Making CAR T cells in vivo: Cardiac fibrosis is the stiffening and scarring of heart tissue and can be fatal. Rurik et al . designed an immunotherapy strategy to generate transient chimeric antigen receptor (CAR) T cells that can recognize the fibrotic cells in the heart (see the Perspective by Gao and Chen). By injecting CD5-targeted lipid nanoparticles containing the messenger RNA (mRNA) instructions needed to reprogram T lymphocytes, the researchers were able to generate therapeutic CAR T cells entirely inside the body. Analysis of a mouse model of heart disease revealed that the approach was successful in reducing fibrosis and restoring cardiac function. The ability to produce CAR T cells in vivo using modified mRNA may have a number of therapeutic applications. —PNK

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual’s lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP–messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti–human CD117/LNP–based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

Hepatic fibrosis is a key predictor of mortality in liver disease, driven by fibrogenic hepatic stellate cells (HSCs). Targeting these fibrogenic cells may therefore offer a therapeutic approach for hepatic fibrosis. We previously showed that in vivo–generated chimeric antigen receptor (CAR) T cells targeting fibroblast activation protein alpha (FAP) reduced murine cardiac fibrosis. Here, we explored the antifibrotic potential of this in vivo–generated anti-FAP CAR T cell therapy in metabolic dysfunction–associated steatohepatitis (MASH), a highly prevalent disease with no approved antifibrotic therapies. We first established that FAP expression in both human and murine MASH is specific to HSCs. We then used flow cytometry, Sirius Red morphometry, digital pathology analysis, and single nuclear RNA sequencing to assess the impact of anti-FAP CAR T cell therapy on murine MASH. Anti-CD5–targeted lipid nanoparticles carrying anti-FAPCAR messenger RNA transiently generated activated anti-FAP CAR T cells, which substantially reduced fibrosis by depleting profibrogenic HSCs. They also modulated immune cells, endothelial cells, and hepatocytes in a non–cell autonomous manner to mitigate inflammation and restore hepatic homeostasis. These findings highlight the potential of in vivo CAR T therapy to attenuate a highly morbid and pervasive liver disease, not only by directly reducing fibrosis but also through indirect effects on other cell types.

In this study, we aimed to determine the efficacy of in vivo chimeric antigen receptor (CAR) T cell therapy, generated by targeted lipid nanoparticles (t-LNPs), as an anti-fibrotic in metabolic dysfunction-associated steatotic liver disease. Hepatic fibrosis is a key predictor of mortality in liver disease, driven by fibrogenic hepatic stellate cells (HSCs). In heart, chimeric antigen receptor (CAR) T cells targeting fibroblast activation protein alpha (FAP) reduce murine cardiac fibrosis. However, the value of this approach in liver is unknown. We explored the anti-fibrotic potential of in vivo-generated anti-FAP CAR T cells in metabolic dysfunction-associated steatohepatitis (MASH), a highly prevalent disease with no approved anti-fibrotic therapies. We first established that FAP expression in both human and murine MASH is specific to HSCs. We then used flow cytometry, Sirius Red morphometry, digital pathology analysis, and single nuclear RNA-sequencing to assess the impact of anti-FAP CAR T cell therapy on murine MASH. Anti-CD5 targeted-LNPs carrying anti-FAPCAR mRNA generate activated, transient anti-FAP CAR T cells, which significantly reduced fibrosis by depleting pro-fibrogenic HSCs, and by modulating immune cells, endothelial cells and hepatocytes in a non-cell autonomous manner to mitigate inflammation and restore hepatic homeostasis. These findings reinforce the potential of in vivo CAR T therapy to attenuate a highly morbid and pervasive liver disease through integrated, multicellular salutary effects. One Sentence Summary RNA-based treatment transiently reprograms immune cells to target scar-forming cells in fatty liver disease, thus improving liver health overall.

Fibroblast activation protein (FAP), which is highly expressed on cancer-associated fibroblasts (CAF), is a promising therapeutic target to achieve normalization of the tumor microenvironment. We previously established an ex vivo retroviral-transduced FAP-specific chimeric antigen receptor (FAP-CAR) T-cell approach to deplete FAP+ CAFs that resulted in delayed tumor growth associated with disruption of desmoplastic matrix and enhanced immune cell infiltration and reversed immune exclusion and immunosuppression. In this study, we describe an in vivo strategy for generating FAP-CAR T cells using anti-CD5–conjugated targeted lipid nanoparticles (tLNP) encapsulating FAP-CAR mRNA and assessed the efficacy of this approach compared with adoptive transfer of retrovirus-transduced CAR T cells in a preclinical model of pancreatic ductal adenocarcinoma. With transient CAR expression in >45% of splenic, >69% of circulating, and >35% of tumor-infiltrating T cells, the abundance of peripheral and intratumoral FAP-CAR+ T cells detected following a single intravenous dose of FAP-CAR mRNA tLNPs was greater than that detected following administration of 1 × 107ex vivo retrovirally transduced FAP-CAR T cells. Furthermore, in vivo mRNA CAR T-cell engineering resulted in as good or greater inhibition of tumor growth as compared with adoptive transfer of ex vivo retroviral-engineered T cells. Given that in vivo generation of CAR T cells resulted in transient CAR expression and circumvented the need for autologous T-cell isolation, viral vectors, and lymphodepletion, this platform represents a potentially safer, more accessible, and cost-effective method for targeting stromal cells to normalize the tumor microenvironment in desmoplastic tumors and has potential implications for tumor antigen–targeted CAR T cells.