Poster 65 - Engineering probiotic yeast to secrete long-lasting insulin as an oral therapy for diabetes

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Jicheng Feng

Student
Marriotts Ridge High School
West Friendship, Maryland, United States

Co-Author(s)

DY

Di Yu

Post Doctorate
University of Maryland, United States
LN

Lydia Nyasae

Associate Director
FZata, United States
RL

Rachel Lee

Scientist
FZata, United States

Primary Investigator(s)

HY

Hua Yu

Director
FZata, United States

Introduction: Diabetes mellitus is characterized by high blood glucose levels. There are two types: type 1 diabetes (T1DM), caused by insufficient insulin production, and type 2 diabetes (T2DM), where the body becomes resistant to insulin. Insulin, a peptide hormone produced by pancreatic beta cells, regulates blood glucose levels, preventing hyperglycemia and its severe complications, such as retinopathy, nephropathy, and neuropathy. Current insulin treatments for T1DM and some T2DM patients involve injections, which face compliance issues and do not mimic the natural insulin pathway to the liver. Oral insulin delivery could enhance patient compliance and better regulate liver glucose production.

Saccharomyces boulardii, a probiotic yeast, promotes intestinal health and reduces inflammation and oxidative stress, benefiting diabetic patients. This project aims to engineer S. boulardii to secrete long-lasting insulin as an oral biotherapeutic, combining probiotics and oral insulin in one capsule. We engineered a long-lasting insulin by fusing mutated insulin with the human IgG Fc fragment to increase serum half-life and enable FcRn-mediated transcytosis through intestinal epithelial cells. Besides probiotic benefits, S. boulardii offers efficient expression of complex proteins, resistance to antibiotics and bacterial phages, and does not transfer antibiotic resistance genes. Moreover, S. boulardii can be produced in large quantities, lyophilized, and formulated into capsules or pills, making it a cost-effective and convenient solution. This innovative approach holds the potential to significantly improve diabetes management and patient outcomes.

Materials and

Methods: Plasmids containing a mutated insulin gene linked to the human IgG Fc fragment were constructed for expression in S. boulardii and Chinese hamster ovary (CHO) cells. The fusion genes, optimized for yeast expression, were synthesized and cloned into vectors using gold gate assembly. Four plasmids (P1-P4) were generated with either insulin on the N-terminus (Ins-Fc) or C-terminus (Fc-Ins) under TEF1 or TDH3 promoters. For CHO cell expression, the Ins-Fc gene was cloned into the pSEC vector under a CMV promoter.

E. coli DH5a was used for gene construction. HeLa cells, grown in Eagle's Minimum Essential Medium with supplements, were utilized to assay the function of insulin/Fc fusion proteins purified from yeast supernatants. CHO cells were used to express the Ins-Fc fusion protein as a control. S. boulardii, grown on YPD medium, served as the vector for oral delivery of insulin/Fc.

To engineer S. boulardii, final insertion cassettes (FICs) containing the insulin/Fc genes and a selection marker were amplified from the plasmids P1-P4 and transfected into S. boulardii via electroporation. Colonies of transformed yeast on selection agar plates were screened for the highest expression of the insulin/Fc proteins by ELISA.

Quantitative PCR was used to determine the copy numbers of insulin/Fc genes in engineered yeast strains. Insulin/Fc proteins from the yeast and CHO culture supernatants were purified via Protein A affinity column. The bioactivity of the proteins was determined through insulin-mediated AKT phosphorylation in HeLa cells. Human wild-type insulin protein purchased from Sigma was used as a control for the bioassay.

Results, Conclusions, and Discussions: The experimental goal was to generate probiotic yeast strains secreting functional insulin/Fc fusion proteins. Figure 1 shows the diagram of the experimental procedure for gene cloning and yeast engineering. The insulin gene was fused to either the N-terminus (Ins-Fc) or C-terminus (Fc-Ins) of the Fc fragment, and their expression in S. boulardii was driven by a strong constitutive promoter, TEF1 (PTEF) or TDH3 (PTDH). Plasmid constructs containing the insulin/Fc gene cassettes were successfully generated and validated by restriction enzyme digestion and sequencing. Electroporation was used to integrate the insulin/Fc gene cassettes, along with a selection marker, into the yeast genome. Transformed yeast colonies expressing the highest levels of insulin/Fc fusion proteins were identified by ELISA screening. Final lead clones, designated as FZ030 and FZ032, were evaluated and demonstrated the stable expression of insulin-Fc (Ins-Fc) or Fc-insulin (Fc-Ins) fusion proteins respectively. Quantitative PCR revealed that FZ030 contained multiple copies of Ins-Fc genes under PTEF and PTDH promoters, while FZ032 had multiple copies of Fc-Ins gene under PTDH (Figure 2). ELISA indicated that Fc-Ins was expressed at higher levels than Ins-Fc. Protein purification via Protein A affinity chromatography showed that Fc-Ins and Ins-Fc had different molecular weights, suggesting post-translational modifications (Figure 3).

Bioactivity assays (Figure 4) using HeLa cells revealed that Ins-Fc had higher bioactivity in inducing AKT phosphorylation as compared to Fc-Ins (Figure 5). This indicates that the position of the Fc fragment influences both expression and bioactivity of the insulin/Fc fusion proteins. Despite higher expression levels, Fc-Ins exhibited significantly lower bioactivity than Ins-Fc. Therefore, while Fc-Ins showed higher expression, Ins-Fc purified from FZ030 displayed the expected bioactivity determined by AKT phosphorylation in Hela cells (Figure 5).

These findings demonstrate that S. boulardii can be engineered to secrete insulin/Fc fusion proteins with varying levels of expression and bioactivity. In the future, we will perform in vivo studies to determine the pharmacokinetic properties and therapeutic efficacy of the final yeast strains. Our ultimate goal is to develop a patient-friendly and cost-effective biotherapeutic against diabetes for the benefit of millions of people across the globe.

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