Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene

Abstract

Specifically targeting genomic rearrangements and mutations in tumor cells remains an elusive goal in cancer therapy. Here, we used Cas9-based genome editing to introduce the gene encoding the prodrug-converting enzyme herpes simplex virus type 1 thymidine kinase (HSV1-tk) into the genomes of cancer cells carrying unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135-CCDC67 and MAN2A1-FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9^D10A and guide RNAs targeting the breakpoint sequences, and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed decreased tumor burden and no mortality during the study. Thus, Cas9-mediated suicide-gene insertion may be a viable genotype-specific cancer therapy.

Figure 1. Schema of strategy to introduce EGFP-tk into the breakpoint of TMEM135-CCDC67 fusion gene

(A) Diagram representation of TMEM135 and CCDC67 recombination in Chr.11and Sanger sequencing of TMEM135-CCDC67 chromosome breakpoint. Direction of transcription is indicated by arrow. (B) Diagram of the gRNA design and location. The locations of gRNA− and gRNA+ are indicated by boxes. The directions of gRNA are indicated by arrows. (C) Stepwise schematic diagram of genome therapy strategy to introduce EGFP-tk into the breakpoint of TMEM135-CCDC67. These gRNAs were ligated with Cas9^D10A into VQAd5-CMV shuttle vector and recombined into pAd5 virus. Separately, 584 bp of TMEM135 intron 13 sequence and 561 bp of CCDC67 intron 9 sequence are designed to sandwich a promoterless EGFP-tk cDNA, ligated into PAdlox shuttle vector and recombined into adenovirus. A splice acceptor and a splice donor from exon 14 of TMEM135 are inserted between TMEM135 intron 13 and EGFP-tk, and between EGFP-tk and CCDC67 intron 9, respectively, to allow proper EGFP-tk RNA splicing to occur. Cells containing TMEM135-CCDC67 chromosome breakpoint are infected with these recombinant viruses. After nicking at the sites corresponding to gRNA sequences by Cas9^D10A, donor DNA containing EGFP-tk is integrated into the breakpoint region of TMEM135-CCDC67 through homology directed recombination (HDR). The integrated EGFP-tk is transcribed by the fusion head gene (TMEM135) promoter in these cells, spliced and translated into protein product of EGFP-tk, which in turn blocks DNA synthesis by converting ganciclovir (Gan) to ganciclovir triphosphate.

Figure 2. EGFP-tk integration and expression in cells expressing TMEM135-CCDC67 fusion breakpoint transcript

(A) gRNA mediated cleavage of pCMV-TMEM135^int13-CCDC67^int9. In vitro cleavage assays were performed on PVUI linearized pCMV-TMEM135^int13-CCDC67^int9 vector using recombinant Cas9, S. pyogenes and in vitro transcribed gRNA− or gRNA+ as indicated. The cleavage generated 4317 and 3206 bp fragments of pCMV-TMEM135^int13-CCDC67^int9 vector for gRNA−, and 4414 and 3109 bp for gRNA+, indicated by arrows. (B) Genome integration and expression of TMEM135^int13-CCDC67^int9 breakpoint in PC3 and DU145 cells. Top panel: PCR products of TMEM135^int13-CCDC67^int9 breakpoint from the genome of indicated cells; Second from the top: PCR products of genomic β-actin from the genome of indicated cells. Third from the top: RT-PCR products of TMEM135^int13-CCDC67^int9 breakpoint from the mRNA of the indicated cells. Bottom panel: RT-PCR products of β-actin from the mRNA of the indicated cells. PC3 BP denotes PC3 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; DU145 BP denotes DU145 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; PC3 CMV denotes PC3 transfected with pCMVscript; DU145 CMV denotes DU145 cells transfected with pCMVscript. Primer sequences are listed in Supplementary table 2. (C) Infection of PC3 or DU145 cells containing TMEM135-CCDC67 breakpoint led to expression of EGFP-tk. PC3 or DU145 cells transformed with pCMV-TMEM135^int13-CCDC67^int9 were infected with pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9 and pAD-TMEM135^int13-EGFP-tk-CCDC67^int9 (Ad-TC). Expression of Cas9^D10A-RFP is indicated by red fluorescence, while expression EGFP-tk is indicated by green. PC3 or DU145 cells transformed with pCMVscript were used as controls. Selected images were shown. (D) Quantification of EGFP-tk integration/expression by flow cytometry as of (C).

Figure 3. Treatment with nucleotide analogue ganciclovir kills cancer cells expressing EGFP-tk

(A) PC3 or DU145 cells containing TMEM135-CCDC67 breakpoint were infected with pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9/pAD-TMEM135^int13-EGFP-tk-CCDC67^int9 (Ad-TC). These cells were then incubated with various concentrations of ganciclovir for 24 hours. Cell deaths were then quantified with phycoerythrin labeled Annexin V through flow cytometer. PC3 or DU145 cells harboring no TMEM135-CCDC67 breakpoint were used as controls. PC3 BP denotes PC3 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; DU145 BP denotes DU145 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; PC3 CMV denotes PC3 transfected with pCMVscript; DU145 CMV denotes DU145 cells transfected with pCMVscript. (B) Representative sample of cell death induced by ganciclovir on cells infected with Ad-TC, and treated with 5 μg/ml ganciclovir. PC3 or DU145 cells harboring no TMEM135-CCDC67 breakpoint were used as controls. Apoptosis was indicated by Annexin V staining. PC3 BP denotes PC3 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; DU145 BP denotes DU145 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; PC3 CMV denotes PC3 transfected with pCMVscript; DU145 CMV denotes DU145 cells transfected with pCMVscript.

Figure 4. Treatment of ganciclovir induced partial remission of xenografted prostate cancers in SCID mice

(A) PC3 cells harboring TMEM135-CCDC67 breakpoint were xenografted into the subcutaneous regions of SCID mice. These tumors were allowed to grow for 3 week before the treatment. The indicated drugs were applied through intraperitoneal injections 3 times a week until all the mice from control treatments died off. The tumor volumes were measured weekly. PC3 BP denotes PC3 cells transformed with pCMV-TMEM135^int13-CCDC67^int9; PC3 CMV denotes PC3 cells transformed with pCMVscript; Ad-TC denotes treatment of pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9 and pAD-TMEM135^int13-EGFP-tk-CCDC67^int9; Gan denotes Ganciclovir; PBS denotes phosphate buffer saline. (B) DU145 cells harboring TMEM135-CCDC67 breakpoint were xenografted into the subcutaneous regions of SCID mice. These tumors were allowed to grow for 3 week before the treatment. The indicated drugs were applied through intraperitoneal injections 3 times a week until all the mice from control treatments died off. The tumor volumes were measured weekly. DU145 BP denotes DU145 cells transformed with pCMV-TMEM135^int13-CCDC67^int9; DU145 CMV denotes DU145 cells transformed with pCMVscript; Ad-TC denotes treatment of pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9 and pAD-TMEM135^int13-EGFP-tk-CCDC67^int9; Gan denotes Ganciclovir; PBS denotes phosphate buffer saline. (C) Mice treated with TMEM135-CCDC67 breakpoint therapy are free of cancer metastasis. (D) Mice treated TMEM135-CCDC67 breakpoint therapy had no mortality.

Figure 5. Genome therapy targeting at MAN2A1-FER breakpoint

(A) Design of gRNA and recombination donor adenoviruses for MAN2A1-FER fusion gene. Upper panel: Sanger sequencing diagram of MAN2A1-FER chromosome breakpoint of HUH7 cells; Middle panel: Design of gRNA for pAD5-Cas9^D10A-gRNA^MAN2A1int13-gRNA^FERint14; Lower panel: Design of homologous DNA sequences and EGFP-tk for pAD-MAN2A1^int13-EGFP-tk-FER^int14. The splicing acceptor and donor sequences correspond to the juncture sequences of intron13-exon 14 of MAN2A1and exon15-intron 15 of FER. (B) Expression of MAN2A1-FER in HUH7 cells. Lanes 1 and 2: immunoblots of protein extracts from HUH7 and HEP3B cells with antibodies specific for FER or GAPDH. MAN2A1-FER (MF) and FER protein are indicated. Lanes 3 and 4: RT-PCR of RNA from HUH7 and HEP3B cells with primers specific for MAN2A1-FER (MF) or β-actin. (C) In vitro cleavage assays were performed on BamH1 linearized pTA-MAN2A1^int13-FER^int14 vector using recombinant Cas9, S. pyogenes and in vitro transcribed gRNA− or gRNA+ as indicated. The cleavage generated 2446 and 1944 bp fragments of pTA-MAN2A1^int13-FER^int14 vector for gRNA−, and 2484 and 1906 bp for gRNA+. (D) Infection of HUH7 or HEP3B cells led to expression of EGFP-tk in HUH7 but not HEP3B cells. HUH7 and HEP3B cells were infected with pAD5-Cas9^D10A-gRNA^MAN2A1int13-gRNA^FERint14 (ad-gMF) and pAD-MAN2A1^int13-EGFP-tk-FER^int14 (Ad-MF-EGFP-tk). Expression of Cas9^D10A-RFP is indicated by red fluorescence, while expression EGFP-tk is indicated by green. HUH7 cells infected with pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9 (Ad-gTC) and pAD-TMEM135^int13-EGFP-tk-CCDC67^int9 (Ad-TC-EGFP-tk) were used as specificity control. Selected images were shown. (E) Quantification of EGFP-tk integration/expression by flow cytometry as of (D). (F) Killing of HUH7 cells with ganciclovir. HUH7 or HEP3B cells were infected with pAD5-Cas9^D10A-gRNA^MAN2A1int13-gRNA^FERint14/pAD-MAN2A1^int13-EGFP-tk-FER^int14 (Ad-MF). These cells were then incubated with various concentrations of ganciclovir for 24 hours. Cell deaths were then quantified with phycoerythrin labeled Annexin V through flow cytometer. HUH7 cells infected with pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9/pAD-TMEM135^int13-EGFP-tk-CCDC67^int9 (Ad-TC) were used as specificity controls. (G) HUH7 and HEP3B cells were xenografted into the subcutaneous regions of SCID mice. These tumors were allowed to grow for 2 weeks before the treatment. These mice were treated with the indicated viruses plus ganciclovir (G, 80mg/kg) or PBS (P). The indicated drugs were applied through intraperitoneal injections 3 times a week until all the mice from control treatments died off. The tumor volumes were measured weekly. (H) Mice treated with MAN2A1-FER breakpoint therapy are free of cancer metastasis. (I) Mice treated MAN2A1-FER breakpoint therapy had no mortality.