Analysis on How CRISPR Technology Facilitates Anticancer

Therapeutics

Sitian Wang

Faculty of Medicine, Imperial College London, SW7 2AZ, London, U.K.

Keywords: Component, CRISPR, Immunotherapy, Cancer, TCR Therapy, CAR-T Therapy.

Abstract: As a novel treatment modality, immunotherapy, based on the principle of boosting the antitumour response,

help patients to fight against cancer. Out of many different types of immunotherapies, adoptive T cell therapy,

characterized by enhancing immunity and specificity by ex vivo manipulation on patient-derived T cells, has

aroused great attention of scholars, who expect to apply it to future cancer treatment. Several CAR-T therapies

have been officially approved in clinical use. Nevertheless, post-transfer T cell exhaustion and

immunosuppression within the tumour region still constitute the major technical limitations. As an

indispensable gene-editing tool with strong capacity in both biomedical research and clinical fields, CRISPR

technology has strong potentials in facilitating adoptive T cell therapies to overcome the current barriers

through full gene knockout on the engineered T cells. In this paper, the clinical feasibility and future prospect

of the combined use of CRISPR-Cas9 and adoptive T cell therapies are analyzed using two latest studies for

discussion and comparison. The studies indicate that CRISPR-Cas9 has facilitated to increase T cell

persistence and potency in TCR therapy and CAR-T therapy respectively with acceptable safety profile, and

it has shed the light for clinical use of CRISPR-Cas9 to increase the therapeutic effectiveness of adoptive T

cell therapy. Future investigations are still needed to further assess its clinical safety and to understand the

underlying mechanism of how CRISPR-Cas9 helps to extend the survival and increase anti-tumour response

of the T cells within the tumour region.

1 INTRODUCTION

CRISPR, which stands for Clustered Regularly

Interspaced Short Palindromic Repeats, is an

indispensable tool in biological research. It was

firstly found in archaea by Mojica et al. in 1995

(Mojica, Ferrer, Juez and Rodríguez-Valera 1995),

and then later experimentally verified by Barrangou

et al. in 2007 as the adaptive immune system of

bacteria to fight against the invading viruses

(Barrangou, Fremaux, Deveau, Richards, Boyaval

and Moineau et al 2007). After years of development,

CRISPR technology can now be modified to target

specific sequence of the genetic code and perform

gene-editing at a relatively precis location, and it has

been widely applied in various field including

biomedicine and agriculture. The function of

CRISPR technology was mainly dependent on the

CRISPR-associated (Cas) genes flanked by the

sequence of CRISPR. Out of the various types of

CRISPR-technology, the most widely applied one is

CRISPR-Cas9, where Cas9 is an endonuclease

guided by sgRNA (single guide RNA).

When Cas9 enters the nucleus, sgRNA facilitates

the recognition of the PAM sequence (protospacer

adjacent motif) and the target sequence, which

consequently leads to the activation of PAM-

dependent Cas9 nuclease. The consequent induced

DNA cleavage will generate double strand breaks

(DSB) and activate homologous recombination (HR)

or non-homologous end joining (NHEJ) to achieve

gene-editing (Sternberg, Redding, Jinek, Greene and

Doudna 2014). Compared with previous gene-

editing tools like Zinc Finger Nucleases (ZFNs) and

Transcription activator-like effector nucleases

(TALENs), CRISPR technologies allow rapid

retargeting of DNA sequence without the

requirement for manufacturing novel proteins for

each target site. Such advantage and the high

genome-editing efficiency of CRISPR allows it to

facilitate the progress in oncology research and

anticancer therapies through different modalities. In

addition to genetic screening to discover potential

128

Wang, S.

Analysis on How CRISPR Technology Facilitates Anticancer Therapeutics.

DOI: 10.5220/0011197300003444

In Proceedings of the 2nd Conference on Artiﬁcial Intelligence and Healthcare (CAIH 2021), pages 128-133

ISBN: 978-989-758-594-4

therapeutic targets, CRISPR technologies can be

used to generate cell lines with specific gene

deletions or to manipulate multiple genes to explore

human malignancies. Compared with the traditional

method of manipulations of germline cells to

introduce driver mutation, CRISPR-Cas 9 can

establish cancers more directly in animal models,

which is less time consuming. More importantly, the

combination of CRISPR-Cas9 and cancer

immunotherapy can be a powerful therapeutic

strategy in increasing clinical safety and efficacy

(Yin, Xue and Anderson 2019). The aim of this

review is to analyse the clinical feasibility of using

CRISPR-Cas9 in cancer immunotherapy, especially

in adoptive T cell therapy, and discuss the current

limitation and the prospect of this direction in the

future. Two latest studies are analyzed in this review

to discuss how CRISPR-Cas9 has facilitated

adoptive T cell therapy, where they used CRISPR-

Cas9 to ex vivo knock out the genes of T cells to

increase its therapeutical effectiveness. With the

progress in gene-editing technologies and advances

in immunotherapy, the investigations so far show that

the combined use of CRISPR-Cas9 and

immunotherapy has high translational potential for

clinical use, and some of them have even been

proved safe and effective in incipient clinical pilot

study. Future studies still need to be carried out to

expand our understanding in the underlying

mechanism of the engineered T cells in tumour

microenvironment and further prove its feasibility

for wide clinical use.

2 CRISPR-CAS9 IN TCR

THERAPY AND CAR-T

THERAPY

2.1 CRISPR-Cas9 Knockout of the

Gene Encoding PD-1 And

Endogenous TCR Increases the

Persistence of the Engineered T

Cells in TCR Therapy

Adoptive T cell therapy is a type of immunotherapy

that involves the direct extraction of T cells from the

patient and conduct certain manipulation to increase

its anti-tumour effectiveness. One of the adoptive T

cell therapy called engineered T cell receptor (TCR)

therapy, where T cells from the patients are isolated

and genetically manipulated in vitro to express the

synthetic T cell receptor that specifically target the

cancer cells. However, previous studies have

illustrated that the expression of α and β chains in

endogenous TCR is related with the reduced the

expression of therapeutic TCR due to the competitive

expression, and programmed cell death protein 1

(PD-1) is negatively associated with the antigen

response and persistence of the engineered T cells in

the tumour region (Hamilton, Doudna 2020). The

consequently reduced therapeutic efficacy

constituted the major limitation of TCR therapy, and

CRISPR-Cas9 may be a promising approach to

overcome it through disrupting the genes associated

with T cell exhaustion and the reduced antigen

response.

In an article published in Science in 2020,

Stadtmauer et al. conducted the first-in-human phase

1 clinical trial, where they aimed to investigate the

use of CRISPR-Cas9 in improving the effectiveness

and safety of TCR therapy on patients with advanced

and refractory cancer (Stadtmauer, Fraietta, Davis,

Cohen, Weber and Lancaster et al 2020). They

hypothesised that the deletion of the genes encoding

PD-1 and the α and β chain in endogenous TCR

would improve the persistence of the engineered T

cells and increase the feasibility of the initial TCR

therapy. Referring to the graphical abstract (see

Fig.1), CRISPR-Cas9 was used to knock out TRAC,

TRBC, and PDCD1 over isolated T cells from the

patients, and then they introduced the synthetic TCR

transgene NY-ESO-1, which can specifically target at

myeloma, melanoma, and sarcoma, through

lentiviral transduction into the cells. The CRISPR-

Cas9-engineered T cells were later infused back into

the 3 patients with advanced and refractory cancer.

The T cells were then tracked and monitored in vivo

to determine if they could persist longer with better

safety profile after the CRISPR-Cas9 modification.

The results of the In vitro assessment indicated that

cells modified by CRISPR-Cas9 has higher

cytotoxicity than those retained the endogenous

TCR, suggesting a higher potency. After the cell

infusion, there is no evidence of T cell genotoxicity

or overt side effect observed in the patients, and high

level and sustained persistence of the engineered

cells were found with antigen-specific cytotoxicity.

Overall, the results of this trial overall indicated the

acceptable safety profile of the CRISPR-modified

transgenic T cells with higher sustained level and

higher specificity targeting the tumour. However, the

potential mechanism of the extended survival of the

T cells was not investigated in this study, and whether

the longer half-life of the engrafted transgenic T cells

is attributed to PD-1 deficiency was not explained.

Hence the transcriptional state of the modified T cells

Analysis on How CRISPR Technology Facilitates Anticancer Therapeutics

129

within the tumour micro-environment can be

investigated next step to see how the cytotoxicity and

persistence are increased. In addition, only one

patient out of the total three had the highest level of

engraftment, which restricted the in vivo single-cell

analysis, herein more patients for infusion of the cells

with higher editing efficiencies and engraftment

level are needed to fully assess the safety and

feasibility in the use of CRISPR-Cas9 in TCR

therapy in the future.

Figure 1: Graphical Abstract of using CRISPR-Cas9 to knock out the gene encoding PD-1 and endogenous TCR (Stadtmauer,

Fraietta, Davis, Cohen, Weber and Lancaster et al 2020).

2.2 CRISPR-Cas9 Improves the

Efficacy of CAR-T Therapy via

Knockout of Adenosine Receptor

Similarly, CRISPR-Cas9 also shows great potential

in improving the effectiveness of CAR-T therapy,

which is another type of adoptive T cell therapy.

Similar to TCR therapy, CAR-T therapy involves the

process of T cell extraction with ex vivo transduction

of the T cell with chimeric antigen receptor (CAR),

which can specifically recognize a defined tumour

antigen. Compared to TCR therapy, the introduction

of CAR transgene in TRAC gene can knock out the

gene encoding TCR simultaneously (Roth, Puig-

Saus, Yu, Shifrut, Carnevale and Li et al 2018).

However, one of the major barriers of CAR-T

therapy is the effect of immunosuppression. Out of

the multiple immunosuppressive pathways, the

hypoxia-adenosine link is relatively prominent in

tumour region, where adenosine binding to the

receptor A2AR reduces the accumulation of

intracellular cAMP and increases the production of

anti-inflammatory factors in immune cells to

suppress the immune response (Raker, Becker and

Steinbrink 2016). Previous studies indicated that

pharmacological blockade of A2AR is able to

enhance the T-cell-mediated antitumour effect

(Halpin-Veszeleiova, Hatfield 2020). Hence, these

findings suggest the potential of targeting the

immunosuppressive pathway via CIRSPR-Cas9 to

improve the efficacy of adoptive T-cell therapy.

In May 2021, an article published in Nature by

Giuffrida et al. It indicated that A2AR deletion can

enhance the efficacy of CAR-T therapy via CRISPR-

Cas9, which further proves the strong potential of the

use of CRISPR technology in improving anticancer

therapies compared with other methodologies

(Giuffrida, Sek, Henderson, Lai, Chen and Meyran et

al 2021). They delivered the recombinant Cas9 and

sgRNA targeting at the gene encoding A2AR into

naïve splenocytes via electroporation, followed by

retroviral transduction of CAR targeting human Her2

cells. With the aim to investigate whether CRISPR-

Cas9-mediated A2AR deletion could enhance CAR-

T cell function, the level of cAMP signalling, in vivo

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antitumour efficacy in mice models, and

transcriptional profile of the engineered T cells were

all assessed and analysed. It was then found the

editing efficiency of CRISPR-Cas9 on the gene

encoding A2AR could achieve more than 75% in

human CAR-T cells and result in potent attenuation

in the level of intracellular cAMP. Because of the

high sensitivity of A2AR towards adenosine, A2AR

knockdown via short hairpin RNA (shRNA) or

pharmacological blockade is not effective enough in

attenuating the conversion of ATP to cAMP. Instead,

the full knockout of A2AR is sufficient to

significantly suppress the immunosuppressive

pathway mediated by adenosine in the function of

CAR-T cells. The survival of the tumour bearing

mice was significantly prolonged due to the

enhanced inhibition of tumour growth by CAR-T

cells after A2AR deletion, and the memory recall

responses was able to get evoked. Through the

analysis of the transcriptional profile, the suppressive

effect on the production of pro-inflammatory

cytokines like IFNγ and TNF by CD4+ and CD8+

mediated by hypoxia-adenosine pathway was also

significantly reduced, indicating the enhanced

therapeutic efficacy. It has been found that the

increased CAR-T cell activation mediated by the

knockdown or knockout of A2AR could enhance the

expression of several effector-related genes

including PD-1, granzyme B and Ki-67, which may

compromise the persistence the T cells. Surprisingly,

the deletion of A2AR by CRISPR-Cas9 had minimal

effect in the persistence of the CAR-T cells compared

with the control group, unlike knockdown or

pharmacological blockade. However, the

mechanisms that full knockout of A2AR by

CRISPR-Cas9 uses to circumvent the reduction in

persistence is unknown yet, and it may be related

with the production of pro-survival factors in

memory T cells. For next step, it would be interesting

to analyse the difference in the expression of the

memory associated genes between knockdown and

knockout CAR-T cells to investigate the underlying

mechanisms of the uncompromised persistence in

CRISPR-modified CAR-T cells.

3 DISCUSSIONS

As discussed above, T-cell exhaustion and

immunosuppression are major technical barriers that

result in reduced efficacy and potency of the adoptive

T cell therapy when the cells are infused back into

the patients. Gene-editing technology like CRISPR-

Cas9 can be an influential tool to overcome these

limitations with increasing editing efficiencies and

precisions over these years. Herein, two latest studies

from 2020 and 2021 were discussed in this review to

demonstrate how CRISPR-Cas9 helps with

improving the effectiveness of CAR-T therapy and

TCR therapy.

In these 2 studies, different pathways were

targeted with similar aims, and both show promising

future for clinical application. The first study used

CRISPR-Cas9 to prevent the engineered T cell

exhaustion through suppressing the apoptosis

pathway via PD-1 knockout and to improve the

expression of the synthetic TCR through disrupting

the expression of endogenous TCR, and it has been

proved safe and effective to improve the cell

persistence in the first-in-human pilot study.

Whereas the second study distinctively targeted the

immunosuppressive pathway via A2AR knockout to

increase the antitumour response, and it has been

proved effective in improving the potency of the

CAR-T cells in animal models. This has shed the

light of utilizing CRISPR-Cas9 to target multiple

immunosuppressive genes to improve the therapeutic

efficacy of CAR-T therapy in the future. In contrast

to the first study, the second study showed that A2AR

deletion via CRISPR-Cas9 in T cells has increased

the expression of PD-1. However, the persistence of

the CAR-T cells is not significantly affected but with

increased cytokine production, and this achieved a

well-balanced trade-off between cell persistence and

therapeutic efficacy. Compared with other

immunosuppressive pathways, the adenosine-

activated pathway is more prominent in hypoxia

tumour microenvironment, which also equips it with

advantageous efficacy profile. In terms of frequency

of editing, the editing efficiencies of TRAC, TRBC,

and PDCD in the first study are 45%, 15%, and 20%

respectively, and it might be due to the limited

progress in the CRISPR-based technology back in

2016 when their clinical trial application was

approved, leading to higher off-target effect.

Whereas the editing efficiency of A2AR reached

over 75% in the latest paper here, suggesting the

strong potential of CRISPR technology in facilitating

anticancer therapies with increasing on-target editing

efficiency over the years.

However, safety considerations are still important

considerations regarding the permanent deletion of

certain genes using CRISPR-Cas9. There are still

limited studies using gene-editing technologies to

target immunosuppressive pathways in CAR-T cells.

Despite the in vivo assessment in mice models

proved that A2AR-edied CAR-T cells are well

tolerated with good safety profile through liver and

Analysis on How CRISPR Technology Facilitates Anticancer Therapeutics

131

kidney toxicity analysis. Whether permanent A2AR

deletion will induce excessive immune response

against the host still needs further assessment and

observation. In addition, PD-1 is not the only

indicator of T-cell exhaustion which is also related

with other immunoregulatory pathways like soluble

factors IL-10 and regulatory T cells (Wherry 2011).

Moreover, the underlying mechanism of increased

persistence and improved potency of the modified T

cells after PD-1 and A2AR knockout are not clear

yet. Hence, despite the positive conclusion in early

clinical trials and incipient in vivo assessment, the

wide feasibility and long-term safety of CRISPR-

modified engineered T cells still needs future

investigations.

Nowadays, CRISPR-based technology has

shown promising future in improving the

effectiveness of immunotherapy to enhance its

efficacy and reduce the toxicity. Immunotherapy is

mainly based on the innate and adaptive immune

system of the host to activate the specific immune

response or reduce the immunosuppressive effect,

including monoclonal antibodies, vaccine therapies,

checkpoint inhibitors, and adoptive T cell therapies.

The general principle of adoptive T cell therapy is to

boost the antitumour response through ex vivo

manipulation of the T cells to increase their ability

and specificity targeting the tumour when they are

infused back into the patient. The modalities include

ex vivo expansion of tumour-infiltrating

lymphocytes (TILs), the introduction of transgenic

TCR that targets the major histocompatibility

complex (MHC) to eradicate tumour cells, and gene

transfer of chimeric antigen receptors that target

specific antigen presented on the surface of the

tumour cells . With the high response rate compared

with conventional chemotherapy, adoptive T cell

therapy has made huge progress in recent years.

Several therapies have been approved for clinical use

with large number of them in the stage of clinical

trials. Notwithstanding such success in its early

clinical application, adoptive T cell therapy are still

facing various challenges. For example, CAR-T

therapy is mainly used in haematological cancers,

because of the high heterogeneity of the solid

tumours leading to challenges for transgenic TCR

targeting the tumours. Moreover, the nature of

adoptive T cell therapy being highly personalized

also make the manufacturing cost unexpectedly high

and difficult to get industrialized. Hence allogeneic

CAR T cells from have become a promising direction

to reduce the manufacturing cost and simplify the

procedure. However, the main barrier of using

allogeneic T cells from healthy donors are the

rejection by the immune system of the recipient and

the toxicity resulted from the non-self antigen grafted

from the recipient to the donor cells (Graham,

Jozwik, Perpper and Benjamin 2018). Thus, in

addition to improving the efficacy of the current

adoptive T cell therapies, CRISPR-Cas9 can also be

a useful tool to knock out the related genes expressed

on the surface of the allogeneic T cells to reduce the

rejection and toxicity (see Fig.2) and make the

therapies “off -the-shelf” for most of the patients.

Figure 2: Graphical Abstract of using CRISPR-Cas9 on allogeneic T cells for CAR-T cells (Yin, Xue and Anderson 2019).

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4 CONCLUSIONS

To sum up, this paper discusses the clinical feasibility

of CRISPR-Cas9 being applied into adoptive T cell

therapy and addresses the limitations of the current

investigations. Gene-editing technology has overall

showed high clinical translational potential in

overcoming T cell exhaustion and

immunosuppression to improve the therapeutical

effectiveness of adoptive T cell therapy. Moreover, it

is also promising to contribute to the development of

allogeneic CAR-T therapy through reducing the

toxicity and rejection effect via gene knockout and to

further simplify the manufacturing process.

However, safety considerations are still major

concerns regarding the application of gene editing

technologies. The underlying mechanisms of

CRISPR-Cas9 in improving adoptive T cell therapies

are still not fully understood yet, and the clinical

feasibility and safety of CRISPR-Cas9 application in

anticancer therapies still need further investigations,

despite the incipient success.

REFERENCES

C.Graham, A. Jozwik, A. Pepper, R. Benjamin. Allogeneic

CAR-T Cells: More than Ease of Access?. Cells.

2018;7(10):155.

E. Stadtmauer, J. Fraietta, M. Davis, A. Cohen, Weber,

Lancaster E et al. CRISPR-engineered T cells in

patients with refractory cancer. Science. 2020;

367(6481): eaba7365.

E.Wherry. T cell exhaustion. Nature Immunology. 2011;

12(6):492-499.

F. Mojica, C. Ferrer, G. Juez, F. Rodríguez-Valera. Long

stretches of short tandem repeats are present in the

largest replicons of the Archaea Haloferax

mediterranei and Haloferax volcanii and could be

involved in replicon partitioning. Molecular

Microbiology. 1995;17(1):85-93.

H. Yin, W. Xue, D. Anderson. CRISPR–Cas: a tool for

cancer research and therapeutics. Nature Reviews

Clinical Oncology. 2019;16(5):281-295.

J. Hamilton, J. Doudna. Knocking out barriers to

engineered cell activity. Science. 2020;367(6481):976-

977.

K.Halpin-Veszeleiova, S. Hatfield. Oxygenation and

A2AR blockade to eliminate hypoxia/HIF-1α-

adenosinergic immunosuppressive axis and improve

cancer immunotherapy. Current Opinion in

Pharmacology. 2020; 53:84-90.

L.Giuffrida, K. Sek, M. Henderson, J. Lai, A. Chen, D.

Meyran et al. CRISPR/Cas9 mediated deletion of the

adenosine A2A receptor enhances CAR T cell efficacy.

Nature Communications. 2021;12(1).

R. Barrangou, C. Fremaux, H. Deveau, M. Richards, P.

Boyaval, S. Moineau et al. CRISPR Provides Acquired

Resistance Against Viruses in Prokaryotes. Science.

2007;315(5819):1709-1712.

S. Sternberg, S. Redding, M. Jinek, E. Greene, J. Doudna.

DNA interrogation by the CRISPR RNA-guided

endonuclease Cas9. Nature. 2014;507(7490):62-67.

T. Roth, C. Puig-Saus, R.Yu , E.Shifrut , J. Carnevale, Li P

et al. Reprogramming human T cell function and

specificity with non-viral genome targeting. Nature.

2018;559(7714):405-409.

V. Raker, C. Becker, K.Steinbrink. The cAMP Pathway as

Therapeutic Target in Autoimmune and Inflammatory

Diseases. Frontiers in Immunology. 2016;7.

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