155 Human proinsulin and insulin production in the milk of transgenic cattle

Large pharma has controlled the price of insulin, driving the costs up more than 600% in the past 20 years. Because of the projected insulin shortage and growing demand for human insulin to treat diabetes, the goal of this work was to make transgenic cattle producing insulin in their milk. In that regard, pseudo-lentivirus containing the bovine β-casein promoter and human insulin DNA sequence was used to modify adult female bovine fibroblasts, which were used as nucleus donors for somatic cell nuclear transfer. The pLenti6.2-GW/EmGFP (7.833 kb) was used to construct the expression vector. The P_CMV promoter and EmGFP gene from vector pLenti6.2-GW/EmGFP were replaced with the bovine Pβcas5 promoter and a fragment from the hINS gene, respectively. Modified bovine fibroblasts, screened with 8 µg mL⁻¹ blastocidin containing the transgene were used as nucleus donors for somatic cell nuclear transfer (SCNT). One round of SCNT was performed. The blastocyst rate in the cloned embryos was 26.2% (11 blastocysts from 42 fusions). Embryo transfer was performed on 10 synchronized recipient cows, with each cow receiving only one non-hatched blastocyst-stage embryo. At 60 days, one recipient was pregnant and she gave birth to a live calf at term. The presence of the transgene was confirmed by PCR on the calf’s fibroblasts. Once the heifer started lactating, recombinant protein in milk was evaluated using western blotting and mass spectrometry. Milk analysis showed two bands by western blotting with molecular masses corresponding to proinsulin and insulin. Mass spectrometry showed the presence of human insulin more than proinsulin, proteases that could convert proinsulin into insulin, and insulin-degrading enzymes that could degrade the recombinant protein were also detected. Our results suggest that endogenous milk proteases convert recombinant human proinsulin to insulin in milk because mass spectrometry detected human C-peptide. The methodologies used for generating the transgenic cow were successful in producing recombinant protein in the milk. Endogenous milk proteases appear to convert recombinant proinsulin to functional insulin. For the first time, we demonstrate that mature bioactive human insulin can be produced in cows milk, which could potentially revolutionize how insulin is administered to diabetic patients.