INSIGHTS | PERSPECTIVES
GRAPHIC: A. MASTIN/SCIENCEscience.org SCIENCEmouse model of hypertensive cardiac injury
( 6 ). In this prior work, T cells were stably
integrated ex vivo with the CAR-encoding
transgene through retroviral transduction,
resulting in long-term CAR expression.
Extensive clinical experience in the context
of cancer therapy has shown that autolo-
gous CAR T cells can persist for months or
years after adoptive transfer into the pa-
tient, and this long-term persistence plays
an important role in therapeutic efficacy
and durability of response ( 7 ). However,
cardiac injury has a different temporal pro-
file compared with cancer. Although FAP is
generally expressed at low levels in healthy
tissue, it is up-regulated during the normal
wound-healing process, which involves fi-
broblast activation ( 8 ). As such, long-term
persistence of FAP-targeting CAR T cells
could present a safety risk if the patient
sustains injury after treatment.
To balance the need for robust CAR T
cell activity against pathogenic fibroblasts
while minimizing long-term safety risks,
Rurik et al. developed a method to gener-
ate transient CAR T cells in vivo, bypassing
the need for ex vivo cell manufacturing (see
the figure). In this approach, mRNA that is
chemically modified for stability and en-
codes the FAPCAR is packaged into LNPs
decorated with CD5-targeting antibod-
ies to facilitate selective uptake by T cells.
Upon LNP uptake, the mRNA is translated
to generate CAR T cells. Given the inher-
ent instability of mRNA, CAR expression
is transient, and no T cell will remain per-
sistently CAR+ by design. Rurik et al. con-
firmed the emergence of anti-FAP CAR T
cells in mice 24 hours after LNP injection,
as well as the decline of these CAR T cells
to undetectable levels within 7 days. Using
a mouse model of hypertensive cardiac in-
jury and fibrosis, the authors demonstrated
that transient FAPCAR T cells substantially
reduce ventricular fibrosis and improve
various cardiac functions, as assessed by
echocardiography. These findings provide a
rationale for the use of transient CAR T cells
and demonstrate the applicability of CAR T
cell therapy beyond oncology applications.
Heart disease remains the leading cause
of death globally, requiring therapies that
can be manufactured at large scale and at
reasonable cost. The success of the COVID-19
mRNA-LNP vaccines ( 9 ) suggests that LNP-
based mRNA delivery could be a viable path
to the development of scalable immunother-
apies, providing a more economical alterna-
tive to the conventional method of ex vivo
CAR T cell manufacturing that requires ex-
tensive infrastructure and high-cost reagents
( 10 ). In vivo CAR T cell generation had previ-
ously been reported with the use of adeno-
associated viruses instead of mRNA-LNPs
( 11 ). A potential advantage of the mRNA-LNP
platform is the ability to flexibly decorate the
LNP with antibodies to increase target speci-
ficity, as well as greater ease of clinical-grade
reagent production. Furthermore, compared
with conventional ex vivo CAR T cell manu-
facturing methods, the virus-free, in vivo
CAR T cell manufacturing method detailedby Rurik et al. eliminates the risk of unin-
tended host-genome alterations mediated by
lentiviral or retroviral integration ( 12 ) as well
as the need for lymphodepleting chemother-
apy before adoptive transfer, thus removing
a source of considerable toxicity that is typi-
cally associated with CAR T cell therapies.
FAP-targeting CAR T cells were previously
evaluated, with some groups reporting mini-
mal side effects ( 13 ) and others reporting le-
thal bone toxicity and cachexia (muscle wast-
ing) in mice due to interactions with bone
marrow stem cells ( 14 ). No overt toxicity was
observed by Rurik et al., and LNP-mediated
anti-FAP CAR expression was largely re-
stricted to the spleen. Nevertheless, these dis-
crepancies in toxicity must be carefully evalu-
ated before translation into the clinic, and a
more in-depth characterization of the inter-
action between FAPCAR T cells and activated
fibroblasts within the fibrotic tissue will be
necessary to demonstrate antigen-specific
CAR T cell activity at the disease location.
Additionally, although Rurik et al. demon-
strated that interstitial fibrosis was largely
reduced with mRNA-LNP treatment, it was
noted that perivascular fibrosis persisted
beyond the treatment duration owing to the
presence of activated fibroblasts that do not
express FAP. Perivascular fibrosis and associ-
ated inflammation can decrease the contact
area of neighboring cardiac tissue with blood
vessels and reduce oxygen and nutrient avail-
ability that may exacerbate pathological car-
diac remodeling ( 1 ). Further investigation
into the biology of perivascular fibrosis could
uncover additional therapeutic targets.
The work of Rurik et al. provides a strong
rationale for the broadening of immunother-
apies into disease areas with unmet needs,
and FAPCAR T cells have already been evalu-
ated in the context of cancer ( 15 ). Other fi-
brotic diseases or associated disorders, such
as chronic inflammatory diseases with FAP+
fibroblasts, may benefit from this approach
as well. This work represents an exciting
step toward translating personalized immu-
notherapies into accessible and affordable
“off-the-shelf ” immunotherapies. jREFERENCES AND NOTES- J. G. Travers et al., Circ. Res. 118 , 1021 (2016).
- J. G. Rurik et al., Science 375 , 91 (2022).
- M. Hong et al., Cancer Cell 38 , 473 (2020).
- M. C. O’Leary et al., Clin. Cancer Res. 25 , 1142 (2019).
- M. Seif et al., Front. Immunol. 10 , 2711 (2019).
- H. Aghajanian et al., Nature 573 , 430 (2019).
- L. Jafarzadeh et al., Front. Immunol. 11 , 702 (2020).
- P. Bainbridge, J. Wound Care 22 , 407, 410 (2013).
- W. C. Koff et al., Sci. Transl. Med. 13 , eabd1525 (2021).
- S. Rafiq et al., Nat. Rev. Clin. Oncol. 17 , 147 (2020).
- W. Nawaz et al., Blood Cancer J. 11 , 119 (2021).
- R. M. David, A. T. Doherty, Toxicol. Sci. 155 , 315 (2017).
- L. C. S. Wang et al., Cancer Immunol. Res. 2 , 154 (2014).
- E. Tran et al., J. Exp. Med. 210 , 1125 (2013).
- S. Kakarla et al., Mol. Ther. 21, 1611 (2013).
10.1126/science.abn0851T cellFAPCARCAR expressionRibosomeEndosomal
escapeEndocytosisT cell
trafficking
to the heartFA PActivated
fibroblastmRNA degradation and dilution
during cell divisionNatural loss of CAR expressionActivated fibroblast lysisReduced cardiac brosis1 day after
LNP injection7 days after
LNP injectionLNPInjected LNPsmRNATranslation24 7 JANUARY 2022 • VOL 375 ISSUE 6576
In vivo generation of transient engineered T cells
Messenger RNA (mRNA) that encodes fibroblast activation protein (FAP)–targeted chimeric antigen receptor
(CAR) is carried by lipid nanoparticles (LNPs). These are injected into mice and delivered to T cells, resulting
in transient anti-FAP CAR expression. The anti-FAP CAR directs T cells to eliminate activated fibroblasts and
alleviate cardiac fibrosis in a mouse model of hypertensive cardiac injury.