Associate Investigator, TIGEM
Head of Cell Culture and Cytogenetics Core, TIGEM
Associate Professor, Department of Translational Medicine, University of Naples "Federico II', Italy
Hepatocyte Gene Therapy
The overall goal of our research is to provide solutions to biologically relevant questions and to generate preclinical data that will lead to the development of new clinical therapies for patients with inborn errors of metabolism. Gene transfer and small molecules are hopeful strategies for future therapies, and our research focuses on investigating both of these approaches.
The liver is an attractive organ for gene therapy; hepatocyte genes have long been appreciated because their long-term expression provides a major opportunity for treatment, or perhaps, even a cure for several inborn errors of metabolism. Our efforts are currently focused on developing safe and effective gene therapy for primary hyperoxaluria type 1, Crigler-Najjar syndrome, and alpha1-antitrypsin (AAT) deficiency. To achieve these goals, we are investigating multiple gene therapy platforms, including helper-dependent adenoviral (HDAd) and adeno associated viral (AAV) vectors.
Small Molecules for Inborn Errors of Metabolism
Another one of our laboratory’s research interests is investigation of small molecules, orally bioavailable drugs, for the treatment of inborn errors of metabolism. We are evaluating the use of small molecules for the treatment of two diseases, AAT deficiency, one of the most common genetic causes of liver disease in children, and pyruvate dehydrogenase complex (PDHC) deficiency.
The most common genetic lesion in AAT deficiency results in a mutant protein, which forms aggregates that accumulate within the endoplasmic reticulum, ultimately causing liver injury. We are investigating drugs to increase the cellular clearance of toxic undegraded proteins. For PDHC deficiency, we are currently testing drugs acting on PDHC regulation to enhance the residual enzymatic activity. In collaboration with Dr. di Bernardo we are also searching for candidate drugs for both disorders by using a novel systems biology approach based on the analysis of drug expression profiles
Cell Culture Facility
The Cell Culture Facility provides fully-equipped cell culture facilities and technical support for cellular and cytogenetic studies to TIGEM investigators.
In the last years, research at TIGEM has confirmed that collection of cell lines from individuals with genetic disorders is essential in order to test candidate genes and to perform functional studies. These cell lines have been collected and organized into a database, which includes a wide variety of immortalized cell lines, as well as fibroblast and lymphoblast cell lines from patients with various genetic diseases. This collection has been useful in providing readily available sources of DNA/RNA for projects organized for the investigation of pathogenetic mechanisms.
The Core is responsible for the establishment, maintenance, and centralized database storage of these cell lines. In addition, the Core offers cytogenetic services such as chromosome analyses (standard and high resolution chromosome analysis) and fluorescence in situ hybridization (FISH) on cell lines, peripheral blood samples, embryonic stem cells, induced pluripotent stem (iPS) cells, as well as directly on tissue samples. Lastly, the Core provides mouse primary hepatocytes cultures and performs screening and treatment processes for detection and removal of Mycoplasma contamination, respectively.
Gene transfer vectors are widely used in preclinical studies in disease animal models and as research tools to achieve gene transfer in vitro and in vivo. There is no single vector that is adequate for all applications, and the gene transfer vector has to be carefully chosen depending on disease, targeted cell type, number of treatments required, and the size and nature of the gene to be delivered.
Viral vectors were the first to be developed, as they exploit the natural property of viruses to transfer their genetic material into the nucleus of infected cells. Vectors derived from DNA viruses do not usually integrate into the host genome and are not replicated at cell division. Therefore, long-term transgene expression from DNA virus-derived vectors is restricted to post-mitotic cells.
Our Vector Core is devoted to providing research grade adeno-associated viral (AAV) vector and adenoviral (Ad) vectors to TIGEM researchers at low cost with a short turn-around time.
Pastore N, Nusco E, Piccolo P, Castaldo S, Vaníkova J, Vetrini F, Palmer D, Vitek L, Ng P, Brunetti-Pierri N (2013). Improved efficacy and reduced toxicity by ultrasound-guided intrahepatic injections of helper-dependent adenoviral vector in Gunn rats. Hum Gene Ther Methods. 24(5):321-7. doi: 10.1089/hgtb.2013.108.
Piccolo P, Vetrini F, Mithbaokar P, Grove NC, Bertin T, Palmer D, Ng P, Brunetti-Pierri N (2013). SR-A and SREC-I Are Kupffer and Endothelial Cell Receptors for Helper-dependent Adenoviral Vectors. Mol Ther. 21(4):767-74. doi: 10.1038/mt.2012.287.
Ferriero R, Manco G, Lamantea E, Nusco E, Ferrante MI, Sordino P, Stacpoole PW, Lee B , Zeviani M, Brunetti-Pierri N (2013). Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis. Sci Transl Med. 5 (175): 175ra31. doi: 10.1126/scitranslmed.3004986.
Pastore N, Blomenkamp K, Annunziata F, Piccolo P, Mithbaokar P, Maria Sepe R, Vetrini F, Palmer D, Ng P, Polishchuk E, Iacobacci S, Polishchuk R, Teckman J, Ballabio A, Brunetti-Pierri N (2013). Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency. EMBO Mol Med. 5(3):397-412. doi: 10.1002/emmm.201202046.
Pastore N, Nusco E, Vaníkova J, Sepe RM, Vetrini F, McDonagh A, Auricchio A, Vitek L, Brunetti-Pierri N (2012). Sustained reduction of hyperbilirubinemia in Gunn rats after adeno-associated virus-mediated gene transfer of bilirubin UDP-glucuronosyltransferase isozyme 1A1 to skeletal muscle. Hum Gene Ther. 23(10):1082-9. doi: 10.1089/hum.2012.018.