Project Q&A

LACMA(Lactate-responsive Module and Application) is a functional synthetic biology modular system designed to address lactic acid accumulation in the tumor microenvironment. This system detects excessively high lactate concentrations in the microenvironment through its built-in lactate-sensing module, thereby activating downstream regulatory pathways. This drives the expression and secretion of lactate metabolic enzymes (sLOx, lactate oxidase), ultimately degrading excess lactate through sLOx's catalytic action. This process maintains lactate concentrations within the tumor microenvironment at relatively normal levels.

LACMA's core innovation lies in its synthetic biology-inspired engineering approach, which systematically integrates a "lactic acid sensing module" with a "regulatory module for lactate oxidase (LOx) expression." This creates a closed-loop regulatory system featuring "automatic detection - precise response - targeted clearance" functionality. This design overcomes the limitations of traditional lactate intervention methods—namely, "lack of specificity in regulation" and "reliance on external intervention"—providing an innovative functional solution to address the series of pathological issues caused by lactate accumulation in the tumor microenvironment.

The lactate clearance process in LACMA follows the following regulatory pathway: First, split lactate-sensing proteins in the system (such as the N- and C-terminal fragments of LIdR) dimerize upon detecting excessively high lactate concentrations in the tumor microenvironment. This brings the fused N-terminal (1–118) and C-terminal (119–242) fragments of the TEV protease closer together, restoring its cleavage activity. Subsequently, the active TEV protease specifically cleaves the TEV cleavage site within the GV-2ER plasmid, releasing the nuclear-localizing GV transcription factor. Upon entering the nucleus, the GV transcription factor initiates expression of the downstream lactate metabolic enzyme LOx; Finally, the expressed LOx is secreted into the extracellular space, where it catalyzes the degradation of lactate accumulated in the tumor microenvironment, thereby achieving lactate clearance.

Since the beginning of our research, we have launched three generations of products :

1. First Generation (LS1.0): Constructed by transfecting HEK293T cells with LIdR-TEV fusion plasmids, GV-2ER plasmid, and pGL4.35 to construct a lactate-responsive sensor. Sensitivity was validated using a dual luciferase system, while transcription, expression, and metabolic functions of sLOx were verified with UAS-SLOX and PASS-SLOX plasmids. However, excessive TEV activity was observed in the absence of lactate, likely due to the physically close proximity of the TEV functional fragments.
2. Second Generation (LS2.0): Based on this, LIdR was split into N- and C-termini. New plasmids were constructed by fusing TEV's N-terminus (1-118) and C-terminus (119-242) separately. The intent was to restore TEV activity via lactate-induced LIdR dimerization, yet elevated background activity persisted.
3. Third Generation (LS3.1-LS3.8): Further designed eight fusion combinations of TEV and LIdR fragments. The modeling team pre-screened these using multiple SPGM system parameters (SCI, OMF, BES, KEC), eliminating three ineffective combinations and predicting LS3.5 as optimal. Subsequent screening for background activity in sugar-free medium and verification via 0, 1, 5 mM lactic acid gradient. This process ultimately confirmed LS3.5 as the optimal sensor, establishing a closed-loop optimization system of "dry-lab prediction - wet-lab validation."