A novel immunotherapy strategy that generates transient engineered chimeric antigen receptor (CAR) T cells in vivo can reduce fibrosis and restore cardiac function in a mouse model of heart failure, according to newly reported research headed by scientists at the Perelman School of Medicine at the University of Pennsylvania. The experimental immunotherapy approach is based on the delivery of modified mRNA encapsulated in T cell-targeting lipid nanoparticles (LNPs) to temporarily reprogram the T cells to attack activated heart fibroblast cells. The newly reported mouse studies showed that the reduction in cardiac fibroblasts caused by the reprogrammed CAR T cells led to a dramatic reversal of fibrosis.

The study results suggest that the approach could be useful as a personalized therapeutic platform to treat various other fibrotic diseases or associated disorders. LNP-mRNA technology has also been key to enabling recent COVID-19 vaccine development, the researchers pointed out in their published paper in Science. “Standard CAR T cell technology involves modifying patients’ T cells outside the body, which is expensive and difficult to scale for common diseases or for use in less wealthy countries,” said Drew Weissman, MD, PhD, the Roberts Family professor in vaccine research at Penn. “Making functional CAR T cells inside the body greatly extends the promise of the mRNA/LNP platform.” Weissman is co-author of the researchers’ report, which is titled “CAR T cells produced in vivo to treat cardiac injury,” in which the investigators concluded, “… 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.” The senior study author is Jonathan A. Epstein, MD, CSO for Penn Medicine and executive vice dean and the William Wikoff Smith professor of cardiovascular research in the Perelman School of Medicine.

Cardiac fibrosis is a hallmark of heart disease and plays a critical role in heart failure and death for millions worldwide. Fibrosis occurs when fibroblast cells respond to heart injury and inflammation by chronically overproducing fibrous material that stiffens the heart muscle, impairing heart function. However, therapies targeting cardiac fibrosis remain limited, and demonstrate only a modest positive effect at best. “Fibrosis both stiffens the myocardium and negatively affects cardiomyocyte health and function,” the authors noted. “Despite in-depth understanding of activated cardiac fibroblasts, clinical trials of antifibrotic therapeutics have only demonstrated a modest effect at best. Moreover, interventions have been designed to limit fibrotic progression, and are not designed to remodel fibrosis once it is established.

The new technology developed by Epstein and colleagues is based on CAR T cell technology, which, until now, has required the harvesting of a patient’s T cells and their genetic reprogramming in the lab to recognize markers on specific cell types in the body. These specially targeted T cells can then be multiplied using cell culture techniques and re-infused into the patient to attack a specific cell type. The first CAR T cell therapy was developed by researchers from Penn and Children’s Hospital of Philadelphia and approved by FDA in 2017 for use against certain leukemias—and later approved for lymphoma.

CAR T cell technology is currently used for treating cancers—with dramatic results in many otherwise hopeless cases—though scientists have long envisioned harnessing the approach for other diseases. Epstein and colleagues showed in a 2019 study that the standard CAR T cell approach can be used to attack overactive cardiac fibroblasts and restore heart function in a mouse model of heart failure. However, the researchers noted in their new report, “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.”

Standard CAR T cell therapeutic approaches would be problematic when directed against heart failure or other fibrotic diseases in humans because activated fibroblasts also have an important role in wound healing. CAR T cells that are reprogrammed genetically to attack fibroblasts could survive in the body for months or even years, suppressing the fibroblast population and impairing wound healing over the longer term. As the authors commented, “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.”

For their new strategy, Epstein and colleagues devised a technique for generating a temporary, controllable, and procedurally much simpler type of CAR T cell therapy. They designed mRNA that encodes a T-cell receptor targeting activated fibroblasts and encapsulated the mRNA within lipid nanoparticles that are themselves covered in molecules that home in on T cells. 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/LNPFAPCAR),” they explained. LNP-mRNA technology is also crucial to the mRNA COVID-19 vaccines now in use across the globe, the scientists noted. “LNP-mRNA technology underlies recent successes in COVID-19 vaccine development and holds exceptional promise for additional therapeutic strategies.”

In vivo studies showed that, when injected into mice, the encapsulated mRNA molecules were taken up by T cells and acted as templates for the production of the fibroblast-targeting receptor, effectively reprogramming the T cells to attack activated fibroblasts. This reprogramming strategy is temporary, however. The mRNAs survive within T cells for only a few days—after which the T cells revert to normal and no longer target the fibroblasts.

The scientists found that, despite this brief duration of activity, injections of the mRNA in mice that model heart failure successfully reprogrammed a large population of mouse T cells, causing a major reduction of heart fibrosis in the animals and a restoration of mostly normal heart size and function with no evidence of continued anti-fibroblast T cell activity one week after treatment.

“These experimental results provide a proof of concept that modified mRNA encapsulated in targeted LNPs can be delivered intravenously to produce functional engineered T cells in vivo,” the investigators claimed. “The marked success and safety of modified mRNA/LNP severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has stimulated broad efforts to extend this therapeutic platform to address numerous pathologies.”

“Fibrosis underlies many serious disorders, including heart failure, liver disease, and kidney failure, and this technology could turn out to be a scalable and affordable way to address an enormous medical burden,” Epstein pointed out. “But the most notable advancement is the ability to engineer T cells for a specific clinical application without having to take them out of the patient’s body.”

The researchers are continuing to test this mRNA-based, transient CAR T cell technology, with the hope of eventually starting clinical trials. “By targeting LNPs to specific cell types, as we demonstrate here for lymphocytes, modified mRNA therapeutics are likely to have far-reaching applications,” they further stated. The team acknowledged that further studies will be needed to optimize the dosing strategy, LNP composition, and targeting approaches to optimize therapeutic effects and limit any potential toxicities. Nevertheless, they concluded, “… the possibility of an ‘off-the-shelf’ universal therapeutic capable of engineering specific immune functions provides promise for a scalable and affordable avenue to address the enormous medical burden of heart failure and other fibrotic disorders.”