We have developed a yeast based three-component system called “chemical complementation” that links the survival of cells to the presence of a small molecule. In chemical complementation a small molecule enables yeast to grow by binding a nuclear receptor, triggering expression of an essential gene. The interaction between the small molecule ligand and the receptor provides an effective, general system for genetic selection. We use chemical complementation to engineer new receptors that activate transcription in response to chosen small molecules. Structure-guided codon randomization produces libraries with hundreds of thousands of variant receptors. Chemical complementation evaluates all the library members in parallel- only yeast harboring functional receptors grow. This system is tunable, i.e. the stringency can be varied. Variable stringency chemical complementation is used to adjust the transcription activation threshold required for yeast growth. Using this method while varying the ligand concentration permitted identification of receptors with high efficacy (high levels of gene expression) and low EC50s. Data from several libraries each comprising hundreds of thousands of members will be presented. We are developing the engineered orthogonal ligand-receptor pairs for use in gene therapy. Our overall objective is to develop recombinant gene transfer systems with two features: tight control of transgene expression and inducible transgene expression. Specifically, we are developing vectors that expand populations of multipotent hematopoietic stem cells (HSCs) both ex vivo and in vivo while maintaining their undifferentiated state. Using the methods described above we have generated a series of fusion proteins that combine the genetically engineered ligand binding domain specific for a synthetic "near drug" and a DNA binding domain specific for a DNA sequence not found in the mammalian genome (i.e. the Gal4 response element). By cloning cDNA sequences downstream of the response element we have achieved control over transcription of several cDNA sequences. Our strategy has many advantages over currently available systems, including tight control over transgene expression and high selectivity of the engineered ligand binding domain. Using this system, we have designed and tested first generation retroviral constructs that will allow controlled transgene expression in genetically engineered cells. Our goals are to determine the extent that control of proviral sequences can be genetically engineered into retroviral constructs and to test the effectiveness of our inducible systems to regulate the controlled expression of cDNA sequences that regulate the growth of multipotent HSCs. We have successfully controlled the expression of luciferase and GFP from retroviral vectors in NIH3t3 cells, 293 cells, and HeLa cells. We continue to develop and assess the effectiveness of orthogonal promoters that express reporter genes (luciferase and GFP) as well as HoxB4, which has been shown to be important in the expansion of HSCs. Our future aim is to test regulated expression of HoxB4 in HSCs transduced with our inducible vectors to enforce HSC expansion in vivo.

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