Single Vector Dual Gene Therapy

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DESCRIPTION (Applicant's Abstract): Primary pulmonary hypertension (PPH) is uniformly fatal and has no known cure. A major pathogenic factor in PPH is decreased synthesis of the vacillator prostaglandin 12 (PG 12) and excess synthesis of the vasoconstrictor prostanoid, thromboxane A2 (TxA2). The investigators have shown in an animal model of acute pulmonary hypertension that aerosolized liposome's complexed with plasmids encoding the cyclooxygenase (COX) gene (which is a proximal enzyme in the arachidonate pathway for PGI2 synthesis) results in increased synthesis of PGI2, decreased synthesis of TxA2. and significant attenuation of the pulmonary hypertension. However, since COX is also a proximal enzyme for synthesis of TxA2, COX gene therapy might cause increased TxA2 production in PPH patients and thus worsening pulmonary hypertension. The investigators propose to use a viral internal ribosome entry sequence (IRES), which permits translation of two proteins from a single strand of mRNA, to solve this problem. The investigators hypothesize that transfecting cells using a plasmid containing the COX gene upstream and the PG12 synthase (PGIS) gene downstream to the IRES will result in significantly more synthesis of prostacyclin and less synthesis of TxA2 compared to cells transfected with the COX and the PGIS plasmid. Based on this hypothesis the investigators aim to: 1) Construct two CMV driven plasmids. one with the human COX gene, the IRES sequence, and the human PGIS gene (pCMVCOX-IRES-PGIS) and the other with the IRES sequence and the PGIS gene (pCMV-IRES-PGIS). 2. Transfect human airway epithelial cells, smooth muscle cells, and endothelial cells with pCMVCOX-IRES-PGIS and compare basal and stimulated prostanoid synthesis to cells transfected with either pCMVCOX or pCMV-IRES-PGIS or co-transfected with both pCMVCOX and pCMV-IRES-PGIS. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE