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John A. MacKay, Wan Wang, Benjamin Droese, Mihir Shah, Pang-Yu Hsueh, Guoyong Sun, Sarah F. Hamm-Alvarez; Ocular Drug Delivery Using A Thermo-responsive Lacritin Fusion Protein. Invest. Ophthalmol. Vis. Sci. 2012;53(14):492.
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A fusion protein between recombinant human lacritin and an elastin-like-polypeptide (ELP) was designed as a therapeutic for dry eye disease(DED). Fusion of these domains imparts thermo-sensitive assembly to lacritin, which is a candidate biopharmaceutical. Affecting over 3.2 million Americans, predominantly women, DED is characterized by decreased tear production, which leads to discomfort and visual impairment. To develop biopharmaceuticals for DED, better approaches are required to retain proteins on the eye. Protein polymers, such as ELPs, are an emerging solution to this problem composed of an amino acid repeat (VPGXG). ELPs have characteristic inverse phase transition temperatures (Tt), below which they are soluble and above which they self-associate. By adjusting Tt between room and body temperature ELP fusion proteins may act as a therapeutic depot. The hypothesis tested here is that the fusion protein of ELP and lacritin will undergo thermally-induced phase separation at ocular temperatures without compromising the mitogenic and secretagogue signaling functions of lacritin.
Multiple libraries of synthetic ELP genes were engineered with a range of assembly properties. Genetic engineering enables precise control over their molecular weight, orientation, and Tt. ELP phase behavior was used to purify these biopolymers from bacteria. Fusion protein, free lacritin, and free ELP were characterized for assembly, biodegradation, and induction of secretion from primary rabbit lacrimal gland acinar cells (LGACs).
ELP lacritin fusion proteins were developed with three properties: 1) a control that is soluble at body temperature; 2) a thermo-responsive fusion that forms viscous microparticles at body temperature; and 3) a polypeptide nanoparticle that assembles ~200 nm diameter particles at body temperature. Phase behavior and self-assembly was observed over a range of concentrations (5-100 uM) using UV-Vis spectrophotometry and dynamic light scattering.
ELP lacritin fusion proteins were developed that assemble between 25 and 30 °C. All fusion constructs induce significant secretion of beta-hexosaminidase from primary LGACs. These results suggest that ELP-mediated assembly may be a useful platform to control ocular drug delivery and efficacy.
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