Lactic acid bacteria as microbial cell factories for the in vivo delivery of therapeutic proteins as secretable TAT fusion products

Protein replacement therapy has garnered increasing interest in all areas of medicine for its many advantages to human health. In fact, for a monogenic disease, the delivery of wild-type copies of the mutated protein to cells represents a feasible curative approach. However, the failure of proteins to penetrate mammalian cells restricts their value as therapeutic tools in a variety of diseases. Existing strategies for intracellular protein delivery, such as cell-penetrating peptide (CPP)-based protein delivery approaches, which include the use of the trans activator of transcription (TAT) peptide, have shown promising results, but also limitations, mainly due to their requirement for recombinant protein manufacturing and daily invasive administration. To overcome such hindrances, we engineered the lactic acid bacterium Lactococcus lactis to express and secrete TATκ-GFP protein with the aim of developing a delivery tool that is able to foster the production of proteins directly inside the body of patients and their subsequent internalization by target cells, thanks to the transduction properties of the TATκ peptide. We firstly demonstrated that, despite the presence of the high-charge TATκ peptide, L. lactis can produce and secrete TATκ-GFP protein both in vitro and in vivo. Additionally, we showed that, after oral administration of the recombinant L. lactis in mice, TATk-GFP protein can be detected not only in the intestinal wall but also in the mouse liver, heart, and brain. This provides the first evidence that a recombinant TATκ-fused protein, secreted by L. lactis in the mouse host gut, by virtue of the presence of the TATκ peptide, can efficiently pass from the intestinal wall to other organs. With future research and development, this technology based on LAB as a platform for the production and delivery of therapeutic proteins could offer significant advancements in the field of therapeutic protein delivery.