/w) neonates and G6pc deletion was induced by tamoxifen treatment at weaning. Magnetic resonance imaging was then performed to follow up the development of hepatic tumours. Lentiviral gene therapy restored glucose-6 phosphatase activity sufficient to correct fasting hypoglycaemia during 9 months. Moreover, lentivirus-treated L-G6pc(-/-) mice presented normal hepatic triglyceride levels, whereas untreated mice developed steatosis. Glycogen stores were also decreased although liver weight remained high. Interestingly, lentivirus-treated L-G6pc(-/-) mice were protected against the development of hepatic tumours after 9 months of gene therapy while most of untreated L-G6pc(-/-) mice developed millimetric HCA. Thus the treatment of newborns by recombinant lentivirus appears as an attractive approach to protect the liver from the development of steatosis and hepatic tumours associated to GSD1a pathology.

Correction of hyperbilirubinemia in gunn rats by surgical delivery of low doses of helper-dependent adenoviral vectors.

Helper-dependent adenoviral (HDAd) vectors are attractive for liver-directed gene therapy because they can drive sustained high levels of transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared with intravenous delivery and multiyear transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. After vector delivery into the surgically isolated liver, we show phenotypic correction at the low and clinically relevant vector dose of 1 × 10(11) vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.

Evolution of exchangeable copper and relative exchangeable copper through the course of Wilson's disease in the Long Evans Cinnamon rat.

BACKGROUND: Wilson's disease (WD) is an inherited disorder of copper metabolism leading to liver failure and/or neurological impairment. Its diagnosis often remains difficult even with genetic testing. Relative exchangeable copper (REC) has recently been described as a reliable serum diagnostic marker for WD. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to validate the use of REC in the Long Evans Cinnamon (LEC) rat, an animal model for WD, and to study its relevance under different conditions in comparison with conventional markers. Two groups of LEC rats and one group of Long-Evans (LE) rats were clinically and biologically monitored from 6 to 28 weeks of age. One group of LEC rats was given copper-free food. The other groups had normal food. Blood samples were collected each month and different serum markers for WD (namely ceruloplasmin oxidase activity, exchangeable copper (CuEXC), total serum copper and REC) and acute liver failure (serum transaminases and bilirubinemia) were tested. Every LEC rat under normal food developed acute liver failure (ALF), with 40% global mortality. Serum transaminases and bilirubinemia along with total serum copper and exchangeable copper levels increased with the onset of acute liver failure. A correlation was observed between CuEXC values and the severity of ALF. Cut-off values were different between young and adult rats and evolved because of age and/or liver failure. Only REC, with values >19%, was able to discriminate LEC groups from the LE control group at every time point in the study. REC sensitivity and specificity reached 100% in adults rats. CONCLUSIONS/SIGNIFICANCE: REC appears to be independent of demographic or clinical data in LEC rats. It is a very simple and reliable blood test for the diagnosis of copper toxicosis owing to a lack of ATP7B function. CuEXC can be used as an accurate biomarker of copper overload.

Specific micro RNA-regulated TetR-KRAB transcriptional control of transgene expression in viral vector-transduced cells.

Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.

Priming of hepatocytes enhances in vivo liver transduction with lentiviral vectors in adult mice.

Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.

Retroviral vector-mediated gene therapy for metabolic diseases: an update.

Retroviral vectors have been used for several decades for the transfer of therapeutic genes to various cells or organs including the liver. Initial studies aimed at treating inherited liver deficiencies were carried out with murine oncoretroviral vectors either delivered directly to the organ or using an ex vivo strategy that entailed harvest of the hepatocytes, transduction during a culture phase and further reinfusion to the patient. However, although a clinical trial was performed in the early 1990s, a complete cure of animal models of metabolic diseases was rarely achieved. The advent of lentiviral vectors derived from HIV1 profoundly changed the field and this vector type now appears to be of the most attractive for liver directed gene therapy. Indeed, lentiviral vectors do not require complete cell division to transduce the target cells. There are however still bottlenecks that limit the clinical development of gene therapy using retroviral vectors. In the present review we will specifically focus on specific aspects such as the risk of insertional mutagenesis, the potential requirement of cell cycle activation to enhance transduction and the major issue of an immune response directed against the transgene as well as some specific aspects of ex vivo gene transfer. Finally we will briefly consider the future developments of these vectors made possible by the availability of new techniques in cell and molecular biology.