Single Vector Dual Gene Therapy

描述(申请人摘要)：原发性肺动脉高压(PPH)为 都是致命的，而且还没有已知的治疗方法。PPH的一个主要致病因素是 摇摆性前列腺素12(PG 12)合成减少和过剩 血管收缩激素前列腺素A2(TXA2)的合成。这个 研究人员在一种急性肺动脉高压的动物模型中显示 该雾化脂质体与编码环氧合酶的质粒形成复合体 (COX)基因(它是PGI2的花生四烯酸途径中的一种近端酶 合成)导致PGI2合成增加，TXA2合成减少。 对肺动脉高压有明显缓解作用。然而，由于考克斯 也是合成TXA2的近端酶，COX基因治疗可能引起 PPH患者TXA2产生增加，从而使肺功能恶化 高血压。研究人员建议使用病毒内部核糖体进入 序列(IRES)，允许从单链翻译两个蛋白质 来解决这一问题。研究人员假设，将基因 使用含有COX基因上游和PG12合成酶的质粒的细胞 IRES下游的(PGIs)基因将导致显著更多的合成 与转染组比较，前列环素和TXA2的合成减少 COX和PGIS质粒。基于这一假设，研究人员的目标是 目的：1)构建两个CMV驱动的重组表达载体。一种带有人类COX基因的IRES 序列和人PGIS基因(pCMVCOX-IRES-PGIS)，另一个与 IRES序列和pGIS基因(pCMV-IRES-pGIS)。2.转染人呼吸道 上皮细胞、平滑肌细胞和内皮细胞 PCMVCOX-IRES-PGIs并比较基础和刺激的前列腺素合成 PCMVCOX、pCMV-IRES-PGIs或共转染细胞 PCMVCOX和pCMV-IRES-PGIS。 建议的商业应用：不可用

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