Thomson, E. A. | bioRxiv Science

bioRxiv2024

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

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.

bioengineering↗

bioRxiv2023

Pressure-Driven Insulin Release Overcomes Limitations of Diffusion for Encapsulated Islet Cell Therapy

Cadaveric islet and stem cell-derived transplantations hold promise as treatments for type 1 diabetes. To tackle the issue of immunocompatibility, numerous cellular macroencapsulation techniques have been developed that utilize diffusion to transport insulin across an immunoisolating barrier. However, despite several devices progressing to human clinical trials, none have successfully managed to attain physiologic glucose control or insulin independence. Based on empirical evidence, macroencapsulation methods with multilayered, high islet surface density are incompatible with homeostatic, on-demand insulin delivery and physiologic glucose regulation, when reliant solely on diffusion. An additional driving force is essential to overcome the distance limit of diffusion. In this study, we present both theoretical proof and experimental validation that applying pressure at levels comparable to physiological diastolic blood pressure significantly enhances insulin flux across immunoisolation membranes--increasing it by nearly three orders of magnitude. This significant enhancement in transport rate allows for precise, sub-minute regulation of both bolus and basal insulin delivery. By incorporating this technique with a pump-based extravascular system, we demonstrate the ability to rapidly reduce glucose levels in diabetic rodent models, effectively replicating the timescale and therapeutic effect of subcutaneous insulin injection or infusion. This advance provides a potential path towards achieving insulin independence with islet macroencapsulation. One Sentence SummaryTowards improved glucose control, applying sub-minute pressure at physiological levels enhances therapeutic insulin transport from macroencapsulated islets.

bioengineering↗