E-mail | Formatted Cover Letter |
TO WHOM IT MAY CONCERN
Please allow me to take this opportunity to introduce myself. I am Dr. Adriana Zingone, currently working at the National Cancer Institute at the National Institutes of Health (NIH) in Bethesda, Maryland.
I obtained my medical doctoral degree (M.D.) from The University of Reggio Calabria at Catanzaro in Italy with the highest mark. During my residence in the Unit of Internal Medicine, I was awarded a fellowship for clinical research by the Department of Education, Calabria Region. In 1988 I was admitted to the School of Specialty in Internal Medicine. From 1988 to 1991 I was a clinical staff fellow at the Department of Clinical and Experimental Medicine. During these years I was involved in several research projects. In particular, I studied the distribution of genetic risk factors for atherosclerosis in a southern Italian population, defining the association between different DNA polymorphisms and metabolic abnormalities such as hyperglycemia and hyperlipoprotemia. This experience further shaped my goals towards life sciences research, especially for the molecular mechanisms involved in human diseases. At that time I won a Fellowship awarded by the National Research Council (CNR). In 1992 I became a specialist in Internal Medicine.
In 1995 I was admitted to the Ph.D. program at the Department of Clinical and Experimental Medicine. With this opportunity I was trained under the supervision of Professor Nicola Perrotti and Professor Enrico Avvedimento to become a researcher in the field of Molecular Pathology. I developed a deep interest for molecular diagnosis and physiopathologic aspects of metabolic and genetic diseases including diabetes, dyslipidemias and cystic fibrosis. I was able to optimize a reverse P.C.R.-based method for genotype detection of Hepatitis C virus (HCV) in serum specimens from patients. This pathogenic virus is prevalent in several regions in the world, including the United States. By means of denaturing gel gradient electrophoresis, I was also able to identify mutations and polymorphisms in the gene coding for cystic fibrosis transmembrane regulator (C.F.T.R.) in patients with cystic fibrosis.
In the spring of 1997, I was proposed by several professors in my department to come to the United States and do research at the NIH while completing my PhD. This work was done from 1997 to 2000 in the laboratory of Dr. Janice Chou at the NICHD (National Institute of Child Health and Human Development), the National Institutes of Health (NIH). I was responsible for a challenging project concerning the feasibility of gene replacement therapy in a murine model of Glycogen Storage Disease type 1a (GSD-1a).
Glycogen Storage Disease is an autosomal recessive disorder with an overall frequency of approximately 1 in 100,000 live births, caused by a deficiency of glucose-6-phosphatase (G6Pase). The enzyme, G6Pase, is necessary for glucose to be released from the liver's glycogen stores into the blood and therefore, to maintain the glucose homeostasis in the body. Patients affected with this disease can die by hypoglycemic coma if they do not receive continuous infusion of glucose or frequent oral administration of uncooked cornstarch. The liver is the organ most damaged, which with time can also be invaded by tumors. Thus, the glucose-6-phosphatase deficient homozygous mouse was a perfect model to test a novel therapeutic approach by gene therapy to change the prognosis and the survival of these patients.
I was able to generate a high-titer recombinant defective adenovirus vector expressing the coding region of glucose-6-phosphatase and inject it into two-week old G6Pase-deficient mice. The therapy was successful, as determined by improved growth, longer lifespan and reversal of the metabolic abnormalities of the disease. An interesting experience of this period was mice PET imaging, in collaboration with Dr. Mike Green of the Nuclear Medicine Unit at NIH. We were able to monitor the ability of the livers of glucose-6phosphatase-deficient mice to release the labeled glucose before and after treatment. Based on the work I have done in Dr. Chou's lab, I published two first-author and one second-author peer-reviewed research papers in highly respected journals, including the Journal of Biological Chemistry, Life Science and European Journal of Pediatrics. These results were also included in the thesis for my doctoral degree. In addition, I was invited to give seminars at the annual meeting of the American Glycogen Storage Disease Association, as well as at the Italian Glycogen Storage Disease Association meeting. At this time we established a collaboration with Dr. Barry Byrne (University of Florida - Gainesville), who provided us with an Adeno-Associated Virus (AAV), which I successfully tested in newborn mice. I also started a collaboration, which is still active, with Dr. Suresh Arya at the National Cancer Institute (NCI) to design a lentiviral vector carrying the glucose-6-phosphatase cDNA and also a control vector expressing a reporter gene (EGFP).
In 2000 I obtained my Ph.D. degree in Oncology and returned to Italy, where I worked as a scientist at the G. Gaslini Institute in Genoa. I was responsible for setting up a section of the Molecular Biology Laboratory, directed by Dr. Luigi Varesio, orientated to continuing gene therapy in mouse models using lentiviral vectors. In addition, I trained and supervised students and a post-doc assigned to the project. My position in the laboratory also included the preparation of a grant proposal that we submitted in 2001 to the "Telethon" organization. In addition, my work in the laboratory contributed to publication of a Gene Therapy manuscript.
Since I enjoyed my experience at the NIH, in 2002 I decided to return in order to improve my knowledge on new generations of viral vectors and transgenic mouse skills. I was accepted as a Visiting Fellow in the laboratory of Dr. Michael Kuehl at the National Cancer Institute where I am currently working. In 2003 I was promoted to Research Fellow. Under the guidance of Dr. Michael Kuehl, an internationally recognized expert on the molecular pathogenesis of multiple myeloma, I have been involved in a series of projects to understand the genetic abnormalities causing multiple myeloma (MM). This malignant tumor of terminally differentiated B lymphocytes (plasma cells) is located at multiple sites in the bone marrow, where it can cause severe bone lesions, anemia, and enhanced susceptibility to infectious agents. Multiple myeloma continues to be an incurable tumor that is responsible for approximately 12,000 deaths each year in the United States, or nearly 2% of all cancer deaths. Despite a number of new therapies, the three to four year median survival of patients with myeloma has increased very little during the past several decades.
My research has involved a number of related projects that are attempting to identify and characterize somatic genetic abnormalities that are found in MM tumor cells. For these studies, I have taken advantage of my previous experience with retroviruses. The first set of projects attempts to determine which genetic abnormalities affect the survival and growth of human myeloma tumors. To accomplish this, I have used lentiviral vectors to increase or inhibit expression of specific genes in human multiple myeloma cell lines, and have then determined the resultant changes in survival and growth of the cells including in vivo experiments in xenograft models.
A second set of projects is designed to create cell lines from a specific group of myeloma tumors for which it has not yet proven possible to generate cell lines. We expect that this will be possible by transducing the tumor cells with lentiviruses containing genes that have been shown to inactivate tumor suppressor genes. Starting with bone marrow aspirate of patients with MM, a number of genes are being introduced into purified tumor cells (singly or in combination, such as hTERT, mutant c-Myc, RAS, HPV16E6/E7, SV40 Large T and SV40 small T. Our working hypothesis is that MM tumor cells would have a high probability of generating cell lines if they can express the oncogenes and viral genes that generally have been able to transform many kinds of cultured primary cells. The availability of cell lines from this group of myeloma tumors should prove to be an invaluable resource for improving our understanding of how this particular type of myeloma tumor has developed the ability to survive and proliferate. I am currently characterizing the MM tumor cells and transduced cell lines by Flow Cytometry Analysis, Immunocytochemestry, RT-PCR and Immunoblot.
A third set of projects is designed to develop a model that faithfully reproduces all of the abnormalities seen in human MM tumor cells. We are pursuing this goal by culturing normal human peripheral blood cells so that they differentiate into plasmablasts, which can then be transduced with a retrovirus to generate an immortalized normal plasmablast cell line. The plasmablast cell line would then be transduced with additional retroviruses, each of which includes a gene that is altered in myeloma, to determine how many and which genes need to be altered to convert a normal plasmablast into a multiple myeloma tumor cell. Knowing the molecular events underlying this cancer is a critical step for developing new therapeutic strategies to improve survival from this presently incurable malignancy.
One of the rewarding experiences working in Dr. Kuehl's laboratory has been my involvement in collaborations with several distinguished investigators including Dr. Herbert Morse (NIAID) and Dr. Janz Siegfried (University of Iowa) to engineer and analyze transgenic mice reproducing some of the features of the myeloma tumors (manuscript in preparation).
Sincerely,
Adriana Zingone, MD, PhD