Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.

Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

Molecular weight control of poly-γ-glutamic acid reveals novel insights into extracellular polymeric substance synthesis in Bacillus licheniformis.

BACKGROUND: The structural diversity of extracellular polymeric substances produced by microorganisms is attracting particular attention. Poly-gamma-glutamic acid (γ-PGA) is a widely studied extracellular polymeric substance from Bacillus species. The function of γ-PGA varies with its molecular weight (Mw). RESULTS: Herein, different endogenous promoters in Bacillus licheniformis were selected to regulate the expression levels of pgdS, resulting in the formation of γ-PGA with Mw values ranging from 1.61 × 103 to 2.03 × 104 kDa. The yields of γ-PGA and exopolysaccharides (EPS) both increased in the pgdS engineered strain with the lowest Mw and viscosity, in which the EPS content was almost tenfold higher than that of the wild-type strain. Subsequently, the compositions of EPS from the pgdS engineered strain also changed. Metabolomics and RT-qPCR further revealed that improving the transportation efficiency of EPS and the regulation of carbon flow of monosaccharide synthesis could affect the EPS yield. CONCLUSIONS: Here, we present a novel insight that increased pgdS expression led to the degradation of γ-PGA Mw and changes in EPS composition, thereby stimulating EPS and γ-PGA production. The results indicated a close relationship between γ-PGA and EPS in B. licheniformis and provided an effective strategy for the controlled synthesis of extracellular polymeric substances.

Improved gene therapy for spinal muscular atrophy in mice using codon-optimized hSMN1 transgene and hSMN1 gene-derived promotor.

Physiological regulation of transgene expression is a major challenge in gene therapy. Onasemnogene abeparvovec (Zolgensma®) is an approved adeno-associated virus (AAV) vector gene therapy for infants with spinal muscular atrophy (SMA), however, adverse events have been observed in both animals and patients following treatment. The construct contains a native human survival motor neuron 1 (hSMN1) transgene driven by a strong, cytomegalovirus enhancer/chicken ß-actin (CMVen/CB) promoter providing high, ubiquitous tissue expression of SMN. We developed a second-generation AAV9 gene therapy expressing a codon-optimized hSMN1 transgene driven by a promoter derived from the native hSMN1 gene. This vector restored SMN expression close to physiological levels in the central nervous system and major systemic organs of a severe SMA mouse model. In a head-to-head comparison between the second-generation vector and a benchmark vector, identical in design to onasemnogene abeparvovec, the 2nd-generation vector showed better safety and improved efficacy in SMA mouse model.