Use of non‐canonical amino acids in genetic code expansion‐based therapeutics: Effects on mouse gut microbiota

Vaccines and cell therapeutics based on genetic code expansion are emerging. A crucial step in these therapeutic technologies is the oral administration of non‐canonical amino acids (ncAAs) to control pathogen growth and therapeutic protein levels in vivo. Investigating the toxicity effects of ncAAs can help identify more suitable candidates for developing genetic code expansion‐based vaccines and cell therapeutics. In this study, we determined the effects of three ncAAs, namely, 4‐acetyl‐phenylalanine (pAcF), 4‐iodo‐phenylalanine (pIoF), and 4‐methoxy‐phenylalanine (pMeoF), commonly used in genetic code expansion‐based vaccines and cell therapeutics, on the main organs, serum biochemical parameters, and gut microbiota in mice. We observed that pIoF and pMeoF significantly altered serum biochemical parameters to some extent. Moreover, the alterations in the mouse gut microbial composition were considerably greater after the oral administration of pIoF and pMeoF than after that of pAcF, compared with that in the control mice. These findings suggest that pAcF is more suitable than pIoF and pMeoF for application in genetic code expansion‐based vaccines and cell therapeutics as it disturbs the physiological and gut microecological balance in mice to a lesser extent.