Racz G Z, Zheng C, Goldsmith C M, Baum B J, Cawley N X
Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
Oral Dis. 2015 Mar;21(2):149-55. doi: 10.1111/odi.12217. Epub 2014 Jan 13.
Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis.
We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands.
We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6 , was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway.
This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit.
唾液腺是基因治疗的有用靶点。将基因导入唾液腺后，受调控分泌途径的蛋白质，如人生长激素，会分泌到唾液中，而组成型分泌途径的蛋白质，如促红细胞生成素，则会分泌到血液中。将人生长激素（hGH）分泌到唾液中并无治疗作用。在本研究中，我们试图通过定点诱变将转基因hGH的分泌从唾液重定向至血清。
我们首先在体外使用AtT20细胞（一种表现出受调控分泌途径蛋白质极化分泌的内分泌细胞系模型）对hGH突变体进行测试。对筛选出的突变体，利用腺病毒介导的基因转移至大鼠下颌下腺进行进一步体内研究。
我们鉴定出两个与野生型hGH相比分泌行为不同的突变体。其中一个突变体ΔN1-6在转导大鼠的血清中被检测到，表明该突变体在唾液腺中的表达导致其通过组成型分泌途径分泌。
本研究表明，通常注定进入受调控分泌途径的治疗性蛋白质发生诱变可能会使其通过组成型分泌途径分泌到循环系统中，从而获得潜在的治疗益处。
