C. Anthony Blau
Associate Professor of Medicine, Hematology
Adjunct Associate Professor of Genome Sciences

Our lab has focused on developing gene therapy strategy known as “in vivo selection”. The backdrop to this work is that the inefficiency of gene transfer into stem cells is a bottleneck for most applications of gene therapy. In vivo selection attempts to give genetically modified cells a preferential growth or survival advantage relative to unmodified cells. Historically, many different labs have tried to accomplish in vivo selection by transferring a drug resistance gene into hemopoietic cells. After the cells are transplanted back into the animal, cytotoxic chemotherapy is administered to produce a selective killing of the non-transduced cells, and a concomitant rise in the transduced cells. Many attempts using this approach have been described, including the use of genes encoding MDR1, dihydrofolate reductase, methylguanine methyltransferase or other drug resistance genes. A major problem with this approach resides on the hematological and non-hematological toxicities associated with chemotherapy administration.

In 1997 we published a paper (PNAS 94:3076-3081) that described a completely different approach to achieving in vivo selection. In contrast to the negative selection strategy described above, our approach employed positive selection, using modified growth factor receptors to induce cell growth in response to a class of small molecule drugs called chemical inducers of dimerization (CIDs). Our initial report described the creation of a CID-dependent cell line. This paper received a commentary in Science (276:1891, 1997).

Since then we have taken a systematic approach in moving this strategy toward an eventual clinical trial. In 1998 we showed that mouse primary bone marrow cells, equipped with a retroviral vector encoding a derivative of the thrombopoietin receptor, could be stimulated to divide in response to FK1012 (PNAS 95:8093-8097). In 2000 we showed that the same approach allowed for the in vitro outgrowth of transduced primary human cord blood CD34+ cells (Blood 95:430-6). We subsequently demonstrated the utility of this system for in vivo selection using a mouse model (Nature Genetics 26:64-66, 2000) and recently in collaboration with H-P Kiem, in a dog model (Blood 100:2026-2031, 2002).

Defining the signals that specify stem cell self-renewal (Zeng et al., Blood 98:328-334, 2001; Zhao et al., EMBO J 21:2159-2167, 2002). Defining signals that can support the self-renewal of multipotential hemopoietic progenitor cells (MHPCs) is pertinent to understanding leukemogenesis and may be relevant to developing stem cell-based therapies. We have defined a set of signals, JAK2 plus either c-kit or flt-3, which together can support extensive MHPC self-renewal. Phenotypically and functionally distinct populations of MHPCs were obtained, depending on which receptor tyrosine kinase, c-kit or flt-3, was activated. Self-renewal was abrogated in the absence of STAT5a/b, and in the presence of inhibitors targeting either the mitogen activated protein kinase (MAPK) or phosphotidylinositol 3' kinase (PI-3K) pathways. These findings suggest that a simple two-component signal can drive MHPC self-renewal.

The first clinical gene therapy trial using chemical inducers of dimerization. In conjunction with Stan Riddell and our collaborators at ARIAD Pharmaceuticals we are in the process of preparing to perform a clinical gene therapy trial in relapsed leukemia. Donor lymphocyte-mediated anti-tumor effects represent the single most important therapeutic benefit of allogeneic bone marrow transplantation (BMT). Paradoxically, donor lymphocyte-mediated Graft versus Host Disease (GVHD) represents the single greatest toxicity of allogeneic BMT. Over the past decade a large body of research has focused on harnessing the therapeutic potential of donor lymphocyte infusions while avoiding the development of life threatening GVHD. This proposal describes a pilot study that will test a new system that allows the survival of infused donor lymphocytes to come under pharmacological control. Donor lymphocytes are equipped with a suicide gene. In the past, HSV thymidine kinase has been used for this purpose, however the immunogenic nature of the HSV-TK protein will severely impede the use of this gene in future gene therapy trials. In order to reduce the likelihood of immunogenicity, it would be highly desirable to employ a suicide gene encoding a protein that is completely human in origin. Our collaborators at ARIAD Pharmaceuticals have developed such a system based on the human cell surface receptor Fas, which naturally signals apoptosis (programmed cell death) in T lymphocytes. Fas signaling is normally initiated by clustering of the receptor by its ligand, leading to a cascade of cytotoxic events. In the ARIAD system, clustering of an artificial Fas receptor (introduced by gene transfer) and consequent cell death is brought under the control of a small molecule drug. Binding of this "dimerizer" clusters the chimeric Fas receptors and initiates the natural apoptotic cascade. All the protein components of this system are human in origin, therefore the peptide sequences at the fusion sites and a point mutation in FKBP12 represent the only potentially immunogenic sequences.

Other applications of the dimerizer system. We have embarked on a series of collaborative studies to determine the utility of using chemical inducers of dimerization to stimulate expansion of genetically modified liver cells (with Andre Lieber), and muscle cells (with Charles Murry). If successful, these approaches might prove useful for treating liver diseases, diabetes, and other disorders.

Preliminary studies for gene therapy in sickle cell disease and b thalassemia. In order to perform gene therapy for these disorders, we will need to procure stem cells. The most widely used method for obtaining stem cells is to promote their mobilization into the peripheral blood using the cytokine GCSF so that they can then be collected by leukapheresis. We have previously shown that GCSF can produce life-threatening complications in patients with sickle cell disease (Lancet, 1998).

Our experience with GCSF strongly suggests that alternative means will need to be found for the procurement of stem cells in patients with sickle cell disease. A protocol to study the use of hydroxyurea for the mobilization of progenitors in patients with sickle cell anemia has been approved by the GCRC’s Scientific Advisory Committee and the UW Human Subjects Committee, and has enrolled three patients. A manuscript describing our initial findings is in preparation.

Development of a clinical gene therapy program at the University of Washington. As Associate Program Director for Gene and Cell Therapy at the GCRC I have taken part in the effort to establish a world class gene therapy program at UW. In recent months I have been coordinating with gene therapy investigators to implement their programs upon opening of the GCRC’s Gene Therapy Core Laboratory, which opened October, 2000. I am also Director of the Clinical Core for the Program for Excellence in Gene Therapy, and am building the infrastructure for clinical gene therapy trials at UW.

Publications:

Jin L, Siritanaraktul N, Emery DW, Richard RE, Kaushansky K, Papayannopoulou Th and Blau CA: Targeted expansion of genetically modified bone marrow cells. Proceedings of the National Academy of Sciences USA 95:8093-8097, 1998.

Jin L, Zeng H, Otto KG, Richard RE, Emery DW and Blau CA: In vivo selection using a cell growth switch. Nature Genetics 26:64-66, 2000.

Zeng H, Masuko M, Jin L, Neff T, Otto KG and Blau CA: Receptor specificity in the self-renewal and differentiation of primary multipotential hemopoietic cells. Blood 98:328-334, 2001.

Zhao S, Zoller K, Masuko M, Rojnuckarin P, Yang X, Parganas E, Kaushansky K, Ihle JN, Papayannopoulou Th, Willerford DM, Clackson T and Blau CA: JAK2, complemented by a second signal from c-kit or flt-3, triggers extensive self-renewal of primary multipotential hemopoietic cells. EMBO Journal 21:2159-2167, 2002.

Neff T, Horn PA, Valli VE, Gown AM, Wardwell S, Wood BL, von Kalle C, Schmidt M, Peterson LJ, Morris JC, Richard RE, Clackson T, Kiem HP, Blau CA: Pharmacologically regulated in vivo selection in a large animal. Blood 100: 2026, 2031, 2002.

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