Crigler-Najjar syndrome type I is characterized by unconjugated hyperbilirubinemia resulting from an autosomal recessive inherited deficiency of hepatic UDPglucuronosyltransferase (UGT) 1A1 activity. The enzyme is essential for glucuronidation and biliary excretion of bilirubin, and its absence can be fatal. The Gunn rat is an excellent animal model of this disease, exhibiting a single guanosine (G) base deletion within the UGT1A1 gene. The defect results in a frameshift and a premature stop codon, absence of enzyme activity, and hyperbilirubinemia. Here, we show permanent correction of the UGT1A1 genetic defect in Gunn rat liver with site-specific replacement of the absent G residue at nucleotide 1206 by using an RNA͞DNA oligonucleotide designed to promote endogenous repair of genomic DNA. The chimeric oligonucleotide was either complexed with polyethylenimine or encapsulated in anionic liposomes, administered i.v., and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, and DNA sequencing, and confirmed by genomic Southern blot analysis. DNA repair was specific, efficient, stable throughout the 6-month observation period, and associated with reduction of serum bilirubin levels. Our results indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, with consequent improvement in the metabolic abnormality.UDP-glucuronosyltransferases (UGTs) are a family of membrane-bound enzymes that catalyze the conjugation of numerous xenobiotics and endogenous substrates with glucuronic acid. Of the known isoforms, only UGT1A1 is physiologically relevant in bilirubin glucuronidation and biliary excretion of this potentially toxic metabolite (1, 2). Crigler-Najjar (CN) syndrome is the inherited deficiency of hepatic UGT1A1 activity and is characterized by elevated serum levels of unconjugated bilirubin (3). Of the two types of CN syndrome, type I is more severe and is characterized by a nearly complete absence of UGT1A1 activity, whereas incomplete deficiency of the enzyme is associated with the less severe type II form (4, 5).The homozygous Gunn rat, a mutant strain of Wistar rat, is an accurate animal model for CN syndrome type I. Its liver lacks UGT1A1 activity because of the deletion of a single guanosine (G) base in UGT1A1 that results in a frameshift and a premature stop codon (6, 7). Recombinant adenoviral vectors have been used in vivo to correct the hyperbilirubinemia in the Gunn rat with persistent expression of the human bilirubin UGT1A1 enzyme for as long as 2 months (8, 9). Significant progress also has been made in overcoming the immunogenicity of the adenoviral-based vectors (9-11), but their use requires repeated treatments and immunomodulation of the host to maintain therapeutic levels of UGT1A1.A novel approach, based on mechanisms of DNA repair (12), was reported to correct single nucleotide mutations in ...

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

Object Resected brain metastases have a high rate of local recurrence without adjuvant therapy. Adjuvant whole brain radiotherapy (WBRT) remains the standard of care with the rate of local control >90%. However, WBRT is delivered over 10–15 days, which can delay other therapy and is associated with acute and long-term toxicities. Intra-operative permanent Cesium-131 (Cs-131) implants can be performed at the time of surgery, thereby avoiding any additional therapy. We evaluate the safety, feasibility and efficacy of a novel treatment approach of brain metastases with a permanent intra-operative Cs-131 brachytherapy. Methods After IRB approval, 24 patients with a newly diagnosed metastasis to the brain (n=24) were accrued on a prospective protocol between 2010 and 2012. There were 10 frontal, 7 parietal, 4 cerebellar, 2 occipital, and 1 temporal metastases. Histology included lung (16), breast (2), kidney (2), melanoma (2), colon (1), and cervix (1). Cs-131 stranded seeds were placed as a permanent volume implant. Prescription dose was 80Gy at 5mm depth from the resection cavity surface. Distant metastases were treated with stereotactic radiosurgery (SRS) or WBRT, depending on the number of lesions. Primary end point was resection cavity freedom from progression (FFP). Secondary end points included distant metastases FFP, median survival, overall survival (OS), and toxicity. Results Median follow-up was 19.3 months (range, 12.89 – 29.57 months). Median age was 65 years (range, 45–84 years). Median volume of resected tumor was 10.31 cc (range, 1.77 - 87.11 cc). Median number of seeds employed was 12 (range, 4–35) with median activity per seed of 3.82 mCi (range, 3.31–4.83 mCi) and total activity of 46.91 mCi (range, 15.31–130.70 mCi). Local recurrence FFP was 100%. There was 1 adjacent leptomeningeal recurrence, resulting in a 1-year regional FFP of 93.8% (95% CI = 63.2%, 99.1%). Distant metastasis FFP was 48.4% (95% CI = 26.3%, 67.4%). Median OS was 9.9 months (95% CI = 4.8 months, upper limit not estimated) and 1-year OS was 50.0% (95% CI = 29.1%, 67.8%). Complications included cerebrospinal fluid leak (1), seizure (1), infection (1). There was no radiation necrosis. Conclusions Cs-131 post-resection permanent brachytherapy implants resulted in no local recurrences and no radiation necrosis. This treatment approach was safe, well tolerated, and convenient for patients, resulting in a short radiation treatment course, high response rates, and minimal toxicity. These results merit further study with a multicenter trial.

Objective The goal of this study is to describe the complications and toxicities for post-operative radiation therapy in Fanconi anemia patients. Design Cohort study. Setting Patients treated at community and tertiary care hospitals throughout the United States. Patients Fanconi anemia patients enrolled in the International Fanconi Anemia Registry (IFAR) who developed head and neck squamous cell carcinoma and received post-operative radiation. Main Outcome Measures Demographics of Fanconi anemia patients, radiation doses, and radiation toxicities. Results 12 Fanconi anemia patients (7 male, 5 female) were identified. Patients developed cancers at a mean age of 35.5 years (range 20 to 48). Sites of primary cancer were oral cavity (8/12), larynx (2/12), pharynx (1/12), and unknown (1/12). Median radiation dose was 5590 cGy (range: 2500 to 7020). The most common toxicities were mucositis (9/12), dysphagia (8/12), and pancytopenia (6/12). Other complications included esophageal stenosis, laryngeal edema, and wound breakdown. Radiotherapy could not be completed in 5/12 cases. Overall 8/12 patients died, 4 during the course of radiation. The post-operative disease-free survival time ranged from 0 to 55 months. Conclusions Fanconi anemia patients have a high rate of complications to radiation therapy. Common radiation toxicities, particularly mucositis, are especially prevalent and difficult to manage in this population. Pancytopenia is common and may lead to further complications, particularly bleeding and infection. Overall survival is poor. Further study of FA patients’ response to radiation should be attempted in order to establish appropriate doses to balance treating disease while limiting toxicity.

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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.

OBJECTIVE Managing patients whose intraparenchymal brain metastases recur after radiotherapy remains a challenge. Intraoperative cesium-131 (Cs-131) brachytherapy performed at the time of neurosurgical resection may represent an excellent salvage treatment option. The authors evaluated the outcomes of this novel treatment with permanent intraoperative Cs-131 brachytherapy. METHODS Thirteen patients with 15 metastases to the brain that recurred after stereotactic radiosurgery and/or whole brain radiotherapy were treated between 2010 and 2015. Stranded Cs-131 seeds were placed as a permanent volume implant. Prescription dose was 80 Gy at 5-mm depth from the resection cavity surface. The primary end point was resection cavity freedom from progression (FFP). Resection cavity freedom from progression (FFP), regional FFP, distant FFP, median survival, overall survival (OS), and toxicity were assessed. RESULTS The median duration of follow-up after salvage treatment was 5 months (range 0.5-18 months). The patients' median age was 64 years (range 51-74 years). The median resected tumor diameter was 2.9 cm (range 1.0-5.6 cm). The median number of seeds implanted was 19 (range 10-40), with a median activity per seed of 2.25 U (range 1.98-3.01 U) and median total activity of 39.6 U (range 20.0-95.2 U). The 1-year actuarial local FFP was 83.3%. The median OS was 7 months, and 1-year OS was 24.7%. Complications included infection (3), pseudomeningocele (1), seizure (1), and asymptomatic radionecrosis (RN) (1). CONCLUSIONS After failure of prior irradiation of brain metastases, re-irradiation with intraoperative Cs-131 brachytherapy implants provides durable local control and limits the risk of RN. The authors' initial experience demonstrates that this treatment approach is well tolerated and safe for patients with previously irradiated tumors after failure of more than 1 radiotherapy regimen and that it results in excellent response rates and minimal toxicity.