The long term goal of this work is to create effective therapies for patients with acute promyelocytic leukemia (APML) who have failed conventional treatment protocols. To accomplish this goal, we will attempt to better understand the biology and immunology of APML by refining and expanding our """"""""first generation"""""""" transgenic mouse model of APML as follows:
Specific Aim 1. We will develop a """"""""knock-in"""""""" system for inserting novel cDNAs into the mouse cathepsin G (CG) locus, and precisely define the myeloid progenitor/precursor compartment in which targeted transgenes first are expressed. We will target an enhanced green fluorescent protein (eGFP) cassette into the murine CG 5' untranslated region (UTR) using homologous recombination, and create transgenic animals bearing this mutation. We will sort GFP positive and negative cells using flow cytometry, and examine these populations for their ability to form hematopoietic colonies in vitro and reconstitute hematopoiesis in vivo.
Specific Aim 2. We will create and analyze mice that express both PML- RARalpha and a GFP """"""""tag"""""""" in the same early myeloid cells, so that the biology and immunology of these cells can be characterized. We will use homologous recombination to insert a breakpoint 1-derived PML- RARalpha cDNA into the 5-HTR of the cathepsin expressing PML- RARalpha will therefore carry a fluorescent tag that will permit their purification and biological characterization. We will characterize the latency, penetrance, and phenotype of tumors developing in these animals, and characterize them immunologically in Specific Aim 4.
Specific Aim 3. We will create and analyze mice that express both RARalpha-PML and a GFP tag in early myeloid cells, and intercross them with the PML-RARalpha mice of Specific Aim 2. We will insert a reciprocal breakpoint 1 RARalpha-PML-IRES-eGFP cDNA into the murine cathepsin G 5'UTR, and define the phenotype of expressing cells. We will intercross these mice with animals containing the cathepsin G targeted PML-RARalpha- cDNA, and compare the latency, penetrance, and phenotype of tumors arising in PML-RARalpha, RARalpha-PML, or double transgenic mice.
Specific Aim 4. We will determine whether tumor specific immunity can be developed against APML cells derived from the mouse models. Our preliminary data suggest that immunity can be induced in vivo against APML tumors with a plasmid that expresses PML-RARalpha. In this Aim, we will further explore the type of immunity conferred by DNA vaccines, and will attempt to determine whether immunity can be developed against peptides spanning the PML-RARalpha-PML breakpoint regions.
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