This application proposes an inquiry into the causes of the recurrent problems which have frustrated the application of gene correction therapies to the treatment of hemophilia as well as other genetic disorders which might be responsive to somatic gene therapy approaches. Some of these problems, such as limitations in vector capacity and titer, are rooted in the retrovirus-based gene delivery systems which represent the current standard for gene replacement therapy. Others, such as the inability to achieve a stable high rate of synthesis of an exogenous gene product, may be a consequence of a poorly understood general mechanism leading to extinction of ectopic gene expression. In this proposal we have tried to identify and address the underlying causes of obstacles to both gene delivery and expression which have obstructed the application of gene therapy to the treatment of hemophilia B today. A newly developed class of episomal expression vectors, based on the Epstein- Barr virus latent origin of replication, will be used to express multiple gene products from independent transcription units to facilitate expression and regulation of factor IX and the processing proteins that influence its biological activity. To further enhance gene expression, a synthetic factor IX gene will he created with the optimal codon usage of highly expressed human genes. Experiments will be conducted to improve the frequency of transfection of cells in culture to the level found in retrovirally infected cells (on the order of 20-80% of cells transfected). These experiments will also explore strategies for facilitating the nuclear import of expression vectors into mitotically quiescent cells. The initial studies will aim to establish whether sufficient expression of factor IX can be achieved in lymphocytes, as these are the cells that are presently most amenable to genetic therapy. The expression levels of engineered human lymphocytes will be evaluated following their introduction into immunodeficient SCID mice.
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