How to conduct the genome editing of TCR?

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How to conduct the genome editing of TCR?

01. Introduce

Today, many T cell engineering methods still use viral vectors to deliver transgenes of interest. However, the field of precision genome editing now offers the possibility of precise integration of transgenes into specific genomic sites, while reverse transcription or lentiviral transduction produces random integration with variable transgene copy numbers and expression levels.

The interest of genome editing in the field of engineered T cell therapy is manifold, including

(1) elimination of endogenous TCR and HLA features (for example, universal donor CD19 CAR T cells or UCART19),

(2) elimination of endogenous TCR to enhance the expression and function of the introduced transgenic TCR and avoid cross-reactions,

(3) eliminate immunosuppressive receptors to design resistance to immune checkpoint ligands,

(4) insert CAR or TCR transgene into the TCR locus, use Physiological regulation of transgene expression while eliminating endogenous TCR specificity,

(5) discovering new targets for enhancing combinatorial immune engineering.

How to conduct the genome editing of TCR?

Figure 3. Engineering strategies to enhance the function of TCR transgenic T cells

02. Eliminate endogenous TCR specificity

Several programmable nuclease-based genome editing tools have been developed for targeted destruction of endogenous TCR loci through non-homologous end joining (NHEJ) induction of double-strand breaks and DNA repair (Figure 3f).

These tools include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTAL nucleases, and more recently clustered regulatory interval short palindrome repeats/CRISPR-associated protein 9 (CRISPR /Cas9).

Cas nuclease is guided to the target DNA sequence by a short guide RNA, recognizes the target DNA through Watson-Crick base pairing, and allows multiplexing, while other enzymes rely on protein-DNA interactions to achieve target specificity.

Based on the destruction of the two endogenous TCRβ constant regions (TRBC1 and TRBC2) and the TCRα constant region (TRAC) of ZFN, the tumors generated by the transgenic WT-1 specific TCR were subsequently transduced with lentivirus to redirect T cells without Endogenous TCR specificity.

Compared with TCR+T cells produced by lentiviral transduction alone, TCR+T cells edited by TRAC/TRBC are characterized by enhanced affinity, anti-tumor function, and sharply reduced non-specific alloreactivity in vitro and in vivo.

This seminal paper provides a proof of concept that the destruction of the endogenous TRAC and TRBC loci significantly enhances the safety (by eliminating TCR mismatches), affinity, and anti-tumor functions of T cells edited by the TCR locus.

Subsequently, the same team showed that ZFN-based single editing of the TRAC locus and lentiviral TCR transduction is sufficient to generate safe and highly active tumor-targeting T cells, and its solution is more suitable for clinical transformation.

TALEN, meganuclease, megaTAL nuclease, and CRISPR/Cas9 systems have also been explored to efficiently generate TRAC and/or TRBC locus-edited T cells.

Comparing various platforms, clinical-grade manufacturing scalability, efficiency, and off-target activities were evaluated for TRAC locus disruption, and further optimized by others.

Compared with lentiviral transduction alone, CRISPR/Cas9-mediated TRBC1/2 destruction and lentiviral transduction using transgenic γδ-TCR also resulted in γδ-TCR redirected cells for more effective anti-tumor functions.

03. Genome editing based on multiple CRISPR/Cas9

In order to generate allogeneic universal donor TCR or CAR T cells resistant to PD1-mediated inhibition, a CRISPR-based system was developed by simultaneously destroying TRAC, TRBC, β2-microglobulin (B2M) and PDCD1 loci. /Cas9 multiple editing method (Figure 3f), combined with CAR lentiviral transduction. In preclinical models, combined with PD1 destruction further improved the anti-tumor activity of the edited CAR T cells.

The destruction of other immune checkpoints has been studied in CAR T cells, including CTLA-4 alone and in combination with PD-1 and LAG-3.

Recently, the first human phase I clinical trial of using CRISPR/Cas9 engineered NY-ESO-1 TCR-specific T cells to treat patients with advanced refractory sarcoma and multiple myeloma was reported.

Compared with the previous NY-ESO-T trial, the two main goals of the trial are (1) to improve the safety and effectiveness of NY-ESO-1 TCR T cells, and (2) to limit the development of T cell depletion, TCR and conventional lentivirus transduction.

To this end, CRISPR/Cas9 multiple methods were used to disrupt the TRAC, TRBC, and PDCD1 loci to reduce the possibility of mixed TCR heterodimer formation and to evade checkpoint ligand-mediated T-cell immunosuppression in TME.

The trial proved in three patients that it is feasible and safe to use CRISPR/Cas9 and lentiviral transduction to perform multiple genome editing on T cells.

In the absence of editing, the persistence of infused T cells exceeds the previous clinical results of NY-ESO-1 TCR+ T cells transduced with lentivirus, and no pre-existing immune response to Cas9 was observed.

Potential rejection. So far, off-target editing has not produced safety-related side effects, but patients will need longer follow-up. These promising results are clearly worthy of further investigation.

04. Targeted delivery of transgenes to defined gene loci

Homologous Directed DNA Repair (HDR) can be used to specifically insert a donor DNA template (encoding a sequence of interest) into a target genomic site.

For example, the targeted integration of the CRISPR-based CD19 CAR with the adeno-associated virus 6 (AAV6) TRAC locus resulted in consistent CAR expression, enhanced the anti-tumor function of CAR T cells in vitro and in vivo, and at the same time eliminated endogenous TCR specificity sex.

Compared with constitutively active promoters, placing CAR transgenes under the control of endogenous TCR promoters will result in more physiological CAR expression and antigen-stimulated turnover, thereby delaying the development of terminally differentiated exhausted CAR T cells.

There are also reports that MegaTAL and AAV6 mediated CAR insertion into the TRAC or CCR5 locus.

The use of a non-viral DNA donor template achieves the insertion of tumor-targeted TCR into the TRAC locus (Figure 3f), which significantly improves the flexibility of the method compared with the AAV6 vector.

However, HDR has a low success rate and requires additional optimization. Five different TCRs with antiviral or antitumor specificity were studied.

Interestingly, using this method, TRAC locus editing alone does not completely eliminate the formation of mixed TCR dimers and requires additional TRBC locus destruction. Importantly, the in situ insertion of the transgenic TCR produced nearly physiological TCR regulatory dynamics in all five different TCRs evaluated, underlining the high reproducibility of these results in different TCRs.

CRISPR tools can also be used to design artificial T cell signaling pathways to enhance anti-tumor T cell functions by using endogenous transcriptional regulation of specific responses (Figure 3f).

For example, inserting IL-12p70 into the IL-2Rα or PDCD1 locus will generate T cells with antigen-dependent IL-12p70 production, thereby enhancing the anti-tumor function without significant toxicity.

Therefore, the transcription regulation system that hijacks a specific locus can achieve strict control of transgene expression, and can improve the safety and specificity of functional T cell output.

05. Conclusion

Utilize genome engineering tools to discover and validate new methods faster. Precise modification of gene circuits will open up new possibilities for controlling the function of transgenic T cells, and the first therapeutic genome editing application for identified gene loci in T cells has entered the clinic.

We believe that as we learn how to best manipulate the human immune system to fight cancer, some of these new developments may lead to clinical breakthroughs.

How to conduct the genome editing of TCR?

Original Source: Cells 2020, 9, 1485; doi:10.3390/cells9061485

https://en.wikipedia.org/wiki/Transgene

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