β-Cells as a Cell Factory for On-Demand Recombinant Protein Dosing: Harnessing the Neuroendocrine Cell Secretory Pathway for Controlled Release

Abstract

This study explores the potential of utilizing {beta}-cells, exemplified with R7T1 {beta}-cell pseudoislets, as a transplantable cell factory for on-demand recombinant protein therapeutic delivery. While mammalian cell lines are widely used for in vitro protein production, the commonly utilized constitutive secretion pathway poses challenges to in vivo cell therapy, especially for delivering proteins requiring precise exposure kinetics. The proposed approach capitalizes on unique aspects of {beta}-cells, including substantial vesicular protein storage capacity and electrochemically-regulated protein release, to facilitate timely and titratable in vivo therapeutic delivery. Examining a variety of strategies to acheive {beta}-cell glucagon or glucagon-like peptide 1 (GLP-1) storage and secretion, we devised a flexible {beta}-cell-based expression platform for efficient cellular peptide production and on-demand release. This platform utilizes the preproinsulin coding sequence as a template, wherein therapeutic peptides of interest (glucagon or GLP-1) are substituted for C-peptide while the A- and B-peptide insulin chains are mutated to prevent bio-active insulin production. This approach overcomes the challenge of efficient bio-active peptide expression by leveraging the endogenous {beta}-cell peptide expression, translation, processing, storage and secretion machinery. Furthermore, {beta}-cells provide a mechanism for scalable electyrochemnically-triggered peptide delivery. This transformative strategy, which may be extended to other proteins and peptide expression cassettes, holds significant promise for targeted and temporally controlled in vivo production and release of recombinant protein therapeutics. The study suggests potential applications in addressing challenges in metabolic disorders, blood disorders, and oncology. Future refinements may focus on optimizing vector design, peptide production, and in vivo adaptation.