*Purpose: Stem cell-derived β-cells not only promise a renewable supply of insulin-producing cells with negligible immunosuppression but may also provide a substrate for the identification of personalized therapies for individuals with type-1 or 2 diabetes. Though innovative, the development of stem cell-derived β-cells is cumbersome and requires a seven-step protocol with very meticulous procedures. We designed a simple and single-shot approach to generate insulin-producing cells directly from somatic cells via targeted epigenetic activation of β-cells signature genes leveraging CRISPR/Cas9 technology.

*Methods: We have developed multiplex epigenetic engineering vector (MEE-β) with fusion protein (dCas9.P300^core domain of histone acetyl transferase). MEE-β contains 5 cloned oligonucleotides under separate U6 promoter targeting the DNase I hypersensitive sites of PDX1, NKX6.1, MAFA, Insulin, and Glut2 genes (Figure 1A). Human somatic cells (lung endothelial cells, lung fibroblasts and PBMCs, 1x 10⁶) were transfected in triplicate with MEE-β and Glut2⁺ cells were flow-sorted after 14 days post-transfection. Glucose-stimulated insulin secretion (GSIS) was measured in Glut2⁺cells following stimulation with 2.8mM (low) and 28 mM (high) glucose and measurement of insulin was done using an ultrasensitive ELISA platform. Glut2⁺ cells were evaluated for expression of β-cell-specific genes using SYBR-green based qRT-PCR.

*Results: Cloning of aforementioned oligonucleotides and correct fusion of P300^core in MEE-β vector was confirmed by PCR and Sanger’s sequencing (Fig.1B). Total of 2×10⁴, 2.8×10⁴ , 4.7x 10⁴ and 1.7×10⁴ Glut2⁺ cells were recovered after flow sorting of human lung endothelial cells, human lung fibroblasts and PHA activated human PBMCs, respectively (Fig.1C). These cells responded to high and low concentration glucose challenge and produced insulin (Fig.1D). In addition, Glut2⁺ cells not only demonstrated expression of targeted genes by MEE-β (PDX1, NKX6.1. MAFA, Insulin, Glut2), but also upregulation of other downstream β-cell genes, including NUROD1, NKX2.2, SLC03A08 (Fig.1E).

*Conclusions: This is a proof-of-concept study to demonstrate the conversion of somatic cells into insulin-secreting cells by a user-friendly, less cumbersome, and less expensive strategy. The activation of other downstream genes related with β-cell development besides targeted genes by MEE-β suggests the triggering of β-cell-specific cascades.

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