Genetic modification of non-β cells to produce insulin is a promising therapeutic strategy for type 1 diabetes, however, it is associated with issues including biosafety and precise regulation of insulin supply. In this study, a glucose-activated singlestrand insulin analogue (SIA) switch (GAIS) was constructed to achieve repeatable pulse activation of SIA secretion in response to hyperglycemia. In the GAIS system, the CAD-FCS-SIA fusion protein was encoded by the intramuscularly delivered plasmid and temporarily kept in the endoplasmic reticulum (ER) as it binds to the GRP78 protein, then upon hyperglycemia, SIA was released and secreted into the blood. In vitro and in vivo experiments systematically demonstrated the effects of the GAIS system, including glucose-activated and repeatable SIA secretion, long-term precise blood glucose control, recovered HbA1c level, improved glucose tolerance, and ameliorated oxidative stress. Additionally, this system offers sufficient biosafety as evidenced by the assays of immunological and inflammatory safety, ER stress, histological evaluation, etc. Compared with the viral delivery/expression system, the ex vivo implantation of engineered cells, and the exogenous inducer system, the GAIS system combined the advantages of biosafety, effectiveness, persistence, precision, and convenience, providing therapeutic potential for the treatment of type 1 diabetes. Article Highlights We undertake this study to establish a glucose-responsive SIA self-supply system in vivo. Whether ER can serve as a safe and temporary repository to store designed fusion proteins and release SIAs under hyperglycemia condition for efficient blood glucose regulation. Intramuscularly expressed plasmid-encoded CAD-FCS-SIA fusion protein can be temporarily stored in ER and SIA can be released under the stimulation of hyperglycemia, resulting in efficient and long-term regulation of blood glucose stable in T1D mice. GAIS system provides applicable potential for type 1 diabetes therapy integrating regulation and monitor of blood glucose level.

中文翻译：

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调节胰岛素类似物分泌的葡萄糖激活开关可实现 1 型糖尿病小鼠的长期精确血糖控制

对非 β 细胞进行基因改造以产生胰岛素是一种很有前途的 1 型糖尿病治疗策略，但它与生物安全和胰岛素供应的精确调节等问题有关。在这项研究中，构建了葡萄糖激活的单链胰岛素类似物 (SIA) 开关 (GAIS)，以实现 SIA 分泌的可重复脉冲激活以响应高血糖。在 GAIS 系统中，CAD-FCS-SIA 融合蛋白由肌内递送的质粒编码，并在与 GRP78 蛋白结合时暂时保留在内质网 (ER) 中，然后在高血糖时，SIA 被释放并分泌到血液中. 体外和体内实验系统地证明了 GAIS 系统的作用，包括葡萄糖激活和可重复的 SIA 分泌、长期精确的血糖控制、恢复了 HbA1c 水平，改善了葡萄糖耐量，并改善了氧化应激。此外，该系统提供了足够的生物安全性，免疫学和炎症安全性、ER 应激、组织学评估等检测证明了这一点。与病毒递送/表达系统、工程细胞的离体植入和外源诱导系统相比，该系统GAIS系统结合了生物安全性、有效性、持久性、精确性和便利性等优点，为1型糖尿病的治疗提供了治疗潜力。文章要点 我们进行这项研究是为了在体内建立葡萄糖响应性 SIA 自供给系统。ER 是否可以作为一个安全的临时储存库来存储设计的融合蛋白并在高血糖条件下释放 SIA 以实现有效的血糖调节。肌内表达的质粒编码的CAD-FCS-SIA融合蛋白可以暂时储存在ER中，在高血糖刺激下释放SIA，从而实现T1D小鼠血糖稳定的高效长期调控。GAIS 系统为 1 型糖尿病治疗提供了集成血糖水平调节和监测的应用潜力。