INSERM, U872 Physiopathology of Ocular Diseases: Therapeutic innovations, Paris, France.
J Gene Med. 2010 Nov;12(11):904-19. doi: 10.1002/jgm.1517.
We have developed a nonviral gene therapy method based on the electrotransfer of plasmid in the ciliary muscle. These easily accessible smooth muscle cells could be turned into a biofactory for any therapeutic proteins to be secreted in a sustained manner in the ocular media.
Electrical conditions, design of electrodes, plasmid formulation, method and number of injections were optimized in vivo in the rat by localizing β-galactosidase expression and quantifying reporter (luciferase) and therapeutic (anti-tumor necrosis factor) proteins secretion in the ocular media. Anatomical measurements were performed via human magnetic resonance imaging to design a human eye-sized prototype that was tested in the rabbit.
In the rat, transscleral injection of 30 µg of plasmid diluted in half saline (77 mM NaCl) followed by application of eight square-wave electrical pulses (15 V, 10 ms, 5.3 Hz) using two platinum/iridium electrodes, an internal wire and an external sheet, delivered plasmid efficiently to the ciliary muscle fibers. Gene transfer resulted in a long-lasting (at least 5 months) and plasmid dose-/injection number- dependent secretion of different molecular weight proteins mainly in the vitreous, without any systemic exposure. Because ciliary muscle anatomical measurements remained constant among ages in adult humans, an integrated device comprising needle-electrodes was designed and manufactured. Its usefulness was validated in the rabbit.
Plasmid electrotransfer to the ciliary muscle with a suitable medical device represents a promising local and sustained protein delivery system for treating posterior segment diseases, avoiding repeated intraocular injections.
我们开发了一种基于质粒在睫状肌中电转移的非病毒基因治疗方法。这些容易接近的平滑肌细胞可以变成一个生物工厂，持续地分泌任何治疗性蛋白质到眼内介质中。
通过在大鼠体内定位β-半乳糖苷酶表达并定量报告（荧光素酶）和治疗（抗肿瘤坏死因子）蛋白在眼内介质中的分泌，优化了电条件、电极设计、质粒配方、注射方法和次数。通过人体磁共振成像进行解剖学测量，设计了一个适合人眼大小的原型，并在兔子身上进行了测试。
在大鼠中，经巩膜注射 30 µg 稀释在半盐（77 mM NaCl）中的质粒，然后使用两个铂/铱电极、一根内丝和一个外片施加 8 个方波电脉冲（15 V，10 ms，5.3 Hz），有效地将质粒传递到睫状肌纤维中。基因转移导致不同分子量的蛋白质在玻璃体中持续（至少 5 个月）和质粒剂量/注射次数依赖性分泌，而没有任何系统性暴露。由于成年人的睫状肌解剖学测量值在不同年龄之间保持不变，因此设计并制造了一种包含针电极的集成装置。它在兔子身上的有效性得到了验证。
用合适的医疗设备将质粒电转移到睫状肌中，代表了一种有前途的局部和持续的蛋白质输送系统，可用于治疗后节疾病，避免重复眼内注射。
