*Purpose: To investigate if PEGylated Lacritin N-104 (PEG N-104) prevents the progression of type 1 diabetes (T1D) in the NOD mouse.

*Background: Lacritin is a multifunctional protein in human tears with prosecretory, prosurvival, and mitogenic properties. Lacritin N-104, an NFAT and mTOR agonist, promotes islet viability, insulin secretion, and graft survival following islet transplantation. Polyethylene glycol (PEG) extends the peptide’s half-life by increasing its size and making the surface less sensitive to proteolysis.

*Methods: a) Lacritin and its analogs were PEGylated at the N- or C-terminus. Mouse islets were treated with 4uM PEGylated N-94 or C-95, positive control N-104, or negative control C-95 for 24 hours in DMEM at 37°C. These islets were used in glucose-stimulated insulin secretion (GSIS) assay. Viability of islets was also scored by propidium iodide – fluorescein diacetate staining; and b) PEG N-104 was injected subcutaneously (SQ) or N-104 was injected intraperitoneal (IP) to 9-weeks old NOD mice, thrice a week until they were 16-weeks old. Tail vein blood glucose was measured daily.

*Results: Mouse islets treated with 4uM PEGylated N-94 or C-95, N-104, or C-95 were used in GSIS assay. N-94 activity was superior when N-terminus was PEGylated, GSIS being 1.6-fold high compared to untreated islets. GSIS with both free N-104 and N-terminus PEG N-104 was 2-fold higher compared to the untreated group and the negative control C-95 group. In NOD mice, PEG N-104 (SQ) or free N-104 (IP) completely prevented the development of T1D at week 18 (n=5). In contrast, neither SQ or IP injected C-95 (n=5) nor saline (n=5) could delay the progression of T1D, as 100% of mice became diabetic by SQ injection of C-95 or saline, and 60% became diabetic by IP injection of C-95. Moreover, both PEG N-104 and free N-104, injected at 20, 40, or 80 μM concentrations, completely prevented T1D progression in NOD mice as indicated by blood glucose (BG) below 200 mg/dL. BG was measured until mice were 26-weeks old. No stomach bloating or weight loss was observed in these mice.

*Conclusions: Our data indicates N-terminus PEGylation of N-104 could extend the peptide’s half-life without inhibiting its abilities to promote islet function. In addition, systemic administration of free N-104 or PEG-N-104 by SQ or IP injection could prevent the progression of T1D in NOD mice.

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