Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya, Turkey.
Department of Medical Genetics, Cukurova University, Faculty of Medicine, Adana, Turkey.
Adv Exp Med Biol. 2023;1409:145-159. doi: 10.1007/5584_2022_746.
Since insulin deficiency results from pancreatic beta-cell destruction, all type 1 and most type 2 diabetes patients eventually require life-long insulin injections. Insulin gene synthesis could also be impaired due to insulin gene mutations as observed in diabetic patients with MODY 10. At this point, insulin gene therapy could be very effective to recompense insulin deficiency under these circumstances. For this reason, an HIV-based lentiviral vector carrying the insulin gene under the control of insulin promoter (LentiINS) was generated, and its therapeutic efficacy was tested in a beta-cell transplant model lacking insulin produced by CRISPR/Cas9-mediated genetically engineered pancreatic beta cells. To generate an insulin knockout beta-cell transplant animal model of diabetes, a dual gene knockout plasmid system involving CRISPR/Cas9 was transfected into a mouse pancreatic beta cell line (Min6). Fluorescence microscopy and antibiotic selection were utilized to select the insulin gene knockout clones. Transplantation of the genetically engineered pancreatic beta cells under the kidney capsule of STZ-induced diabetic rats revealed LentiINS- but not LentiLacZ-infected Ins2KO cells transiently reduced hyperglycemia similar to that of MIN6 in diabetic animals. These results suggest LentiINS has the potential to functionally restore insulin production in an insulin knockout beta-cell transplant animal model of diabetes.
由于胰岛素缺乏是由于胰岛β细胞破坏引起的，所有 1 型和大多数 2 型糖尿病患者最终都需要终身胰岛素注射。由于 MODY10 糖尿病患者中观察到的胰岛素基因突变，胰岛素基因的合成也可能受到损害。在这种情况下，胰岛素基因治疗可以非常有效地补偿胰岛素缺乏。出于这个原因，生成了一种基于 HIV 的慢病毒载体，该载体在胰岛素启动子的控制下携带胰岛素基因（LentiINS），并在缺乏 CRISPR/Cas9 介导的基因工程胰岛β细胞产生的胰岛素的胰岛β细胞移植模型中测试了其治疗效果。为了生成胰岛素敲除胰岛β细胞移植糖尿病动物模型，将涉及 CRISPR/Cas9 的双基因敲除质粒系统转染到小鼠胰岛β细胞系（Min6）中。荧光显微镜和抗生素选择用于选择胰岛素基因敲除克隆。将基因工程胰岛β细胞移植到 STZ 诱导的糖尿病大鼠的肾脏包膜下，结果表明，与 MIN6 相比，LentiINS-但不是 LentiLacZ 感染的 Ins2KO 细胞可短暂降低高血糖。这些结果表明，LentiINS 有可能在胰岛素敲除胰岛β细胞移植糖尿病动物模型中恢复胰岛素的产生。
