Asparagine-linked (N-linked) glycosylation is ubiquitous and can stabilize immune inhibitory PD-1 protein. Reducing N-linked glycosylation of PD-1 may decrease PD-1 expression and relieve its inhibitory effects on CAR-T cells. Considering that the codon of Asparagine is aac or aat, we wondered if the adenine base editor (ABE), which induces a·t to g·c conversion at specific site, could be used to reduce PD-1 suppression by changing the glycosylated residue in CAR-T cells. Our results showed ABE editing altered the coding sequence of N74 residue of PDCD1 and downregulated PD-1 expression in CAR-T cells. Further analysis showed ABE-edited CAR-T cells had enhanced cytotoxic functions in vitro and in vivo. Our study suggested that the single base editors can be used to augment CAR-T cell therapy.
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Abkürzungen
ABE
Adenine base editor
CAR
Chimeric antigen receptor
CRISPR
Clustered regularly interspaced short palindromic repeats
DSB
Double-stranded break
gRNA
Guide RNA
IFN-γ
Interferon-γ
IL-2
Interleukin-2
To the Editor,
Chimeric antigen receptor T (CAR-T) cells are not satisfying in treating solid tumors [1]. PD-1 limits CAR-T cell therapy within solid tumors. CRISPR/cas9 can downregulate PD-1 [2] but also potentially leads to carcinogenesis, because success of such tools relies on suppressing DNA damage response [3]. Furthermore, CRISPR/cas9 could bring in missense mutations that might exacerbate T cell dysfunction. Hence, we need safer and more precise gene-editing tools to produce better CAR-T cells. N-linked glycosylation can stabilize PD-1 to compromise anti-tumor immunity [4]. As N-linked glycosylation is restricted on asparagine coded by aac or aat, adenine base editors (ABE) can convert a·t to g·c base pair [5], and may be used to diminish such glycosylation. Herein, we explored the potentials of ABE to edit and downregulate PD-1 in CAR-T cells.
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Mutated PD-1 at N74 had decreased surface expression (Fig. 1a). Therefore, N74 in PD-1 is a good target for ABE. Three types of amino acids may be produced after base editing at N74 coded by aac (Fig. 1b). All 3 types of mutations into D74 (gac), S74 (agc), and G74 (ggc) comparably downregulated the surface and total PD-1 (P < 0.001) (Fig. 1c and Additional file 1: Figure S1c). Next, we investigated whether ABE was able to decrease PD-1 in CAR-T cells. The delivery of gRNA using lentivirus is efficient [6], so we constructed lentiviral vectors simultaneously expressing mesothelin-directed CAR and gRNA targeting non-specific sites (scramble) or N74 of PDCD1 (gRNA), under 2 independent promoters (Additional file 1: Figure S1a). T cell transduction efficacies were over 85% (Additional file 1: Figure S1b). Then the commercially synthesized ABE proteins were delivered into CAR-T cells by electroporation. Sequencing data showed the conversion to g majorly happened from the first adenine within N74 codon of PDCD1 in CAR-T cells expressing specific gRNA (Fig. 1d). Conversion was also noticed at the second adenine with lower frequencies (Fig. 1d). This editing pattern is consistent with previous report [7].
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In following experiments, the ratios of CAR-expressing cells were comparably adjusted to 85%. In gRNA CAR-T cells, PD-1 expression was decreased at protein level but not at mRNA level (Fig. 1e and f). Consistently, surface PD-1 was remarkably decreased in resting and activated gRNA CAR-T cells (P < 0.01) (Fig. 1g). Further analysis suggested that ABE editing did not impair the proliferation and activation of CAR-T cells (P > 0.05) (Fig. 1h–j) when PD-L1 was absent. Then mesothelin-positive cells with high PD-L1 expression were prepared (Fig. 2a). After washing out exogenous cytokines, CAR-T cells and target cells were co-incubated. Upon target cell engagement, CAR-T cells divided efficiently (Fig. 2b). Compared with gRNA counterparts, the proliferations of CAR-T cells expressing scramble RNA were significantly suppressed (P < 0.05) (Fig. 2b). gRNA CAR-T cells had enhanced cytolytic capacities (P < 0.05) and increased secretions of IL-2 and IFN-γ (P < 0.05) after activation by tumor cells (Fig. 2c and d). To further confirm the effectiveness of ABE in relieving T cell inhibition, we checked the anti-tumor functions of CAR-T cells in vivo. Consistently, CAR-T cells expressing N74-targeted gRNA attained greater expansion (P < 0.05) (Fig. 2e and Additional file 2: Figure S2). Decreased surface PD-1 (P < 0.01) and upregulated activation markers (CD69 and CD27) (P < 0.05) were noticed on gRNA CAR-T cells (Fig. 2f and g). gRNA CAR-T cells more efficiently delayed tumor growth and improved overall survival when compared with scramble counterparts (P < 0.05) (Fig. 2h–j) (Additional files 3 and 4).
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Single base editing can modulate the stability and function of target protein by changing a single residue [8]. Our work further uncovered the potential of such editing tool in T cells. Compared with CRISPR/cas9, ABE has narrower editing window and much less frequent off-target events [9], representing a safer and more precise approach for gene editing. ABE-mediated point mutation can downregulate the inhibitory PD-1, therefore providing an alternative approach to augment T cell immunotherapy.
We thank Ning Wang for his help in in vivo image. We thank Guohui Qin and Huiyun Yang for their help in animal experiments. We also thank Ting Li for her help in experiment performing.
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Ethics approval and consent to participate
All experiment procedures were followed the protocol approved by institute committee on ethics. And all animal experiments were performed in accordance with the ethics approval obtained from the First Affiliated Hospital of Zhengzhou University Animal Ethics Committee.
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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