structure, we observe constitutive engage-
ment of the RGS7-Gb5 complex by GPR158 in
the absence of a G protein. We speculate that
binding of a ligand to the ECD would acti-
vate GPR158 rearrangement of the cytoplas-
mic domains that engage RGS to alter its
activity. Given that RGS binding precludes
GPR158 from canonical activation of G pro-
teins, one can describe it as an RGS-coupled
receptor.
In addition to providing information on
the GPCR-RGS structure, we show the role
of two phospholipids in organizing the dimer-
ization interface of GPCRs. These lipids staple
the protomers and provide intriguing possi-
bilities for GPCR modulation. We also iden-
tify a Cache domain, raising the possibility
that GPR158 detects a small-molecule li-
gand that could regulate the RGS module—an
avenue to be explored in future studies. We
hope our findings will spur further progress
in understanding the regulatory and signal-
ing mechanisms of GPR158 by facilitating the
structure-based discovery of its ligands and by
guiding the exploration of GPR158-mediated
control of RGS proteins in the endogenous
neuronal setting.
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ACKNOWLEDGMENTS
We are indebted to our colleagues at The Scripps Research
Institute, E. S. Rangarajan and M. Candido Primi, for their daily
help throughout the project, which led to the generation of
homogeneous samples required for high-resolution cryo-EM single-
particle analyses as well as generous and unlimited access to their
dedicated equipment that was necessary to obtain the cryo-EM
structure reported here. We thank A. Wier, T. Edwards, and U. Baxa of
the NCI National CryoEM Facility for data collection, and D. Kumar
Jaijyan for help with the cryo-EM sample.Funding:Supported by
NIH grants MH105482 (K.A.M.) and GM127883 (J.F.H.); the National
Cancer Institute’s National CryoEM Facility at the Frederick
National Laboratory for Cancer Research under contract
HSSN261200800001E; grants from the US Department of
Defense, NSF, NIH, and start-up funds provided to The Scripps
Research Institute from the State of Florida (T.I.); and Wellcome
Trust grant 104633/Z/14/Z. A.K.S. is a IYBA and Ramalingaswamy
DBT fellow and is supported by the SERB-SRG funding agency
(SERB/SRG/2020/000266). For the purpose of Open Access,
the author has applied a CC BY public copyright license to any Author
Accepted Manuscript version arising from this submission.Author
contributions:D.N.P. and K.A.M. conceived the project; D.N.P.
performed the constructs’design and cloning, preliminary screening
of constructs, and generation of BacMam viruses, protein expression
and production, protein purification for cryo-EM and biophysical
studies, buffer optimization and preliminary screening of samples for
EM study, model building and structural analysis, and mutagenesis
and biochemical experiments; S.S. performed final cryo-EM grid
preparation; D.N.P. and S.S. performed EM data processing;
T.L. performed functional experiments; T.S.S. performed cross-
linking MS; S.J.N. performed HDX-MS; X.Q. and D.W. performed
lipidomics; J.F.H. supervised cryo-EM studies; T.I. guided
biochemical protein purification experiments and sample
preparation for the EM; P.R.G. supervised cross-linking MS
and HDX-MS; C.V.R. supervised lipidomics studies; A.K.S.
supervised cryo-EM studies and built the atomic models; D.N.P.
and K.A.M. wrote the manuscript with input from all otherauthors; K.A.M. supervised the overall project implementation.
Competing interests:The authors declare that they have no
competing interests.Data and materials availability:The
cryo-EM density maps and coordinates have been deposited in
the Electron Microscopy Data Bank (EMDB) and Protein Data
Bank (PDB), respectively, with accession codes EMD-25125
and 7SHE for GPR158 apo, and EMD-25126 and 7SHF for the
GPR158-RGS7/Gb5 complex. The cross-linking mass spectrometry
proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE ( 24 ) partner repository with the
dataset identifier PXD026603. Raw HDX-MS data are deposited
at https://figshare.com/s/56c03387d510fd39eb08. Raw
lipidomics data are deposited at https://figshare.com/s/
f78c92eca1f90fe485d3.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abl4732
Materials and Methods
Figs. S1 to S13
Tables S1 and S2
References ( 25 – 52 )
Movies S1 and S2
MDAR Reproducibility Checklist15 July 2021; accepted 8 November 2021
Published online 18 November 2021
10.1126/science.abl4732CELL AND GENE THERAPYCAR T cells produced in vivo to treat cardiac injury
Joel G. Rurik1,2,3, István Tombácz^4 †, Amir Yadegari^4 †, Pedro O. Méndez Fernández1,2,3,
Swapnil V. Shewale^2 , Li Li1,2, Toru Kimura^4 ‡, Ousamah Younoss Soliman^4 , Tyler E. Papp^4 ,
Ying K. Tam^5 , Barbara L. Mui^5 , Steven M. Albelda4,6, Ellen Puré^7 , Carl H. June^6 , Haig Aghajanian1,2,3*,
Drew Weissman^4 *, Hamideh Parhiz^4 *, Jonathan A. Epstein1,2,3,4*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.C
ardiac fibroblasts become activated in
response to various myocardial inju-
ries through well-studied mechanisms
including transforming growth factor
b–SMAD2/3, interleukin-11, and other
interactions with the immune system ( 1 – 6 ).
In many chronic heart diseases, these fibro-
blasts 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 demon-
strated a modest effect ( 5 , 7 ) at best. Further-
more, 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 dem-
onstrated 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 mouseSCIENCEscience.org 7 JANUARY 2022•VOL 375 ISSUE 6576 91
(^1) Department of Cell and Developmental Biology, Perelman
School of Medicine at the University of Pennsylvania,
Philadelphia, PA, USA.^2 Penn Cardiovascular Institute,
Perelman School of Medicine at the University of
Pennsylvania, Philadelphia, PA, USA.^3 Institute for
Regenerative Medicine, Perelman School of Medicine
at the University of Pennsylvania, Philadelphia, PA, USA.
(^4) Department of Medicine, Perelman School of Medicine at
the University of Pennsylvania, Philadelphia, PA, USA.
(^5) Acuitas Therapeutics, Vancouver, British Columbia V6T
1Z3, Canada.^6 Center for Cellular Immunotherapies,
Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, USA.^7 Department of Biomedical
Sciences, School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, PA, USA.
*Corresponding author. Email: [email protected]
(H.A.); [email protected] (D.W.); parhizh@
pennmedicine.upenn.edu (H.P.); [email protected] (J.A.E.)
†These authors contributed equally to this work.
‡Present address: Department of General Thoracic Surgery, Osaka
International Cancer Institute, Osaka, Japan.
RESEARCH | REPORTS