Macrophage manufacturing and engineering with 5′-Cap1 and N1-methylpseudouridine-modified mRNA

Abstract

Macrophage-based cell therapeutics are an emerging modality to treat cancer and repair tissue damage. A reproducible manufacturing and engineering process is central to fulfill their therapeutical potential. Here, we establish a robust macrophage manufacturing platform (Mo-Mac), and demonstrate that macrophage functionality can be enhanced by N1-methylpseudouridine (m1Ψ)-modified mRNA. Using single-cell transcriptomic analysis as an unbiased approach, we found that >90% cells in the final product were macrophages, and the rest primarily comprised T cells, B cells, natural killer cells, promyelocytes, promonocytes and hematopoietic stem cells. This analysis also guided the development of flow cytometry strategies to assess cell compositions in the manufactured product to meet requirements by the National Medical Products Administration. To modulate macrophage functionality, as an illustrative example, we examined whether the engulfment capability of macrophages could be enhanced by mRNA technology. We found that efferocytosis was increased in vitro when macrophages were electroporated with m1Ψ-modified mRNA encoding CD300LF (CD300LF-mRNA-macrophage). Consistently, in a mouse model of acute liver failure, CD300LF-mRNA-macrophage facilitated organ recovery from acetaminophen-induced hepatotoxicity. These results demonstrate a GMP-compliant macrophage manufacturing process, and indicate that macrophage can be engineered by versatile mRNA technology to achieve therapeutic goals.