T cells to fix a broken heart
- Making CAR T cells in vivo
- Abstract
- Acknowledgments
- Supplementary Materials
- References and Notes
- 0eLetters
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
Abstract
Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell–targeted lipid nanoparticles (LNPs). The efficacy of these in vivo–reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.Cardiac fibroblasts become activated in response to various myocardial injuries through well-studied mechanisms including transforming growth factor β–SMAD2/3, interleukin-11, and other interactions with the immune system (1–6). In many chronic heart diseases, these fibroblasts fail to quiesce and secrete excessive extracellular matrix, resulting in fibrosis (7). Fibrosis both stiffens the myocardium and negatively affects cardiomyocyte health and function (8). Despite in-depth understanding of activated cardiac fibroblasts, clinical trials of antifibrotic therapeutics have only demonstrated a modest effect (5, 7) at best. Furthermore, these interventions aim to limit fibrotic progression and are not designed to remodel fibrosis once it is established. To address this substantial clinical problem, we recently demonstrated the use of chimeric antigen receptor (CAR) T cells to specifically eliminate activated fibroblasts as a therapy for heart failure (9). Elimination of activated fibroblasts in a mouse model of heart disease resulted in a significant reduction of cardiac fibrosis and improved cardiac function (9). One caveat of that work is the indefinite persistence of engineered T cells similar to CAR T cell therapy currently used in the oncology clinical setting (10). Fibroblast activation is part of a normal wound-healing process in many tissues, and persistent antifibrotic CAR T cells could pose a risk in the setting of future injuries. Therefore, we leveraged the power of nucleoside-modified mRNA technology to develop a transient antifibrotic CAR T therapeutic.Therapeutic mRNAs can be stabilized by the incorporation of modified nucleosides, synthetic capping, and the addition of lengthy poly-A tails, and can be enhanced with codon optimization (11–13). 1-Methylpseudouridine integration also boosts translation (13, 14). Direct introduction of mRNA into T cells ex vivo by electroporation has been used successfully by our group and others to make CAR T cells (15); however, this process carries significant cost and risk and requires extensive infrastructure. Thus, we developed an approach that could be used to avoid removing T cells from the patient by packaging modified mRNAs in lipid nanoparticles (LNPs) capable of producing CAR T cells in vivo after injection. LNP-mRNA technology underlies recent successes in COVID-19 vaccine development and holds exceptional promise for additional therapeutic strategies (16–20). Once in the body, mRNA-loaded LNPs, absent of any specific targeting strategies, are endocytosed by various cell types (especially hepatocytes if injected intravenously) (21, 22). Shortly after cellular uptake, the mRNA escapes the endosome, releasing the mRNA into the cytoplasm, where it is transiently transcribed before degrading (11). Targeting antibodies can be decorated on the surface of LNPs to direct uptake (and mRNA expression) to specific cell types (23, 24). We hypothesized that an LNP directed to T lymphocytes could deliver sufficient mRNAs to produce functional CAR T cells in vivo (Fig. 1A). Because mRNA is restricted to the cytoplasm and is incapable of genomic integration, intrinsically unstable, and diluted during cell division, these CAR T cells will be, by design, transient.
We generated modified nucleoside-containing mRNA encoding a CAR designed against fibroblast activation protein (FAP) (a marker of activated fibroblasts) and packaged it in CD5-targeted LNPs (referred to as “targeting antibody/LNP-mRNA cargo” or CD5/LNP-FAPCAR) (Fig. 1A) (9, 25). CD5 is naturally expressed by T cells and a small subset of B cells and is not required for T cell effector function (26, 27). As a first proof-of-concept experiment, we incubated CD5/LNPs containing modified mRNA encoding either FAPCAR or green fluorescent protein (GFP) with freshly isolated, activated murine T cells in vitro for 48 hours. CD5-targeted LNPs delivered their mRNA cargo to most T cells in culture, where 81% expressed GFP after exposure to CD5/LNP-GFP (Fig. 1B) and 83% expressed FAPCAR after exposure to CD5/LNP-FAPCAR (Fig. 1, C and D), as measured by flow cytometry (fig. S1A). In vitro, CAR expression peaks at 24 hours and rapidly abates over the ensuing days (fig. S1B). LNPs decorated with isotype control [immunoglobulin G (IgG)] antibodies, and thus not explicitly directed to lymphocytes, were only able to deliver mRNA to a small fraction (7%) of T cells in vitro (Fig. 1, C and D). These LNP-generated CAR T cells were able to effectively kill FAP-expressing target cells in vitro (Fig. 1E) in a dose-dependent manner (fig. S1C) similar to virally engineered FAPCAR T cells. Gene transfer through targeted LNPs in vitro is also possible and efficient (89 to 93%) in human T cells, as demonstrated by targeting ACH2 cells with CD5/LNP-GFP (fig. S1D).We next assessed whether CD5-targeted LNP mRNA could also efficiently reprogram T cells in vivo. Mice that were intravenously injected with CD5/LNPs containing luciferase mRNA (CD5/LNP-Luc) were found to express abundant luciferase activity in their splenic T cells, whereas mice injected with isotype control (nontargeting) IgG/LNP-Luc did not (Fig. 2A). Bioluminescence imaging demonstrated spleen targeting only in CD5/LNP-Luc–treated animals (fig. S2A). Liver expression of LNP-delivered mRNA was observed in both CD5/LNP-Luc– and IgG/LNP-Luc–treated animals, as expected mainly due to normal hepatic clearance of LNPs, as reported previously (22, 24). In another experiment, CD5/LNPs were loaded with mRNA encoding Cre recombinase (CD5/LNP-Cre) and injected into Ai6 Cre-reporter mice (Rosa26CAG-LSL-ZsGreen). We found evidence of genetic recombination (ZsGreen expression) specifically in CD3+ T cells (both CD4+ and CD8+ subsets) from CD5/LNP-Cre–injected animals but little evidence of Cre recombinase activity in CD3– (non-T) cells (mainly representing B cells, dendritic cells, and macrophages) or in IgG/LNP-Cre–injected mice (Fig. 2B). We next investigated whether targeted LNPs could deliver FAPCAR mRNA (CD5/LNP-FAPCAR) to T cells in an established murine hypertensive model of cardiac injury and fibrosis produced by constant infusion of angiotensin II/phenylephrine (AngII/PE) through implanted 28-day osmotic mini-pumps (9, 28). Mice were injured for 1 week to allow fibrosis to be established before injecting CD5/LNP-FAPCAR (9). Forty-eight hours after LNP injection, we found a consistent population of FAPCAR+ T cells (17.5-24.7%) exclusively in mice that received CD5/LNP-FAPCAR (Fig. 2, C and D, and fig. S2B). By contrast, nontargeted (IgG/LNP-FAPCAR) and targeted LNPs containing GFP (CD5/LNP-GFP) did not produce FAPCAR T cells (Fig. 2, C and D, and fig. S2B). We observed FAPCAR expression in each major T cell subset with a slight enrichment in CD4+ T cells above their prevalence in the spleen (of all FAPCAR T+ cells, 87% were CD4+ and 9 to 10% CD8+, with most of both classes portraying a naïve phenotype; 25 to 37% of regulatory T cells were FAPCAR+; fig. S2C and table S1). A mixture of CAR+ T cell subtypes has been shown to benefit CAR effectiveness (29). We did not observe significant FAPCAR expression in splenic B cells or natural killer cells (fig. S2C). No FAPCAR expression was found in splenic T cells 1 week after injection, demonstrating the transient nature of FAPCAR expression in this model (table S1)…..
