643. Prolonged Expression of Human Alpha-1-Antitrypsin in C3H/Hej Mice with Helper-Dependent Adenoviral Vectors Irrespective of Tissue Distribution of Gene Expression

Abstract

We have recently constructed a novel HAC vector called 21ΔqHAC, generated by deletion of a distal portion of the long arm of human chromosome 21 and insertion of a loxP sequence to facilitate insertion of any desired gene construct. We intend to use this HAC vector to rescue T and B cell development and the radiosensitive phenotype in the DNA-PKcs-deficient SCID (severe combined immunodeficiency) mice. Our approach will involve introduction of 21ΔqHAC containing the DNA-PKcs gene into DNA-PKcs-deficient ES cells, differentiation of corrected ES cells into blood stem cells and transplantation of these blood stem cells into SCID mice. To evaluate the usefulness of 21ΔqHAC vector for this purpose, we first attempted in vitro studies using CHO cells (V3) exhibiting radiosensitive phenotype due to a DNA-PKcs deficiency. In this experiment, we inserted a ~21 kb doxycycline-inducible DNA-PKcs expression cassette into 21ΔqHAC vector by Cre-mediated recombination. The resulting 21ΔqHAC/DNA-PKcs construct was then introduced into the V3 cells. RT-PCR results from cells containing the 21ΔqHAC/DNA-PKcs vector showed doxycycline-dependent regulation of the DNA-PKcs gene. Furthermore, radiosensitivity assays showed significant phenotype rescue in these cells due to the production of functional DNA-PKcs protein. To our knowledge, our study is the first to report successful artificial control of gene expression by placing the multiple regulatory elements of the tetracycline system in a single expression cassette within a HAC vector. Our results also show that 21ΔqHAC is a very useful tool in the introduction of a therapeutic gene and the correction of a defective phenotype.