CRISPR-Edited Immune Effectors: The End of the Beginning

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Commentary
CRISPR-Edited Immune
Effectors: The End of the
Beginning
Feiyan Mo,
1,2
Helen E. Heslop,
1,2
and Maksim Mamonkin
1,2
https://doi.org/10.1016/j.ymthe.2020.03.009
Genome editing has emerged as a powerful
tool to enhance the potency of effector cell
therapies and potentially improve their
safety.
1
Stadtmauer et al.
2
at the University
of Pennsylvania recently reported prelimi-
nary results of a first-in-man clinical study
using CRISPR/Cas9-edited therapeutic
T cells in patients with advanced malig-
nancies. In this study, peripheral T cells
were obtained from patients with multiple
myeloma or sarcoma. To allow their engi-
neered T cells to be more resistant to tu-
mor-mediated immune suppression, the
cells were then activated and treated with
a mixture of CRISPR/Cas9 ribonucleopro-
teins (RNPs) to disrupt the PDCD1 gene
that encodes inhibitory receptor PD-1 as
well as TRAC and TRBC genes. TRAC and
TRBC encode constant regions of endoge-
nous TCR aand bchains, respectively. By
eliminating endogenous T cell receptor
(TCR) expression, the authors aimed to in-
crease the expression of the transgenic NY-
ESO-1/LAGE-1-specific TCR they subse-
quently incorporated by lentiviral transduc-
tion. The resulting NY-ESO-1 TCR-trans-
genic CRISPR triple-edited cells, or “NYCE
cells,”were manufactured for four patients
enrolled in the study, of whom three were
infused. Even though the number of patients
treated with NYCE T cells remains insuffi-
cient to draw definitive conclusions about
the safety of their approach or the biological
role of the mutations introduced, the study
marks an important milestone in the devel-
opment and clinical application of gene-edi-
ted effector cell therapy.
Using recombinant Cas9 complexed with
four distinct single guide RNAs (sgRNAs),
the authors disrupted the TRAC, TRBC,
and PDCD1 genes with efficiencies ranging
from 15% (for the TRBC gene) to 45% (for
the TRAC gene). The average lentiviral
NY-ESO1 TCR transduction efficiency was
3.1%. Highlighting the inhibitory effect of
endogenous TCR and/or PD-1 expression
on the cytotoxicity of TCR-transgenic
T cells, disruption of these genes modestly
increased in vitro killing of tumor by
NYCE T cells. The selected sgRNAs had
minimal off-target activity, as evaluated us-
ing the iGUIDE method, and the identified
non-specific sites were not mapped to known
tumor suppressor genes or oncogenes in
T cells. Of note, chromosomal translocations
that arose from multiple simultaneous dou-
ble-strand breaks in CRISPR-edited T cells
were also detected in these patients. How-
ever, these genome alterations were unlikely
to confer survival or proliferative advantage
to the modified T cells, as the frequency of
both NY-ESO-1 TCR-positive and -negative
T cells carrying edited genes or transloca-
tions gradually decreased after infusion.
Notably, PD-1-deficient T cells did not
expand or persist better than T cells with un-
altered PD-1 expression, consistent with
previous findings that loss of PD-1 may
reduce the ability of T cells to establish a
long-lived memory population.
3
Upon infusion, NYCE T cells expanded and
tracked to tumor sites, stabilizing disease in
two of three patients treated on the protocol.
Although NYCE T cells remained detectable
in circulation for up to 9 months after infu-
sion, at least in one patient with myeloma,
the authors documented downregulation
of NY-ESO-1 and LAGE-1 antigens recog-
nized by the transgenic TCR. Evidence of
antigen escape, which has also been
frequently observed in studies using CAR
T cells for B cell malignancies,
4
likely
contributed to the limited antitumor activity
of NYCE T cells and may warrant the use of
T cells simultaneously targeting multiple
tumor antigens.
Despite the presence of pre-existing anti-
bodies to the Cas9 protein from Strepto-
coccus pyogenes,
5
a component of the normal
commensal flora in humans, no reactivation
of Cas9-specific humoral immunity was
observed after administration of NYCE
T cells. The authors attributed this lack
of response to the minimal presence of
residual Cas9 protein in T cells at the time
of cryopreservation and the transient im-
mune suppression of patients induced by
the lymphodepleting treatment and prior
lymphotoxic therapies. While the authors
did not measure activation of endogenous
Cas9-specific T cells,
6
these responses, even
if present, did not blunt the expansion of
gene-edited T cells in vivo. Overall, these
results indicate genome editing of thera-
peutic T cells ex vivo using CRISPR/Cas9-
sgRNA RNP complexes results in minimal
additional immunogenicity, largely due to a
very transient persistence of electroporated
Cas9 protein in rapidly dividing T cells prior
to infusion.
Numerous proof-of-concept reports have
utilized genome-editing tools in T cells to
minimize the risk of graft-versus-host dis-
ease,
7
tumor immunosuppression,
8,9
and
T cell fratricide,
10,11
among other applica-
tions. Further, commercial and academic
entities have conducted several clinical
studies using gene-edited T cells, including
a landmark report in 2014 by Tebas et al.
12
utilizing zinc-finger nucleases to create
CCR5-edited T cells resistant to HIV infec-
tion. The latest study by Stadtmauer et al.
2
is the first to report the manufacturing,
safety, and clinical activity of CRISPR/
Cas9-edited effector T cells.
1
Center for Cell and Gene Therapy, Baylor College of
Medicine, Houston Methodist Hospital and Texas
Children’s Hospital, Houston, TX, USA;
2
Graduate
Program in Translational Biology and Molecular
Medicine, Baylor College of Medicine, Houston, TX,
USA
Correspondence: Feiyan Mo, Center for Cell and
Gene Therapy, Baylor College of Medicine, Houston
Methodist Hospital and Texas Children’s Hospital,
Houston, TX, USA.
E-mail: feiyan.mo@bcm.edu
Molecular Therapy Vol. 28 No 4 April 2020 ª2020 The American Society of Gene and Cell Therapy. 995

Recent technological advances could further
improve the efficiency and precision of
multiplexed genome editing and minimize
its side effects. Examples include employing
high-fidelity versions of Cas9 with reduced
off-target activity,
13
utilizing base editing
in lieu of non-homologous end-joining
(NHEJ) repair to minimize CRISPR-induced
double-strand breaks and the resulting
chromosomal translocations,
14,15
or knock-
ing in desired constructs to replace endoge-
nous genes via homology-directed recombi-
nation (HDR).
16
With the first clinical
testing of CRISPR/Cas9-edited therapeutic
T cells, this pioneering study of NYCE
T cells is perhaps “the end of the beginning”
of the era of gene-edited immune effectors.
AUTHOR CONTRIBUTIONS
F.M., H.E.H., and M.M. wrote the
commentary.
DISCLOSURES
H.E.H. is a founder with equity in Allovir
and Marker Therapeutics; has served on
advisory boards for Gilead, Novartis, Tessa
Therapeutics, Kiadis, and PACT Pharma;
and receives research support from Tessa
Therapeutics and Kuur Therapeutics.
ACKNOWLEDGMENTS
The National Cancer Institute
(P50CA126752), the SU2C/AACR (604817)
Meg Vosburg T cell Lymphoma Dream
Team, and the Leukemia and Lymphoma So-
ciety supported the authors.
REFERENCES
1. Bailey, S.R., and Maus, M.V. (2019). Gene editing for
immune cell therapies. Nat. Biotechnol. 37, 1425–
1434.
2. Stadtmauer, E.A., Fraietta, J.A., Davis, M.M., Cohen,
A.D., Weber, K.L., Lancaster, E., Mangan, P.A.,
Kulikovskaya, I., Gupta, M., Chen, F., Tian, L., et al.
(2020). CRISPR-engineered T cells in patients with
refractory cancer. Science 367, eaba7365.
3. Odorizzi, P.M., Pauken, K.E., Paley, M.A., Sharpe, A.,
and Wherry, E.J. (2015). Genetic absence of PD-1
promotes accumulation of terminally differentiated
exhausted CD8+ T cells. J. Exp. Med. 212, 1125–1137.
4. Majzner, R.G., and Mackall, C.L. (2018). Tumor anti-
gen escape from CAR T-cell therapy. Cancer Discov.
8, 1219–1226.
5. Charlesworth, C.T., Deshpande, P.S., Dever, D.P.,
Camarena, J., Lemgart, V.T., Cromer, M.K.,
Vakulskas, C.A., Collingwood, M.A., Zhang, L.,
Bode, N.M., et al. (2019). Identification of preexisting
adaptive immunity to Cas9 proteins in humans. Nat.
Med. 25, 249–254.
6. Wagner, D.L., Amini, L., Wendering, D.J., Burkhardt,
L.M., Akyüz, L., Reinke, P., Volk, H.D., and
Schmueck-Henneresse, M. (2019). High prevalence
of Streptococcus pyogenes Cas9-reactive T cells
within the adult human population. Nat. Med. 25,
242–248.
7. Poirot, L., Philip, B., Schiffer-Mannioui, C., Le Clerre,
D., Chion-Sotinel, I., Derniame, S., Potrel, P., Bas, C.,
Lemaire, L., Galetto, R., et al. (2015). Multiplex
Genome-Edited T-cell Manufacturing Platform for
“Off-the-Shelf”Adoptive T-cell Immunotherapies.
Cancer Res 75, 3853–3864.
8. Menger, L., Sledzinska, A., Bergerhoff, K., Vargas,
F.A., Smith, J., Poirot, L., Pule, M., Hererro, J.,
Peggs, K.S., and Quezada, S.A. (2016). TALEN-
Mediated Inactivation of PD-1 in Tumor-Reactive
Lymphocytes Promotes Intratumoral T-cell
Persistence and Rejection of Established Tumors.
Cancer Res. 76, 2087–2093.
9. Ren, J., Liu, X., Fang, C., Jiang, S., June, C.H., and
Zhao, Y. (2017). Multiplex Genome Editing to
Generate Universal CAR T Cells Resistant to PD1
Inhibition. Clin. Cancer Res. 23, 2255–2266.
10. Gomes-Silva, D., Srinivasan, M., Sharma, S., Lee,
C.M., Wagner, D.L., Davis, T.H., Rouce, R.H., Bao,
G., Brenner, M.K., and Mamonkin, M. (2017).
CD7-edited T cells expressing a CD7-specific CAR
for the therapy of T-cell malignancies. Blood 130,
285–296.
11. Gomes-Silva, D., Atilla, E., Atilla, P.A., Mo, F.,
Tashiro, H., Srinivasan, M., Lulla, P., Rouce, R.H.,
Cabral, J.M.S., Ramos, C.A., et al. (2019). CD7 CAR
T Cells for the Therapy of Acute Myeloid Leukemia.
Mol. Ther. 27, 272–280.
12. Tebas, P., Stein, D., Tang, W.W., Frank, I., Wang,
S.Q., Lee, G., Spratt, S.K., Surosky, R.T., Giedlin,
M.A., Nichol, G., et al. (2014). Gene editing of
CCR5 in autologous CD4 T cells of persons infected
with HIV. N. Engl. J. Med. 370, 901–910.
13. Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai,
S.Q., Nguyen, N.T., Zheng, Z., and Joung, J.K.
(2016). High-fidelity CRISPR-Cas9 nucleases with
no detectable genome-wide off-target effects. Nature
529, 490–495.
14. Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and
Liu, D.R. (2016). Programmable editing of a target
base in genomic DNA without double-stranded
DNA cleavage. Nature 533, 420–424.
15. Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa,
A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson,
C., Newby, G.A., Raguram, A., and Liu, D.R. (2019).
Search-and-replace genome editing without double-
strand breaks or donor DNA. Nature 576, 149–157.
16. Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der
Stegen, S.J.C., Hamieh, M., Cunanan, K.M., Odak,
A., Gönen, M., and Sadelain, M. (2017). Targeting a
CAR to the TRAC locus with CRISPR/Cas9 enhances
tumour rejection. Nature 543, 113–117.
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Commentary
996 Molecular Therapy Vol. 28 No 4 April 2020