Intrapulmonary Gene Transfer Using Compacted DNA

为了开发一种非静脉、无针的基因转移平台，用于治疗包括血友病和α(1)抗胰蛋白酶缺乏在内的系统性缺乏性疾病，哥白尼治疗公司评估了致密DNA(纤溶酶复合体)专利配方的气管内输送。我们的基因转移系统将单分子的质粒DNA压缩成15-25 nm的颗粒，这种配方能够有效地转染未分裂的细胞。在初步研究中，气管内注射非靶向纤溶酶复合体产生的转基因表达水平是裸露DNA的1000多倍。单次气管内注射纤溶酶复合体后，肺上皮细胞衬里液中人凝血因子IX(-300 ng/ml)水平显著升高，纤溶酶复合体与丝氨酸酶复合体受体(SECR)的配基结合，SECR是一种表达在支气管上皮表面的受体。为了将这项技术推向人体试验，我们建议：i)鉴定在非靶向纤溶酶复合体肺内输送后所导入的细胞类型；ii)评估SecR靶向纤溶酶复合体在肺内基因输送方面的作用；iii)测定非靶向和SecR靶向纤溶酶复合体肺内给药后血浆和支气管肺泡灌洗液中人凝血因子IX和α(1)-抗胰蛋白酶的水平。我们有希望的结果表明，全身性遗传缺陷状态的患者可以每天或每周多次雾化给予纤溶酶复合体治疗。拟议的商业应用：尽管基因疗法在治疗人类疾病方面前景看好，但安全性和有效性方面的限制限制了其商业发展。我们的无毒浓缩DNA制剂，称为纤溶酶复合体，在生理液中稳定，可转染有丝分裂后细胞，并在肺内剂量后在肺内产生高水平的基因表达。纤溶酶复合体通过手持吸入器以气雾剂的形式提供，可能为包括血友病和阿尔法(1)-抗胰蛋白酶缺乏症在内的遗传缺陷状态提供一种有效和简单的治疗方法。

To develop a non-intravenous, needle-less gene transfer platform for systemic deficiency disorders, including hemophilia and alpha (1)- antitrypsin deficiency, Copernicus Therapeutics has evaluated intratracheal delivery of proprietary formulations of compacted DNA (PLASmin complexes). Our gene transfer system compacts single molecules of plasmid DNA into 15-25 nm particles, and this formulation is capable of efficiently transfecting non-dividing cells. In preliminary studies, intratracheal administration of non-targeted PLASmin complexes generated over 1,000-fold higher levels of transgene expression than naked DNA. Significant levels of human factor IX (-300 ng/ml) in lung epithelial lining fluid were measured following a single intratracheal dose of PLASmin complexes targeted with a ligand for the serpin enzyme complex receptor (SECR), a receptor expressed at the lumenal surface of bronchial epithelium. To advance this technology toward human trials, we propose to: i) identify the cell type transfected following intrapulmonary delivery of non-targeted PLASmin complexes; ii) evaluate SECR-targeted PLASmin complexes for intrapulmonary gene delivery; and iii) determine plasma and bronchoalveolar lavage fluid levels of human factor IX and alpha(1)-antitrypsin after intrapulmonary administration of non-targeted and SECR-targeted PLASmin complexes. Our promising results suggest that patients with systemic genetic deficiency states could be treated with aerosolized PLASmin complexes administered daily or several times per week. PROPOSED COMMERCIAL APPLICATIONS: Although gene therapy holds great promise for treating human disease, limitations in safety and efficacy have restricted its commercial development. Our non-toxic formulation of condensed DNA, termed PLASmin complexes, are stable in physiologic fluids, transfect post- mitotic cells, and generate high level gene expression in lung following an intrapulmonary dose. Delivered as an aerosol via a hand-held inhaler, PLASmin complexes may provide an effective and simple therapy for genetic deficiency states, including hemophilia and alpha(1)-antitrypsin deficiency.