Abstract
Congenital blood disorders such as hemoglobinopathies and thalassemias are common, and yet clinically challenging glob in disorders. Gene therapy was thought to serve as a therapeutic method to treat these disorders through the introduction and expression of a transgenic globin gene. While tremendous advances have been made in vivo, allowing for sickle cell correction in mouse models, gene delivery protocols and vector prototypes still require modification. Vectors have yet to deliver the appropriate level of expression necessary for complete sickle cell amelioration. Alternative gene delivery vector systems derived from VL30 retroelements have been designed in the case of this study, and have proven to be effective in expressing a tissue- specific transgene expression, in vitro. Activation of the erythropoietin (epo)-induced VL30 elements was monitored in two mouse erythroid progenitor cell lines, MEL 585S and ELM-1-1. Reverse transcription of a variety of VL30 epo-responsive promoters, and ribonuclease protection of two VL30 promoters, ELM5 and MEL/ELM, confirmed the epo responsiveness of these clones. These promoters were inserted into a VL30-derived expression vector and were intended to be delivered through standard gene therapy delivery systems back into the ELM cells. Low titers hindered these viral deliveries, requiring stable transfection into the erythroid progenitor cells. Quantitative analysis of the (3-galactosidase reporter gene activity of ELM 5, a BVL-1 like VL30 promoter, showed that both VL30 promoters showed conservative epo enhanced reporter gene expression, and was capable of expressing sustained levels of the (3 galactosidase transgene over a sixteen week assay period. These findings support previous in vivo studies and delineates the potential incorporation of these retroelement promoters as transcriptionally active, erythroid specific, auxiliary LTR components, for current globin vector constructs.