Engineered IRES-mediated promoter-free insulin-producing cells reverse hyperglycemia

Area of Science:

• Biotechnology
• Genetic Engineering
• Diabetes Research

Background:

• Type 1 diabetes (T1D) necessitates endogenous insulin, but current treatments have limitations.
• Engineered cells secreting insulin present a promising therapeutic strategy for T1D.
• This approach may overcome autoimmune issues seen with beta-cell transplantation or viral vectors.

Purpose of the Study:

• To develop engineered cells capable of secreting insulin using CRISPR/Cas9 gene editing.
• To assess the viability and functionality of engineered cells on microcarriers.
• To evaluate the therapeutic potential of these engineered cells in a mouse model of T1D.

Main Methods:

• CRISPR/Cas9 and homology-directed repair (HDR) were used to insert an insulin cassette into HEK-293T cells.
• Insulin expression, cell viability, and functionality on GelMA and Cytopore I microcarriers were quantified.
• The efficacy of the engineered cells in reversing hyperglycemia in T1D mice was investigated.

Main Results:

• HDR successfully integrated the insulin loop into HEK-293T cells, enabling insulin expression.
• Cytopore I microcarriers supported cell survival and proliferation in vitro and post-transplantation.
• Transplanted cells formed stable structures with no significant immune rejection, reversing hyperglycemia in diabetic mice.

Conclusions:

• Cytopore I microcarriers are biocompatible and enhance cell survival in vivo.
• The promoter-free insulin loop allows non-endocrine cells to secrete insulin, rapidly reducing glucose levels.
• This study presents a novel gene-editing strategy and transplantation method for diabetes treatment, expanding potential cell sources.