Pilot Project Grant Program Awardees

2006-2007 Pilot and Feasibility Grant Winners

• Peter Glazer, M.D., Ph.D.
  Professor of Therapeutic Radiology and Genetics
  Chairman of Therapeutic Radiology
  Targeted Correction of the Human b-Globin Gene
• Melissa Kacena, Ph.D.
  Assistant Professor of Orthopaedics
  Translational study of megakaryocyte GATA-1 missense mutations and bone density
• Peining Li, Ph.D.
  Assistant Professor of Genetics
  Lineage-oriented molecular cytogenetic approach and clone-based genomic analysis for hematologic disorders
• E. Scott Swenson, M.D., Ph.D.
  Instructor in the Department of Internal Medicine, Section of Digestive Diseases
  Liver injury and repopulation by bone marrow stem cells

2006-2007 WINNERS' ABSTRACTS

Peter Glazer, MD, PhD
Professor of Therapeutic Radiology and Genetics
Chairman of Therapeutic Radiology

Targeted Correction of the Human b-Globin Gene
The ability to genetically manipulate human stem cells has the potential to enhance the utility of stem cell therapy for human disease. One emerging approach to targeted genome modification is the use of triplex-forming oligonucleotides (TFOs). These molecules bind to duplex DNA in a sequence-specific manner, and this binding can be used to stimulate recombination in mammalian cells. The overall hypothesis driving this application is that high-affinity, site-specific DNA binding by TFOs can be used as a tool to accomplish gene targeting in stem cells. Our long-range goal is to develop such molecules as reagents for targeted genome modification of disease-related genes in human stem cells. In Aim 1, we will develop TFOs to bind to the human b-globin gene. We have identified two sites in intron 2 of the b-globin gene (IVS2-24 and IVS2-64) that are amenable to triplex formation. We have developed and tested a series of TFO chemical modifications to optimize binding to these sequences under physiologic conditions. Modifications to be tested include 2’-O-aminoethyl (2’AE), 8-oxoadenine (8oxoA), and 7-deaza-8-azaguanine (7c8nG) substitutions. We will also develop peptide nucleic acids(PNAs) specifically targeted to b-globin sequences. In Aim 2, we will test b-globin-directed TFOs and PNAs to target b-globin gene sequences in specially designed reporter gene assays in cell lines as well as in primary cells.

Melissa Kacena, Ph.D.
Assistant Professor of Orthopaedics

Translational study of megakaryocyte GATA-1 missense mutations and bone density
A powerful new regulatory pathway between megakaryocytes and bone cells identified in mice also exists in the human correlate, according to translational research. Mice deficient in GATA-1, a transcription factor required for megakaryocyte development, have increased numbers of immature megakaryocytes (10 to 100-fold increase), a drastic reduction of platelets (15% of control level), and an unanticipated three-fold increase in bone volume. Recently, eight families with GATA-1 missense mutations have been identified, having hematological phenotypes similar to those seen in GATA-1 deficient mice. It is our hypothesis that humans with mutations in GATA-1 develop a high bone mass phenotype similar to that in GATA-1 deficient mice. We intend to evaluate the bone density in GATA-1 patients and characterize their biochemical markers of bone turnover. Next, we will investigate the osteoclastogenic potential of peripheral blood mononuclear cells from these patients. These studies are relevant to megakaryocyte associated diseases such as thrombocytopenia and idiopathic myelofibrosis, new pathways of bone mass regulation, and bone diseases such as osteoporosis and osteopetrosis.

Peining Li, Ph.D.
Assistant Professor of Genetics

Lineage-oriented molecular cytogenetic approach and clone-based genomic analysis for hematologic disorders
This pilot study will develop new FISH (fluorescence in situ hybridization) probes and validate an antibody-captured cell array for lineage-oriented in situ cell culture and FISH testing.

There are more than 250 known chromosome rearrangements in various leukemias and only 40-50 probes are available from commercial sources. With the completion of the Human Genome Project, gene-specific BAC (bacterial artificial chromosome) clones are readily available to cover all major recurrent multi-partner translocations (MPTs) and chromosome deletions. These probes can be organized into test panels for the screening and detection of lineage-specific chromosome rearrangements and clonal evolution pathways.

Recent reports have demonstrated that microarrayed antibodies can be used to capture cells expressing surface antigens. To test the technical feasibility and clinical validity of this cell array technology in in situ karyotyping, FISH screening, and genomic analysis, antibody array prototypes will be constructed in capture lymphoid cells (T and B cells) and myeloid cells. The affinity of antibody-cell binding, specificity of cell capture, and feasibility of in situ cell karyotyping and direct FISH testing will be evaluated.

Successful completion of this pilot study will lead to further development of a lineage-oriented molecular cytogenetic approach and a clone-based genomic analysis for patients with hematological disorders.

E. Scott Swenson, M.D., Ph.D.
Instructor in the Department of Internal Medicine, Section of Digestive Diseases

Liver injury and repopulation by bone marrow stem cells
Cell therapy may be an alternative to liver transplant. Surprisingly, adult bone marrow contains cells capable of engrafting the liver and treating disease. The studies proposed here are designed to distinguish between mechanisms of engraftment and differentiation of bone marrow cells into functional liver cells. Cells from normal bone marrow can replace up to half of the diseased liver in the FAH knockout mouse model of hereditary tyrosinemia. The repair mechanism involves fusion events between marrow-derived cells and diseased liver cells. However, evidence from other models indicates that liver cells can arise from bone marrow cells in the absence of fusion with host cells.

A Cre-Lox reporter system will be used to test the hypothesis that marrow-derived hepatocytes can arise through cell-cell fusion or through direct differentiation, using FAH knockout mice or wild type mice subjected to subsequent injury with a liver toxin or focal liver irradiation. Using this binary reporter system, marrow-derived cells ubiquitously express β-galactosidase. However, fusion of a transplanted, marrow-derived cell with a host (Cre+) cell results in excision of the lacZ cassette and activation of GFP expression. Thus cells arising through direct differentiation will be lacZ+, while cells arising through cell-cell fusion will be GFP+.