Endocrine Abstracts

Background: Therapeutic peptides are medicines with high potency, low toxicity, and, broad disease targets. However, the widespread use of peptides is limited by their easy degradation in human body. The intestinal organoid technology can be utilized to design a novel cell-based peptide delivery system. Intestinal organoids feature intestinal epithelial tissue-like structure, harboring all the expected in vivo epithelial cell types, including enteroendocrine cells (EECs). EECs are collectively recognized as the largest endocrine system.

Objective: This study aims to use EECs as target cells for the manufacture and in vivo delivery of therapeutic peptides. Insulin was used as a representative peptide to generate proof-of-concept data to validate the use of intestinal organoids as a system for delivery of therapeutic peptides.

Methods: Human proinsulin coding sequence was integrated into the genome of murine duodenal organoids. This genetic modification was conducted by electroporation using piggyBac transposon. The human proinsulin gene was driven by NeuroD1 or CMV promoter. The proinsulin gene co-expressed with tdTomato, red fluorescence indicated the expression of insulin. mRNA expression levels of different clones were assessed by qPCR. Clones with the highest transcription were selected to assess insulin secretion using ELISA. Wholemount immunofluorescence staining of insulin was performed to locate insulin-secreting cells.

Results: Transgenic clones with CMV promoter gave rise to a scattered pattern of red fluorescence and significant insulin mRNA expression. Clones with NeuroD1 promoter showed no red fluorescence and low levels of insulin mRNA. Insulin secretion of CMV clones was significantly higher than that of NeuroD1 clones. Immunostaining showed few insulin-positive cells.

Conclusion: Genetically modified insulin-expressing intestinal organoids delivered using the piggyBac transposon system functioned properly at the transcriptional level but not at the peptide level. This model faces challenges such as low levels of insulin secretion, possible misfolding of insulin protein structure. Solving these issues may provide a usable system.