MILK MAKES MEDICINE: COWS PRODUCING HUMAN INSULIN

Diabetes is one of the fastest rising worldwide health problems, with an estimated 537 million cases in 2021 and a predicted 783 million by 2045. Diabetes, a chronic metabolic disease that severely damages multiple organs, is caused by insulin insufficiency (Type 1) or insulin resistance (Type 2). Type 1 diabetes is caused by autoimmune destruction of β-cells in pancreatic islets, leading to insulin deficiency and elevated blood and urine glucose levels. Hyperglycemia in type 2 diabetes is caused by the decrease of insulin production by β-cells, along with insulin resistance. To compensate for insulin resistance, β-cells secrete more insulin, leading to an increase in plasma glucose levels.

Insulin production begins with the translation of insulin mRNA in the cytoplasm. Preproinsulin is then transported across the endoplasmic reticulum to pancreatic β-cells for posttranslational modifications. Signal peptidase converts preproinsulin to proinsulin, generating disulfide linkages, which are then isomerized and reduced in proinsulin monomers, resulting in proinsulin dimers that move from the endoplasmic reticulum to the Golgi complex. Prohormone convertases (PC1/3 and PC2) and carboxypeptidase E convert proinsulin into secretory granules, which produce mature insulin and C-peptide. Insulin synthesis for human therapy has been accomplished utilizing a variety of techniques and model species. Escherichia coli produces insulin using multiple mechanisms,(1) The A and B peptide chains were synthesized independently and then recombined to form biologically active insulin; (2) biosynthesis of proinsulin;  (3) mini-proinsulin.In yeast, insulin is generated and released in the form of mini-proinsulin and proinsulin. Human proinsulin was generated in transgenic mouse milk and transformed to insulin in vitro with the proteases trypsin and carboxypeptidase B. Mature insulin was found to be active in assays evaluating the insulin receptor's autocatalysis activity in CHO cells. Undigested milk, on the other hand, shown no activity in the assays.

Schematic of the lentiviral vector constructed for mammary gland-specific human insulin expression and restriction map analysis.

Adult fibroblasts were transformed using a pseudo-lentivirus that had the bovine β-casein promoter and human insulin sequences. A cloned fragment of 5.335 kb from the promoter region (Pβcas5) of the bovine β-casein gene was used for lentiviral vector construction. The expression vector was created using pLenti6.2-GW/EmGFP (7.833 kb, Invitrogen # V369-20, Carlsbad, CA). The PCMV promoter and EmGFP gene in vector pLenti6.2-GW/EmGFP were replaced with the bovine Pβcas5 promoter (5.335 kb) and the amplified fragment from the hINS gene (1.193 kb), respectively. Following the vector creation of the mammary gland-specific expression. The cells were then employed for nuclear transfer. Transgenic embryos were given to recipient cows .

Ten embryos were non-surgically transferred to synchronized recipients, and the gDNA from the resulting transgenic calf was used to confirm the presence of the transgene. The transgenic cow was hormonally encouraged to produce milk. After 21 days of lactation induction, the milk was collected throughout the next 30 days. For western blot and mass spectrometry analysis. Western blotting revealed two bands with molecular masses corresponding to proinsulin and insulin. The mass spectrometry study revealed that the milk included more human insulin than proinsulin, as well as proteases capable of converting proinsulin into insulin and an insulin-destroying enzyme capable of degrading the recombinant protein. With further research and development, this technology has the potential to revolutionize insulin production. A 100-head herd could meet a country's needs, and a larger herd could address global requirements within a year. This could significantly improve access to affordable insulin for diabetics worldwide.

REFERENCE:

Monzani PS, Sangalli JR, Sampaio RV, Guemra S, Zanin R, Adona PR, Berlingieri MA, Cunha‐Filho LF, Mora‐Ocampo IY, Pirovani CP, Meirelles FV. Human proinsulin production in the milk of transgenic cattle. Biotechnology Journal. 2024 Mar;19(3):2300307.

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