Programmable base editing of mutated TERT promoter inhibits brain tumour growth

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR interference and programmable base editing have transformed the manipulation of eukaryotic genomes for potential therapeutic applications1,2,3,4. Here, we exploited CRISPR interference and programmable base editing to determine their potential in editing a TERT gene promoter-activating mutation, which occurs in many diverse cancer types, particularly glioblastoma5,6,7,8. Correction of the −124C>T TERT promoter mutation to −124C was achieved using a single guide RNA (sgRNA)-guided and catalytically impaired Campylobacter jejuni CRISPR-associated protein 9-fused adenine base editor (CjABE). This modification blocked the binding of members of the E26 transcription factor family to the TERT promoter, reduced TERT transcription and TERT protein expression, and induced cancer-cell senescence and proliferative arrest. Local injection of adeno-associated viruses expressing sgRNA-guided CjABE inhibited the growth of gliomas harbouring TERT-promoter mutations. These preclinical proof-of-concept studies establish the feasibility of gene editing as a therapeutic approach for cancer and validate activated TERT-promoter mutations as a cancer-specific therapeutic target.

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Fig. 1: Programmable base editing of mutated TERT promoter abrogates binding of ETS1 and GABPA to the promoter.
Fig. 2: Programmable base editing of mutated TERT promoter inhibits TERT expression.
Fig. 3: Mutated TERT promoter-targeted PBE reduces telomere length and induces tumour-cell senescence and proliferation inhibition.
Fig. 4: Mutated TERT promoter-targeted PBE inhibits brain tumour growth.

Data availability

High-throughput sequencing data have been deposited to the NCBI Sequence Read Archive database under accession no. PRJNA597961. Source data are available online for Figs. 14 and Extended Data Figs. 14. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank V. Varghese for technical assistance, C. Li (The University of Texas MD Anderson Cancer Center) for providing U87 cells expressing luciferase, and C. Kang and C. Fang (Tianjin Medical University) for providing PDX GBM cells. This work was supported by the Key Program of the Chinese Academy of Sciences grant no. KJZD-SW-L05 (to X.L.). R.A.D. is supported by an R01 grant (grant no. R01CA84628). Z.L. is a Kuancheng Wang Distinguished Chair.

Author information

Z.L. and X.L. conceived the study. Z.L., R.A.D., X.L. and X.Q. designed the study and wrote the manuscript with comments from all authors. X.L., X.Q., B.W., Y.X., Y.Z., L.D. and D.Xu performed the experiments. R.A.D. and D.Xing provided reagents and conceptual advice.

Correspondence to Xinjian Li or Zhimin Lu.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 PBE Corrects the -124 C>T Mutation in TERT Promoter.

a, The DNA region spanning the mutation at chromosome 5, 1,295,113 C>T (-124 C>T) in the TERT promoter locus of the indicated cells was genotyped. Arrows indicate the mutation. b, Diagram of HA–CjABE targeting the -124 C>T mutation under the guidance of a designed sgRNA expressed in an adeno-associated viral vector. HA-tagged CjABE was expressed under the control of the EF-1α core promoter. SgRNA targeting the TERT promoter mutation or a control sgRNA were expressed under the control of the U6 promoter. Both expression cassettes (EF-1α-HA–CjABE and U6-sgRNA) were inserted into an AAV type 2 vector and packaged into virions for cell infection. CjABE, which were expressed by AAVs, bound to the mutated TERT promoter and converted the targeted A base to I via deamination and, subsequently, to C via mismatch repair of tumour cells, leading to correction of the targeted T•A base pair to C•G in the mutant TERT promoter locus, thereby abrogating ETS-driven TERT transcription. c, The indicated cells were infected with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 for 72 h. Results of immunoblot analyses using the indicated antibodies are shown. WB, Western blot. The experiment was repeated three times independently with similar results. d–f, Diagram of infection of GBM cells with AAV virus and calculation of MOI is depicted (d). GBM cells were infected by lentivirus expressing puromycin gene (0.01 MOI). After puromycin selection, the expanded and cloned cells were co-infected with AAV viruses expressing CjABE and sgRNA (control sgRNA or 124 sg RNA). CjABE and sgRNA were driven by EF1α and U6 promoter, respectively. qPCR analyses were used to determine the copy numbers of puromycin gene and AAV genes basing on the indicated standard curves (e). MOIs of AAV were determined by the copy numbers of AAV genes vs the copy numbers of puromycin gene (f). The depicted results are the averages from three independent experiments. Values are means ± s.d. g, The indicated cells were infected with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 for 72 h. The DNA region spanning the mutation at chromosome 5 (1,295,113 C>T [-124 C>T] in the TERT promoter locus) of the indicated cell lines was genotyped. Arrows indicate the TERT promoter mutation or the WT TERT promoter. h, The time points of the AAV infection and DNA sequencing were shown in upper panel of Fig. 1c. The cells were harvested on day 10 after the first infection with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100. The DNA region spanning the mutation at chromosome 5 in the TERT promoter locus of the indicated cell lines was genotyped. Arrows indicate the WT TERT promoter. i, The indicated cells were infected with the indicated AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation at an MOI of 100 following the time points showed in upper panel of Fig. 1b. The DNA region spanning the mutation in the TERT promoter locus at chromosome 5 of the indicated cell lines was analysed by Illumina next-generation sequencing at day 10. The number of sequencing reads are presented. Statistical source data are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 PBE of the Mutated TERT Promoter Abrogates the Binding of ETS1 and GABPA to the Promoter.

a, U87 cells were infected with the AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation at an MOI of 100 following the time points displayed in upper panel of Fig. 1c. One hundred single cells of each group were seeded into 96-well plates for genomic DNA extraction. PCR-products amplified from the DNA region spanning the mutation in the TERT promoter locus at chromosome 5 of U87 cells were analysed by Sanger sequencing, and correction rates of the -124 C>T mutation in one hundred single cells were calculated. b, A unique barcoded DNA containing -124 C>T mutation was integrated into genome of LN18 cells by infection of lentiviruses expressing this DNA at a low MOI (0.01). These cells were infected with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 following the time points displayed in upper panel of Fig. 1b. Barcoded DNA region of the cells was analysed by Illumina next-generation sequencing at day 10. The correction rates were calculated by dividing the number of reads with correction by the combined numbers of reads with correction and unedited -124 C>T mutation. The corrected sequences do not contain indels. c, The indicated cells were infected with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 following the time points displayed in upper panel of Fig. 1b. Immunoblotting analyses were performed with the indicated antibodies at day 10. d, The indicated cells were infected with AAVs expressing HA-nCjCas9 under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 for 72 h. Results of immunoblot analyses performed using the indicated antibodies are shown. The experiment was repeated three times independently with similar results (c,d). Statistical source data are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Mutated TERT Promoter-Targeted PBE Does Not Affect Telomere Length or Proliferation of LN18 and SVG Cells.

a, QFISH analyses of telomere (Tel) lengths were performed using the indicated cells at the indicated time points after repeated infection (as shown in upper panel of Fig. 1b) with AAVs (MOI = 100) expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation (left panels). The immunofluorescence intensity in 10 cells was quantitated using the ImageJ software program (right panels). Values are means ± s.d. DAPI, 4',6-diamidino-2-phenylindole. b, TRF analyses were performed using the indicated cells at the indicated time points after repeated infection (as shown in Fig. 1b) with AAVs (MOI = 100) expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation. The experiment was repeated three times independently with similar results. c, The indicated cells were infected (as shown in Fig. 1b) with AAVs (MOI = 100) expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation. Thirty days after the first AAV infection, these cells (2 × 105) were plated and counted at the indicated time points. The depicted results are the averages from three independent experiments. Values are means ± s.d. Two-sided unpaired t-test (a); ANOVA test (c). n.s., not statistically significant (P > 0.05). Scale bar, 10 μm (a). Statistical source data are provided in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 Brain Tumour Growth inhibited by PBE-mediated correction of mutated TERT Promoter is not caused by off-target effect.

a, Flow chart of AAV injections into tumours. U87 and PDX cells with or without depletion of endogenous TERT and reconstituted expression of Flag-rTERT were intracranially injected into athymic nude mice (n = 8). AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation were injected into the brains of mice at the indicated time points after injection of U87 and PDX cells expressing luciferase. The frequencies of the virus injections and measurements of the luminescence of tumours luminescent measurements are shown. (b,c) TERT shRNA was expressed in Luciferase-expressing U87 and PDX cells, which were then stably transfected with a vector expressing RNAi-resistant (r)Flag-TERT. Immunoblot analyses with the indicated antibodies were performed. WB, Western blot (b). These cells were intracranially injected into athymic nude mice (n = 8). AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation were delivered (as shown in Extended Data Fig. 4a) into mice via intracranial injection. The luminescence intensity of tumour cells in representative mice at the indicated time points after cell injection is shown in the left panel. The bar graphs in the right panel show the relative luminescence intensity. Values are means ± s.d (c). d, Luciferase-expressing U87 and PDX cells with depletion of endogenous TERT and reconstituted expression of Flag-rTERT were intracranially injected into athymic nude mice (n = 8). AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation were delivered (as shown in Extended Data Fig. 4a) into mice via intracranial injection. The survival times of the mice were recorded. (e,f) Luciferase-expressing U87 and PDX cells with depletion of endogenous TERT and reconstituted expression of Flag-rTERT were intracranially injected into athymic nude mice (n = 8). AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation were delivered (as shown in Extended Data Fig. 4a) into the mice via intracranial injection. The mice were sacrificed 34 days after cell injection. The DNA region spanning the -124 C>T mutation in the TERT promoter locus at chromosome 5 of the combined tumour tissues from each group of mice (n = 8) was analysed by Illumina next-generation sequencing (e). The correction rates of mutated TERT promoters in U87 and PDX cells with depletion of endogenous TERT and reconstituted expression of Flag-rTERT were calculated by dividing the number of reads with correction by the combined numbers of reads with correction and unedited -124 C>T mutation (f). The corrected sequences do not contain indels. g, Luciferase-expressing U87 and PDX cells with depletion of endogenous TERT and reconstituted expression of Flag-rTERT were intracranially injected into athymic nude mice (n = 8). AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation were delivered (as shown in Extended Data Fig. 4a) to the mice via intracranial injection. The mice were sacrificed 34 days after cell injection. The DNA region spanning the -124 C>T mutation in the TERT promoter locus at chromosome 5 of the combined tumour tissues from each group of mice (n = 8) was analysed by Sanger sequencing. Arrows indicate the mutations. h, Luciferase-expressing U87 and PDX cells with depletion of endogenous TERT and reconstituted expression of Flag-rTERT were intracranially injected into athymic nude mice (n = 8). AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation were delivered (as shown in Extended Data Fig. 4a) into the mice via intracranial injection. The mice were sacrificed 34 days after cell injection. TRF analyses of the indicated brain tumour tissues were performed. Two-way ANOVA test (c); log-rank test (d). n.s., not statistically significant (P > 0.05). Statistical source data are provided in Source Data Extended Data Fig. 4. Source data

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Li, X., Qian, X., Wang, B. et al. Programmable base editing of mutated TERT promoter inhibits brain tumour growth. Nat Cell Biol 22, 282–288 (2020). https://doi.org/10.1038/s41556-020-0471-6

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