Competent expression of effective and long-acting human α-Gal A-Fc fusion protein in the milk of transgenic mice.

Abstract

Fabry disease is a rare X-linked inherited lysosomal storage disorder caused by a reduction or deficiency in the activity of α-galactosidase A (α-Gal A). The short half-life of α-Gal A necessitates biweekly infusions, thereby imposing significant economic and physical burdens on patients and their families. In this study, a novel long-acting replacement for α-Gal A, termed α-galactosidase A-Fc (α-Gal A-Fc), was designed. Two transgenic founders with an 18.2% transgene rate were obtained to express recombinant human α-Gal A-Fc protein in mouse milk. The α-Gal A-Fc enzyme activity in the milk of high-copy mice were significantly higher than those in low-copy mice and were stably inherited across F1-F3 generations. No significant differences were observed in α-Gal A-Fc concentration or enzymatic activity among high-copy mice of the same generation. During early lactation, the α-Gal A-Fc concentration and enzymatic activity were 2.1-fold and 2.17-fold higher, respectively, compared to late lactation. The expression levels during late lactation did not affect purification efficiency, allowing for the pooling of milk from high-copy mice throughout the entire lactation period for protein purification. The elimination half-life of the purified α-Gal A-Fc protein in mouse serum was 471 min, approximately 43 times longer than that of the commercially available drug Replagal. These findings facilitate the development of an efficient production system for long-acting human α-Gal A-Fc fusion protein and provide valuable insights into the utilization of transgenic large animal mammary gland bioreactors for biopharmaceuticals.