Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt ؊/؊ mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria.gene therapy ͉ knockout mouse ͉ oxalate ͉ urolithiasis ͉ nephrocalcinosis

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BackgroundRadiation-induced gastrointestinal syndrome (RIGS) results from a combination of direct cytocidal effects on intestinal crypt and endothelial cells and subsequent loss of the mucosal barrier, resulting in electrolyte imbalance, diarrhea, weight loss, infection and mortality. Because R-spondin1 (Rspo1) acts as a mitogenic factor for intestinal stem cells, we hypothesized that systemic administration of Rspo1 would amplify the intestinal crypt cells and accelerate the regeneration of the irradiated intestine, thereby, ameliorating RIGS.Methods and FindingsMale C57Bl/6 mice received recombinant adenovirus expressing human R-spondin1 (AdRspo1) or E.coli Lacz (AdLacz), 1–3 days before whole body irradiation (WBI) or abdominal irradiation (AIR). Post-irradiation survival was assessed by Kaplan Meier analysis. RIGS was assessed by histological examination of intestine after hematoxilin and eosin staining, immunohistochemical staining of BrdU incorporation, Lgr5 and β-catenin expression and TUNEL staining. The xylose absorption test (XAT) was performed to evaluate the functional integrity of the intestinal mucosal barrier. In order to examine the effect of R-spondin1 on tumor growth, AdRspo1 and AdLacZ was administered in the animals having palpable tumor and then exposed to AIR. There was a significant increase in survival in AdRspo1 cohorts compared to AdLacZ (p<0.003) controls, following WBI (10.4 Gy). Significant delay in tumor growth was observed after AIR in both cohorts AdRspo1 and AdLacZ but AdRspo1 treated animals showed improved survival compared to AdLacZ. Histological analysis and XAT demonstrated significant structural and functional regeneration of the intestine in irradiated animals following AdRspo1 treatment. Immunohistochemical analysis demonstrated an increase in Lgr5+ve crypt cells and the translocation of β-catenin from the cytosol to nucleus and upregulation of β-catenin target genes in AdRspo1-treated mice, as compared to AdLacz-treated mice.ConclusionRspo1 promoted radioprotection against RIGS and improved survival of mice exposed to WBI. The mechanism was likely related to induction of the Wnt-β-catenin pathway and promotion of intestinal stem cell regeneration. Rspo1 has protective effect only on normal intestinal tissue but not in tumors after AIR and thereby may increase the therapeutic ratio of chemoradiation therapy in patients undergoing abdominal irradiation for GI malignancies.

The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate preferentially over the host liver cells. We hypothesized that preparative hepatic irradiation ( H epatocyte transplantation (HT) is currently being evaluated as a treatment strategy for patients with acute and chronic liver failure and to replace metabolic liver functions in inherited liver diseases. 1 HT has been used in the treatment of inherited metabolic diseases, such as Crigler-Najjar syndrome type I, 2 and for hepatocyte-based ex vivo gene therapy in experimental animals 3-5 as well as in patients with low-density lipoprotein receptor deficiency. 6 However, the clinical application of HT is limited by the availability of human hepatocytes and the number of liver cells that can be transplanted safely at one time. An important consideration is whether a sufficient number of hepatocytes can be engrafted to achieve the desired metabolic correction without causing portal hypertension or other adverse effects. Therefore, a method to induce preferential proliferation of a relatively small number of engrafted hepatocytes in vivo could markedly enhance the applicability of HT.We hypothesized that preparative irradiation of the liver along with a strong mitotic stimulus provided by a maneuver such as partial hepatectomy (PH) should damage the host hepatocyte DNA, causing cell cycle arrest. Subsequently transplanted normal, nonirradiated hepatocytes should proliferate preferentially in response to the

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

SummaryHepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

Development of new therapies for human hepatitis B virus infection (HBVHepatitis B virus (HBV) is a major cause of morbidity, that affects as many as 350 million people worldwide, resulting in an estimated 2 million deaths per year. 1,2 Currently available therapies for HBV-related hepatitis have limited success. 3 Therefore, new antiviral therapies that inhibit HBV replication or gene expression are being developed. Severe adverse reactions to nucleoside analogues in recent clinical trials 4,5 underscore the need for a small animal model to screen the safety and efficacy of new therapeutic modalities. Because of the narrow host range of HBV, existing animal models are limited to chimpanzees 6 and the recently reported tupaia, 7-10 which are expensive and in short supply. Related hepadnaviridae, duck HBV, 11 and woodchuck HBV, 12 infect their respective natural hosts; however, these viruses are of limited relevance to human HBV infection because of significant structural divergence among the viruses. 13,14 For these reasons, it would be advantageous to have a small animal model for human HBV.In efforts to produce murine models of HBV viremia, other investigators have transferred HBV to mice as a transgene, or by ectopic transplantation of human tumor cell lines producing HBV. HBV transgenic mice produce virus from HBV DNA, which is integrated into mouse chromosomes of all mouse cells. 15 Viral replication and production of all viral antigens have been shown in this model. Disadvantages of transgenic mice include the production of HBV in mouse hepatocytes but not in human hepatocytes, the absense of covalently closed circular DNA (CCC) viral transcription templates, and vertical rather than horizontal transmission of the virus. The other available murine model is the immunodeficient mouse with ectopically transplanted human tumor cell lines containing or producing HBV. 16,17 BALB/c nude mice injected with PLC/PRF/5 tumor cells subcutaneously were shown 2 decades ago to produce tumors, secrete HBsAg, and contain HBV DNA, however their life span and usefulness are restricted by the tumor growth. HepG2.2.15 tumor cell lines subcutaneously injected in SCID mice 17 replicate HBV and secrete viral markers. This model is limited by the ectopic location of the human tumor cells and by their transformed phenotype, both of which prevent normal interactions of engrafted cells with host hepatocytes. Furthermore, human tumor growth in the host mice limits their survival to only a few weeks. 17,18 We developed an immortalized, but nontumorigenic human hepatocyte clone by stable transfection of the HBV genome, which expresses HBV antigens and replicates virus.

The host immune response limits the duration of ex-The use of recombinant retroviruses and adenoviruses for pression of adenovirally transduced genes and precludes somatic gene therapy is being extensively explored. Although long-term gene expression upon re-administration of the retroviruses are able to induce long-term transgene expresvirus. In this study we wished to evaluate whether short-sion because of their integration into the host genome, the term immunosuppression of the host, at the time of recom-utility of these vectors in transferring genes into the liver in binant virus administration, would allow expression of vivo is limited because retroviruses do not transduce nonthe therapeutic gene product upon virus reinjection. Gunn dividing cells.1 Recombinant adenoviral vectors offer a numrats were used as recipients of recombinant adenoviruses ber of advantages for use in gene therapy. These viruses can expressing human BUGT (Ad-hBUGT) or E. coli b-galac-be generated at high titers, transfer foreign genes with high tosidase (Ad-LacZ). Rats were treated with FK506 (1-1.5 efficiency, have a broad host range, and do not require cell mg/kg, per OS daily) for three days beginning 24 hours proliferation for infection to occur.2-6 Despite these advanbefore each virus injection. Control groups did not receive tages, the efficacy of adenoviral vectors in gene therapy has any immunosuppressant. The serum bilirubin level was been severely limited because of lack of integration into the reduced from 7.1 { 0.75 mg/dL to 2.0 { 0.7 mg/dL within host genome, as well as the generation of an anti-viral host two days of viral injection in both FK506 treated and immune response which precludes gene expression upon recontrol groups, and then gradually increased in 6 weeks. injection of the recombinant adenovirus. 7-10 FK506-treated rats had low or undetectable antibody titersWe 11 and others 12 have shown that tolerization of recipient against the recombinant adenovirus and minimal or no animals by administration of recombinant adenovirus in the cytotoxic T lymphocyte (CTL) response against adenovi-neonatal period inhibits the development of antibodies and rus-infected cells. The tolerized rats received two subse-of virus specific cytotoxic T lymphocytes (CTL). Similarly, quent injections 42 and 98 days after the first injection, intrathymic inoculation, 13 or oral administration 14 of adenowhich reduced the bilirubin levels again to 2.0 { 0.56 and viral proteins, can tolerize the host to recombinant adenovi-2.2 { 0.61 mg/dL, respectively. In contrast, control rats ruses, thereby permitting therapeutic gene expression followdeveloped high titer neutralizing antibodies and a CTL ing re-administration of the virus. To explore whether the response, and their serum bilirubin levels were not re-development of an immune response in adult recipients can duced following subsequent injections. We conclude that be prevented by short-term immunosuppression around the short-term FK506 treatment around the time of virus ad-time of virus ...