Stable and Tunable Episomes for Gene Therapy

DESCRIPTION provided by applicant The goal of this SBIR is to develop a novel gene expression plasmid capable of long term non integrated episome maintenance with chemical tunable gene expression The vector system can function in multiple human cell types including rapidly dividing cancer cells progenitor stem cells or post mitotic somatic cells The major innovations are derived from the application of basic discoveries in genome maintenance and gene regulation from studies with human gamma herpes viruses Specifically we incorporate the use of chromatin insulator INS elements consisting of binding sites for the cellular factor CTCF to punctuate key regulatory features derived from Epstein Barr Virus EBV and Kaposiandapos s Sarcoma Associated Herpes virus KSHV We incorporate the EBV chromosome architecture protein EBNA to maintain the episomal features and prevent epigenetic repression of transcription initiation sites at the EBV Q promoter Qp Components of the Qp prevent DNA methylation and epigenetic silencing overcoming a major barrier to previous generations of gene expression vectors In addition we use a version of EBNA protein that limits immune recognition and MHC presentation We also use the KSHV LANA promoter LANAp which functions efficiently in lymphoid and epithelial cells and resists epigenetic suppression Finally we propose to use newly developed small molecules regulators of EBNA to fine tune the episome maintenance and transcriptional control of transgenes cloned into the vector As a proof of principle we will express the cystic fibrosis gene CFTR in EBV plasmid system for long term expression in human lung epithelial cells This vector system will provide long term regulatable expression of transgenes required for in vitro research and ultimately human gene therapy The vector can be further optimized for delivery in liposomes nanoparticles or modified viral like particles for more selective delivery in vivo The proposed plasmid expression system will have significant utility in human gene therapy PUBLIC HEALTH RELEVANCE A major limitation of gene therapy technology is the inability to properly regulate and maintain long term gene expression of transgenes in recipient cells and tissue This SBIR proposed the development of new gene expression vectors to improve the control of long term gene expression and episomal maintenance in human cells A proof of principle expression vector will be designed to optimize expression of the normal Cystic Fibrosis gene CFTR in human lung epithelial cells for ultimate use in gene therapy clinical trials