In Memoriam

Alan M. Gewirtz, MD

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Department: Medicine

Contact information
University of Pennsylvania Medical School
421 Curie Blvd.
Philadelphia, PA 19104
Office: 215-898-4499
Fax: 215-573-2078
A.B. (Marine Biology)
Colgate University, 1971.
M.D., M.A.
State University of New York at Buffalo, 1976.
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Description of Research Expertise

Research Interests

Key words: Oligodeoxynucleotides, Antisense, Hematopoiesis, Human Hematopoietic Cell Development, c-myb, RNA Inference.

Description of Research
The focus of my laboratory has been the cell biology of normal and malignant human hematopoiesis. The laboratory has had a long standing interest in the extracellular regulation of human hematopoietic progenitor cell growth, in particular cells of the megakaryocyte lineage. We have also played a prominent role in the development of "antisense" gene squelching methods which allow the role of specific genes in regulating blood cell development to be investigated. Most recently, we have received funding from the National Space Biomedical Research Institute to study the effects of the deep space radiation environment on hematopoietic cell development. It is our ultimate goal to use the knowledge gained in these studies to advance the development of more effective, and less toxic therapies for human leukemia.

Rotation Projects for 2006-2007

Project #1: Disrupting the expression of hematologically relevant protooncogenes with antisense oligodeoxynucleotides (ODN) can yield functionally informative data with important translational significance. Based on work of this type, we have begun clinical trials with an antisense molecule targeted to the myb gene. Though our trials are still in Phase I, it is clear to us that while myb is a rational target, it is still less than perfect for treatment of hematopoietic malignancies because it is expressed by both normal and malignant cells. We propose to address this problem by developing a more in-depth knowledge of Myb biology in normal and malignant hematopoietic stem/progenitor cells. In so doing, our long term goal of making Myb-directed therapeutic strategies more rational, and more specific will be advanced. We have developed three specific aims which support our goal. These aims are as follows: 1. Identify Myb-regulated genes and the mechanism whereby these targets are transactivated: The myb gene encodes a transcription factor which is of critical importance in the development of both normal and malignant hematopoietic cells. We intend to identify physiologically significant targets of the Myb transcription factor using differential screening, expression assays, deletion and mutation assays, gel shifting/foot printing, and antisense disruption. New, potentially tumor specific targets for antisense mediated inhibition of tumor growth may be identified in this manner; 2. Further define how Myb functions by identifying interacting proteins: The activity of many transcription factors is regulated by interaction with other nuclear proteins. MYC-MAD-MAX, MYO-D-ID, p53-mdm2 are well known examples. Because the Myb protein has domains which are permissive of such interactions, we hypothesize that Myb function is also regulated by interaction with as yet unidentified binding partners. Using standard screening approaches, and a novel method based on flow cytometric screening for protein expression, we will identify physiologically relevant Myb binding partners. Novel therapeutic targets may also be discerned by this line of investigation; 3. Determine the effect of integrin-mediated lymphocyte adherence on c-myb expression: Integrin-mediated cell adhesion, through a process known as "outside-in" signaling, initiates intracellular signal transduction pathways that can modulate gene expression. Based on the important role played by myb in hematopoietic cell development, we hypothesize that integrin mediated interaction of hematopoietic cells with components of the extracellular matrix will modulate myb expression and the consequent downstream events which regulate hematopoietic development.

Project #2: Develop A Rational Method For Targeting Antisense Nucleic Acid (ASNA), and Models for Testing Their Behavior In Vivo. It is likely that improvements in ODN design will be required before highly reproducible and efficient modulation of gene expression is observed. In this context, we are discussing the targeting of ASNA molecules, including siRNA. It is straightforward that ASNA can only be effective if they hybridize with their mRNA target. Hybridization requires a single stranded loop of RNA but the location of such loops in a live cell is virtually impossible to predict because of the complexity of RNA folding in vivo and the association of RNA with intracellular proteins that may also block ODN access. To address this issue, we have developed self-quenching reporter molecules (SQRM) that signal only after hybridization to their target. Preliminary data using this fluorescence based system to probe c-myb mRNA suggest that it may be possible to map hybridization accessible sites in a rapid, "high throughput "manner. Based on RNA mapping studies, antisense nucleic acids will be synthesized and their ability to cleave mRNA targets, in comparison to "random" ODN will be evaluated in vitro, and in vivo using "wild type" leukemia cells obtained from our tissue bank core. This work should lead to development of candidate antisense molecules with enhanced gene modulating activity. We will test the activity of these molecules against wild type leukemia cells obtained from the tissue bank core in a NOD/SCID animal model system. Uptake, distribution, and clearance of the molecules from various fluid and organ compartments will be studied using a variety of hybridization and chromatographic techniques. Data will be analyzed as a function of time, dose, and schedule of drug administration in order to facilitate development of rational drug delivery and treatment protocols. Based on these data, as well as pharmacokinetic and pharmcodynamic data generated by Aim#1, we will then develop and test delivery strategies with the goal of having an improved therapeutic, and the means to deliver it to patients, by the end of the first 5 years of the SPORE's funding.

Lab personnel:
Anna Kalota - Visiting Scholar
Yuji Nakata - Visiting Scholar
Cezary Swider - Visiting Scholar
Ravi Ralla palli - Postdoctoral Fellow
Susan Shetzline - Postdoctoral Fellow
Jyo Swaminathan - Postdoctoral Fellow
Steve Rudnick - PhD Student

Selected Publications

Selina M. Luger, Stephen G. O'Brien, Janina Ratajczak, Mariusz Z. Ratajczak, Rosemarie Mick,Edward A. Stadtmauer, Peter C. Nowell,, John M. Goldman, and Alan M. Gewirtz: Oligodeoxynucleotide Mediated Inhibition of c-myb Gene Expression in Autografted Bone Marrow: A Pilot Study. Blood 99: 1150-1158, 2002.

Rizzo J, Zhang X, Gewirtz AM and Lu P: Chimeric RNA-DNA Molecular Beacon Assay for Ribonuclease H Activity. Molecular Probes. Rizzo J (eds.). 16: 277-283, 2002.

Ptasznik A, Urbanowska E, Chinta S, Costa MA, Katz B, Stanislaus MA, Kokhan D, Linnekin D, Pan ZK, Gewirtz A: Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells. J. Exp. Med 196(5): 667-678, 2002.

Cohen AD, Luger SM, Sickles C, Mangan PA, Porter DL, Schuster SJ, Tsai DE, Nasta S, Gewirtz AM, Stadtmauer EA: Gemtuzumab ozogamicin (Mylotarg) monotherapy for relapsed AML after hematopoietic stem cell transplant: efficacy and incidence of hepatic veno-occlusive disease. Bone Marrow Transplant 30(1): 23-28, 2002.

Opalinska J, and Gewirtz, AM: Nucleic Acid Therapeutics:Basic Principles and Recent Applications. Nat Rev Drug Disc 1(7): 503-514, July 2002.

Opalinska JB, Gewirtz AM: Nucleic acid therapeutics: a work in progress. Curr Opin Investig Drugs 3(6): 928-933, June 2002.

Pertusini E, Ratajczak R, Majka M, Vaughn,D, Ratajczak MZ, Gewirtz AM. : Investigating the Platelet Sparing Mechanism Of Paclitaxel/Carboplatin Combination Chemotherapy. Blood 97: 638-644, 2001.

Choi JK, Hoang N, Vilardi AM, Conrad P, Emerson SG, and Gewirtz AM: Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34+ hematopoietic cells. Stem Cells 19: 236-246, 2001.

Marcin Majka, Anna Janowska-Wieczorek, Janina Ratajczak, Karen Ehrenman, Zbigniew Pietrzkowski, M. Anna Kowalska, Alan M. Gewirtz, Stephen G. Emerson, and Mariusz Z: Ratajczak. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner. Blood 97: 3057-3085, 2001.

Jen KY., Gewirtz AM.: Suppression of gene expression by targeted disruption of messenger RNA: available options and current strategies. [Review] [118 refs] Stem Cells 18(5): 307-19, 2000.

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Last updated: 08/05/2010
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