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Cox et al. J Transl Genet Genom 2021;5:80-8 https://dx.doi.org/10.20517/jtgg.2021.06 Page 84

preferentially expand over time. Three patterns were observed: (1) steady proliferation; (2) immediate
[24]
increase followed by a decrease; or (3) steady decline in number . There was no clinical correlation
[24]
associated with the kinetic patterns observed, nor was there a relationship to the vector integration site .
However, single cell RNA-sequencing revealed gene expression patterns associated with cytotoxicity and
proliferation and enrichment of the AP-1 family, CD69, and glycolysis . This suggested preferential
[24]
[24]
expansion of T cell clones with this gene expression signature. They also saw higher expression of CD27 ,
which correlates with previous transcriptomic analysis by Fraietta et al. , which revealed that an enriched
[21]
+
population of CD27 CD45RO CD8 T cells prior to CART cell production were clinically associated with
-
+
better outcomes in CLL patients. Understanding the phenotype or genetic features of the T cell clones with
a better ability to proliferate or survive could inform the generation of a more effective CART cell therapy.
Several strategies to engineer T cells to express the CAR have been employed in the clinic. These include
lentiviral transduction, retroviral transduction, transposon/transposase editing, and targeted CRISPR
editing . To date, the FDA-approved products are engineered using viral vectors. Lentiviruses integrate
[25]
randomly and efficiently into the cell genome. In order to investigate how genetic alterations following
lentivirus regulate CART cell functions in vivo, lentiviral integration sites in the genome of CD8 T cells
+
were examined from a healthy normal donor on day 9 of CART cell production by Wang et al. . They
[26]
were able to simultaneously assess both the accessibility of the chromatin and the site of CAR integration ,
[26]
which can then be measured on individual cells. They describe that integration of the CAR prefers
[26]
inaccessible chromatin, which then leads to gain in chromatin accessibility at that site . Their results
suggest that lentiviruses prefer introns of transcriptionally active genes whereas retroviruses prefer
accessible chromatin regions . This might explain why tonic signaling is amplified in retrovirally
[26]
transduced CAR compared to lentiviral methods . Differentially accessible regions in the chromatin
[27]
revealed clusters of naïve memory or effector T cells . The AP-1 family (BATF and JUN) was associated
[26]
with effector T cells and HMG proteins (TCF-1 and LEF2) associated with naïve or memory T cells .
[26]
Interestingly, both of these studies associated the AP-1 family to activation and expansion signatures in
CART cells.

Activation and expansion are a delicate balance in CART cell therapy. Studies suggest that CART cells that
undergo potent activation such as using CD28 as a co-stimulatory domain instead of 4-1BB may become
[28]
exhausted, which leads to lack of persistence and loss of CART cells . Understanding the genetic signature
and gene regulation responsible for CART cell activation is a start to unraveling this complex problem.
However, more detailed omics-directed studies will be needed to determine what equilibrium is required to
achieve long-term expansion and persistence of CART cells.

TRANSCRIPTOMIC ANALYSIS TO UNCOVER MECHANISMS OF CART CELL TOXICITIES
While CRS and neurotoxicity after CART cell therapy are typically reversible, refractory cases and deaths
have been reported. One study used single-cell sequencing to look at the association between the CART cell
transcriptome and toxicity in patients with DLBCL . Patients who developed high-grade neurotoxicity also
[22]
had a population of cells at baseline with a gene expression signature similar to monocytes (i.e., CD68, LYZ,
[22]
SPI1, LILRB4, and SIPRA) while not expressing the traditional monocyte markers, CD14 and CD16 . This
population also expressed multiple cytokines and chemokines such as IL1B and IL8 . Understanding the
[22]
origin and function of this subset of cells may either help determine risk prior to infusion or lead to
strategies to prevent neurotoxicity . This study corroborates prior correlative and preclinical studies which
[29]
indicated a role for myeloid cells in the development of CRS and neurotoxicity.