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CRISPR/Cas9 and adoptive T cell therapy
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CRISPR/Cas9 and adoptive T cell therapy.
In recent years, adoptive T cell ( ACT ) immunotherapy has shown great potential in opening up and accelerating the field of tumor treatment, and has achieved unprecedented breakthroughs in clinical practice.
As a multi-purpose gene editing technology, the CRISPR-CAS9 system has laid a solid foundation for effective innovation in tumor research and tumor treatment.
The combination of CRISPR/Cas9 and tumor adoptive T-cell immunotherapy, two revolutionary technologies in tumor research and treatment, may further broaden the application of immunotherapy in more cancer patients.
The CRISPR/Cas9 system is essentially a bacterial defense mechanism against foreign DNA.
CRISPR was first discovered in the late 1980s as an unusual genetic structure composed of alternately repeated and non-repetitive DNA sequences.
Genomic analysis shows that CRISPR and Cas proteins function as an adaptive immune system and protect prokaryotic DNA from phage and plasmid DNA through an RNA-guided DNA cleavage system.
When foreign DNA invades bacteria, its DNA fragments are incorporated into the CRISPR site as spacers.
This site is then transcribed into CRISPR pre-CRISPR RNA ( crRNA ), which is attached to the constitutively formed trans-activating RNA ( tracrRNA ), which is modified into gRNA by CRISPR-related proteins.
When the gRNA binds to the REC I domain of the inactive Cas9 complex, the complex is activated, causing the gRNA and its complementary single-stranded DNA to form a heteroduplex.
The HNH and RuvC nuclease domains of Cas9 then cleave complementary and non-complementary DNA strands, respectively.
Cas9 belongs to type II of the type 1 CRISPR-Cas system and is derived from Streptococcus pyogenes.
The coupling of Cas9 to the target sequence requires a protospacer proximity motif ( PAM ), a 3-8bp DNA sequence in a small invasion genome.
PAM is essential in distinguishing bacteria’s own and non-self DNA and successfully binding Cas9.
Unlike ZFN and TALEN, which rely on the fine protein product of each specific gene sequence, the CRISPR/Cas9 system relies on gRNAs, making it a more flexible platform.
The Cas9 protein remains unchanged, while gRNA can be easily customized for each gene.
Another advantage of the CRISPR/Cas9 system is that it makes it possible to perform gene editing on multiple loci at the same time.
Compared with previous systems, it provides a more efficient and scalable platform.
Application of CRISPR/CAS9 in CAR-T therapy
At present, CAR-T cell therapy has shown unprecedented efficacy in hematological malignancies. However, despite the impressive clinical results, many patients still cannot benefit from CAR-T cell therapy due to various reasons.
First, the personalized method of making T cells is time-consuming and expensive, which prevents many patients, especially those with rapid disease progression, from making full use of this immunotherapy;
Second, during the production process, it is difficult for patients with lymphopenia to produce enough With high-quality T cells, even if patients get enough immune cells, these cells may not be able to complete the entire manufacturing process; finally, the heterogeneity of autologous CAR-T products may lead to unpredictable and variable clinical results.
CRISPR/Cas9 generates universal allogeneic CAR-T cells
In order to overcome the obstacles that limit the wide application of CAR-T cell therapy, a variety of treatment strategies have been proposed.
One of the most feasible and durable methods is to generate allogeneic universal CAR-T cells from healthy donors.
Compared with autologous CAR-T cells, “off-the-shelf” allogeneic CAR-T cells have many potential advantages, including the ability to immediately provide cryopreserved CAR-T cells to patients in urgent need, and enough for the first infusion or re-administration The quantity and production of CAR-T cells are standardized.
Considering the presence of endogenous HLA and TCR on donor T lymphocytes, the biggest challenge for universal products is the potential risk of allogeneic reactions and graft-versus-host disease ( GVHD ).
With the advancement of gene editing technology, people have gradually achieved the eradication of endogenous TCR.
Through zinc finger nucleases ( ZFNs ) and transcriptional activator-like effector nucleases ( TALENs ), people have created CAR-T cells with TCR knockout.
However, the production of fully allogeneic CAR-T cells requires knocking out TCR and HLA molecules and transducing CAR at the same time, which requires a more efficient and precise gene editing technology to achieve.
Compared with ZFNs and TALENs, CRISPR/Cas9 has good flexibility and high efficiency, and has a wide range of applications in the preparation of allogeneic CAR-T cells. CRISPR/Cae9 can knock out multiple gene loci simultaneously and efficiently.
Ren et al. used CRISPR/Cas9 to generate CAR-T cells that simultaneously knock out endogenous TCR, HLA-I and PD-1, which showed strong anti-tumor activity in vitro and in animal models.
CRISPR/Cas9 weakens inhibitory checkpoint molecules
Although significant success has been achieved in hematological malignancies, there are still many patients with unsatisfactory CAR-T cell therapy due to the immunosuppressive tumor microenvironment and T cell failure.
Due to the important role of co-inhibitory molecules ( such as PD-1, CTLA-4, LAG-3 and TIM-3 ) in T cell dysfunction, CRISPR/Cas9 has also been used to destroy these suppressor genes to enhance CAR-T cell function .
CRISPR/Cas9 promotes T cell cytokine production
Cytokines are essential in regulating the function of T cells. The intensity of TCR stimulation greatly affects the secretion of cytokines.
CAR-T cells that structurally express IL-12 and IL-15 can improve anti-tumor activity and long-term durability.
In addition, IL-18 has been shown to promote the production of IFN-γ and the proliferation of CAR-T cells.
Therefore, viral transduction can be used to artificially induce cytokine production; however, cytokine overexpression may lead to T cell exhaustion or autoimmunity.
CRISPR/Cas9 can also be used to knock-in the required cytokine genes at specific sites and make their secretion strictly controlled by the built-in promoter.
Diacylglycerol kinase ( DGK ) is an enzyme that down-regulates diacylglycerol ( DAG ), DAG is the second messenger formed by downstream TCR activation, and the absence of DGK can enhance T cell activity.
Studies have proved how DGKA/DGKB-DKO anti-EGFRvIII CAR-T cells can increase the production of IFN-γ/IL-2 and in vitro cytotoxicity and induce resistance to TGF-β and prostaglandin E2. In the glioblastoma xenograft mouse model, these CAR-T cells also produced promising results in tumor regression.
Application of CRISPR/CAS9 in TCR-T cell therapy
As we all know, CAR-T cells have a limited role in solid tumors. Lack of tumor-specific antigens, tumor antigen heterogeneity and tumor microenvironment suppression are the main reasons.
Compared with CAR-T immunotherapy, engineered TCR-T cell therapy has greater prospects in targeting a wider range of antigens, thereby expanding the scope of cancer treatment.
However, one of the main problems of TCR-T cell therapy is the presence of endogenous TCR on the recipient T cells. Endogenous TCRs compete with transgenic TCRs for CD3 binding.
In addition, the mismatch between endogenous TCR and transgenic TCR may also lead to the formation of mixed TCR dimers.
To solve these problems, the CRISPR/Cas9 system was used to replace the endogenous TCR α and β genes with genes with artificial tumor-specific TCR sequences.
Compared with traditional TRC-T cells, knocking out endogenous TCR can increase the expression and function of transgenic TCR.
In addition, the anti-tumor response of CRISPR/Cas9 modified T cells was enhanced in animal models.
In order to reduce the risk of mismatching, another strategy is to replace it with a stable Vα/Vβ single-stranded TCR (Sc TCR).
According to reports, transduction of Sc-TCRs via CRISPR/Cas9 almost completely eliminates TCR mismatches. In 2020, TCR-T cells edited by CRISPR/Cas9 will be tested in refractory cancer patients for the first time to evaluate their safety and feasibility.
By isolating the patient’s autologous T cells, lentiviral transduction expresses NY-ESO-1 and LAGE-1 specific TCR, and then using CRISPR/Cas9 to destroy the endogenous TCRs and PD-1 genes.
CRISPR-modified T cells were expanded in vitro and then returned to 3 patients. Two patients are in stable condition, and the other patient’s condition is progressing.
The clinical status of CRISPR/CAS9 in the treatment of ACT
In the past decade, many clinical trials have been initiated to evaluate the feasibility, safety and effectiveness of ZFNs, TALENs and CRISPR gene editing platforms in a variety of diseases, such as β-thalassemia, AIDS, and hemophilia B , Mucopolysaccharidosis ( MPS ), sickle cell anemia and various malignant tumors.
ACT treatment of cancer is one of the main focuses of recent gene editing trials.
Researchers at the Chinese People’s Liberation Army General Hospital started the first human phase I clinical trial of CRISPR ( NCT02793856 ) in 2016, which evaluated PD-1-KO primary T cells in patients with stage IV metastatic non-small cell lung cancer. Safety and efficacy.
However, the study showed low non-targeting effects and no serious treatment-related side effects. However, the effectiveness of this study is not clear because the clinical efficacy of the treatment has not been investigated.
In the same year, three other phase I clinical trials were started to evaluate PD-1-KO primary T cells in stage IV bladder cancer ( NCT02863913 ), metastatic renal cell carcinoma ( NCT02867332 ) and hormone refractory prostate cancer ( NCT02867345 ) , But all these trials were later withdrawn.
TIL and primary T cells constitute only a small part of CRISPR-mediated ACT trials, and most clinical trials focus on CAR-T/TCR-T cells.
In an open phase I study ( NCT03398967 ), the safety and effectiveness of CRISPR/Cas9 engineered universal CD19/CD22 CAR-T cells were evaluated in 6 patients with relapsed/refractory acute lymphoblastic leukemia.
Studies have shown that there are no adverse reactions related to GVHD or gene editing, however, all patients have CRS. The therapy also showed significant anti-tumor activity, with 83% of patients achieving complete remission after 28 days of infusion.
In addition, with regard to T cell malignancies, gene editing can be used to eliminate the expression of CD7, an attractive marker that is highly expressed in T cell tumors.
Cooper et al. designed functional UCART7 to eliminate T-ALL in vitro/in vivo while avoiding the risk of GVHD; this has become the basis of current clinical trials for patients with T-cell leukemia or lymphoma ( NCT03690011 ).
CRISPR/Cas9, which weakens immune checkpoints, is also being studied in clinical trials.
Such as the destruction of PD-1 by CRISPR/Cas9 to produce mesothelin-directed CAR-T cells ( NCT03747965 ) and UCART ( NCT03545815 ) are being tested.
A recent clinical trial is investigating the efficacy of using edited endogenous hematopoietic progenitor kinase 1 ( HPK1 ) in CD19-CAR-T cells ; HPK1 is a kinase that negatively regulates TCR activation signals, so it is a A new target for immunotherapy.
Challenges and limitations of CRISPR/CAS9 in ACT treatment
Although CRISPR/Cas9 has shown great potential in improving the effect of immunotherapy, some issues related to safety and effectiveness hinder its transformation into clinical applications.
First of all, ACT-based tumor immunotherapy, including CAR-T cell, TCR-T cell and TILs therapy, its clinical efficacy depends on sufficient T cells. However, most of the CRISPR engineered T cells used in clinical trials are transduced by electroporation, which may cause cell damage and hinder the proliferation of T cells in vitro. Therefore, safer and more efficient delivery methods such as viral vectors need to be explored urgently.
In addition, the immunogenicity of the cas9 protein may be another challenge that limits the clinical transformation of CRISPR/cas9. Anti-SaCas9 and anti-SpCas9 antibodies were detected in 78% and 58% of donors, indicating that humans have a pre-existing adaptive immune response to the Cas9 protein, which may cause adverse reactions when CRISPR/Cas9 treats patients.
Second, although various methods have been reported to improve gRNA design and increase the specificity of Cas enzymes, the risk of off-target effects due to non-specificity is still the main obstacle to the transformation of CRISPR/Cas9 into clinical applications.
Although there are no obvious or only a small number of off-target sites, chromosomal rearrangements or deletions in clinical trials, more patient experience and longer follow-up are still needed to further verify its safety and feasibility.
CRISPR/Cas9 is one of the most important discoveries in the new century. It will completely change the world. Treating and eliminating diseases is only one of its prospects.
Thanks to CRISPR, gene therapy is developing rapidly. ACT is a revolutionary method in fighting cancer.
However, since its introduction, it has been facing many obstacles, such as limited efficacy on solid tumors, T cell exhaustion and loss of function, allogeneic T cell-related GVHD, and manufacturing costs.
Although the versatile CRISPR platform is not perfect, it has the potential to overcome these obstacles faced by ACT.
By using CRISPR technology to eliminate MHC and endogenous TCR expression, the potential GVHD associated with allogeneic T cell therapy is minimized; CRISPR can also be used to optimize T cell function and reduce depletion by destroying immune checkpoint proteins; it will be more effective Therapeutic T cells translate into lower cell doses, lower toxicity, and lower cost.
Although most clinical trials are still in the early stages, as more and more clinical trials are carried out, and the preliminary results of some trials have proved the feasibility and effectiveness, the prospect of CRISPR/Cas9-assisted ACT cancer treatment is worth looking forward to.
1.CRISPR/Cas9 revitalizes adoptive T-celltherapy for cancer immunotherapy. J Exp Clin Cancer Res. 2021; 40: 269.
2. CRISPR/Cas9 Gene-Editing in Cancer Immunotherapy: Promoting the Present Revolution in Cancer Therapy and ExploringMore. Front Cell Dev Biol. 2021; 9: 674467.
CRISPR/Cas9 and adoptive T cell therapy
(source:internet, reference only)