[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes without inducing chronic toxicity. However, vector therapeutic index is narrow because of a toxic acute response with potentially lethal consequences elicited by high vector doses. Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction. We investigated two small peptides (PP1 and PP2) developed by phage display to block scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I), respectively, for enhancement of HDAd-mediated hepatocyte transduction efficiency. Pre-incubation of J774A.1 macrophages with either PP1 or PP2 prior to HDAd infection significantly reduced viral vector uptake. In vivo, fluorochrome-conjugated PP1 and PP2 injected intravenously into mice co-localized with both CD68 and CD31 on KCs and LSECs, respectively. Compared with saline pre-treated animals, intravenous injections of both peptides prior to the injection of an HDAd resulted in up to 3.7-and 2.9-fold increase of hepatic transgene expression with PP1 and PP2, respectively. In addition to greater hepatocyte transduction, compared with control saline injected mice, pre-treatment with either peptide resulted in no increased levels of serum interleukin-6, the major marker of adenoviral vector acute toxicity. In summary, we developed small peptides that significantly increase hepatocyte transduction efficacy and improve HDAd therapeutic index with potential for clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (45 kb) genes. Viral vectors derived from adenovirus (Ad), lentivirus (LV) and herpes virus (HV) can package large DNA sequences, but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium. We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG, albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8.
[Show abstract][Hide abstract] ABSTRACT: Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.
[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral (HDAd) vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.
[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral vectors (HDAd) are attractive vectors 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 due to 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 to intravenous delivery liver and multi-year 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. Following vector delivery into the surgically-isolated liver, we showed phenotypic correction at the low and clinically relevant vector dose of 1x1011 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 compared to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.
Human Gene Therapy Methods 03/2014; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor β (TGF-β) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-β signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-β antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.European Journal of Human Genetics advance online publication, 8 January 2014; doi:10.1038/ejhg.2013.283.
European journal of human genetics: EJHG 01/2014; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rhabdomyolysis may result from various factors, namely trauma, exercise, medications, infections, endocrine disorders, congenital myopathies, and metabolic diseases.(1) Among the latter, mitochondrial fatty acid β-oxidation (FAO) defects frequently cause recurrent rhabdomyolysis. FAO disorders are recessively inherited and have a combined incidence of 1:9,300, estimated after implementation of newborn screening programs by tandem mass spectrometry (MS/MS).(2) Clinical manifestations of these disorders range from sudden infant death to Reye-like syndrome, nonketotic hypoglycemia, skeletal myopathy, peripheral neuropathy, and progressive cardiomyopathy. Here, we describe an 18-month-old child presenting with episodes of recurrent rhabdomyolysis related to mitochondrial trifunctional protein deficiency (MTPD), without additional manifestations of FAO defects. We discuss the diagnosis of MTPD and review the prognosis and treatments.
[Show abstract][Hide abstract] ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
[Show abstract][Hide abstract] ABSTRACT: Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant disorder, caused by heterozygous RASA1 mutations, and manifesting multifocal capillary malformations and high risk for fast-flow lesions. A limited number of patients has been reported, raising the question of the phenotypic borders. We identified new patients with a clinical diagnosis of CM-AVM, and patients with overlapping phenotypes. RASA1 was screened in 261 index patients with: CM-AVM (n = 100), common capillary malformation(s) (port-wine stain; n = 100), Sturge-Weber syndrome (n = 37), or isolated arteriovenous malformation(s) (n = 24). Fifty-eight distinct RASA1 mutations (43 novel) were identified in 68 index patients with CM-AVM and none in patients with other phenotypes. A novel clinical feature was identified: cutaneous zones of numerous small white pale halos with a central red spot. An additional question addressed in this study was the "second-hit" hypothesis as a pathophysiological mechanism for CM-AVM. One tissue from a patient with a germline RASA1 mutation was available. The analysis of the tissue showed loss of the wild-type RASA1 allele. In conclusion, mutations in RASA1 underscore the specific CM-AVM phenotype and the clinical diagnosis is based on identifying the characteristic capillary malformations. The high incidence of fast-flow lesions warrants careful clinical and radiologic examination, and regular follow-up. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Crigler-Najjar syndrome type I is due to mutations of the uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) gene resulting in life-threatening increase of serum bilirubin. Life-long correction of hyperbilirubinemia was previously shown with intravenous injection of high doses of a helper-dependent adenoviral (HDAd) vector expressing UGT1A1 in the Gunn rat, the animal model of Crigler-Najjar syndrome. However, such high vector doses can activate an acute and potentially lethal inflammatory response with elevated serum interleukin-6 (IL-6). To overcome this obstacle, we investigated safety and efficacy of direct injections of low HDAd doses delivered directly into the liver parenchyma of Gunn rats. Direct hepatic injections performed by either laparotomy or by ultrasound-guided percutaneous injections were compared to the same doses given by intravenous injections. A greater reduction of hyperbilirubinemia and increased conjugated bilirubin in bile were achieved with 1x1011vp/kg by direct liver injections compared to intravenous injections. In sharp contrast to intravenous injections, direct hepatic injections did not raise serum IL-6 neither resulted in thrombocytopenia. In conclusion, ultrasound-guided percutaneous injection of HDAd vectors into liver parenchyma resulted in improved hepatocyte transduction and reduced toxicity compared to systemic injections and is clinically attractive for liver-directed gene therapy of Crigler-Najjar syndrome.
Human Gene Therapy Methods 08/2013; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral vectors (HDAd) have been shown to mediate considerably longer duration of transgene expression compared to first generation adenoviral vectors. We have previously shown that transgene expression from HDAd transduced hepatocytes can persist at high levels for up to 2.6 years in nonhuman primates following a single vector administration. Because duration of transgene expression and long-term toxicity are critical for risk:benefit assessment, we have continued to monitor these animals. We report here that transgene expression has persisted for the entire observation period of up to 7 years for all animals without long-term adverse effect. However, in all cases, transgene expression level slowly declined over time to less than 10% of peak values by the end of the observation period but remained 2.3 to 111-fold above baseline values. These results will provide important information for a more informed risk:benefit assessment prior to clinical application of HDAd.
[Show abstract][Hide abstract] ABSTRACT: Hemophilia is an inherited blood clotting disorder resulting from deficiency of blood coagulation factors. Current standard of care for hemophilia patients is frequent intravenous infusions of the missing coagulation factor. Gene therapy for hemophilia involves the introduction of a normal copy of the deficient coagulation factor gene thereby potentially offering a definitive cure for the bleeding disorder. A variety of approaches have been pursued for hemophilia gene therapy and this review article focuses on those that use adenoviral vectors.
Journal of genetic syndromes & gene therapy. 04/2013; 2(Suppl 1):017.
[Show abstract][Hide abstract] ABSTRACT: In summary, the multiple biological effects observed in normal and cancer cells treated with phenylbutyrate can be dependent, at least in part, on the newly recognized activity on PDHC, a key enzyme for metabolism and survival.
[Show abstract][Hide abstract] ABSTRACT: Deficiency of SERPINA1/AAT [serpin peptidase inhibitor, clade A (α-1 antiproteinase, antitrypsin), member 1/α 1-antitrypsin] results in polymerization and aggregation of mutant SERPINA1 molecules in the endoplasmic reticulum of hepatocytes, triggering liver injury. SERPINA1 deficiency is the most common genetic cause of hepatic disease in children and is frequently responsible for chronic liver disease in adults. Liver transplantation is currently the only available treatment for the severe form of the disease. We found that liver-directed gene transfer of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, results in marked reduction of toxic mutant SERPINA1 polymer, apoptosis and fibrosis in the liver of a mouse model of SERPINA1 deficiency. TFEB-mediated correction of hepatic disease is dependent upon increased degradation of SERPINA1 polymer in autolysosomes and decreased expression of SERPINA1 monomer. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease in SERPINA1 deficiency. Moreover, this study suggests that TFEB-mediated cellular clearance may have broad applications for therapy of human disorders due to intracellular accumulation of toxic proteins.
[Show abstract][Hide abstract] ABSTRACT: Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a primary immunodeficiency, predominantly characterized by agammaglobulinemia or hypoimmunoglobulinemia, centromere instability and facial anomalies. Mutations in two genes have been discovered to cause ICF syndrome: DNMT3B and ZBTB24. To characterize the clinical features of this syndrome, as well as genotype-phenotype correlations, we compared clinical and genetic data of 44 ICF patients. Of them, 23 had mutations in DNMT3B (ICF1), 13 patients had mutations in ZBTB24 (ICF2), whereas for 8 patients, the gene defect has not yet been identified (ICFX). While at first sight these patients share the same immunological, morphological and epigenetic hallmarks of the disease, systematic evaluation of all reported informative cases shows that: (1) the humoral immunodeficiency is generally more pronounced in ICF1 patients, (2) B- and T-cell compartments are both involved in ICF1 and ICF2, (3) ICF2 patients have a significantly higher incidence of intellectual disability and (4) congenital malformations can be observed in some ICF1 and ICF2 cases. It is expected that these observations on prevalence and clinical presentation will facilitate mutation-screening strategies and help in diagnostic counseling.European Journal of Human Genetics advance online publication, 13 March 2013; doi:10.1038/ejhg.2013.40.
European journal of human genetics: EJHG 03/2013; · 3.56 Impact Factor