Supplemental figure 1

Transcription

1 Supplemental figure Copy number ratio relative to beta actin Series1 Series2 Series3 0 1 PC3 BP 2 DU145 BP 3 PC3 CMV 4 DU145 CMV Supplemental figure 1. qpcr to quantify the relative copy number of TMEM135 CCDC67 breakpoint and pcmv vector sequence in the genome of transformed cancer cells. One microgram of genome DNA of PC3 BP (PC3 cells transformed with pcmv TMEM135 int13 CCDC67 int9 ), or DU145 BP (DU145 cells transformed with pcmv TMEM135 int13 CCDC67 int9 ), or PC3 CMV (PC3 cells transformed with pcmvscript) or DU145 CMV (DU145 cells transformed with pcmvscript) was quantified for actin or TMEM135 CCDC67 breakpoint through qpcr using the primers listed in Supplemental table 2. The copy numbers of BP and actin were fitted with standard curves generated with serial titrations of known copy numbers of BP and actin, respectively. The BP/ actin ratios were plotted. The CMV/β actin ratios were plotted similarly.

2 Supplemental figure 2 A Virus TMEM intron 13 EGFP tk CCDC67 intron bp 561 bp Genome CMV IE94 Splice acceptor Splice donor TMEM135 int 13 CCDC67 int bp 1234 bp Genome CMV IE94 TMEM intron 13 EGFP tk CCDC67 intron bp 1211 bp Pre integration Pre integration Post integration Post integration Supplemental figure 2A. Schematic diagram for the detection of TMEM135 int13 EGFP tk CCDC67 int9 integration into TMEM135 CCDC67 breakpoint in the PC3 cell genome. Arrows indicate the primer position for PCR. Putative integration sites that generated mutations are indicated by yellow stars. The PCR products obtained from xenografted PC3 cells that contain TMEM135 CCDC67 breakpoint before virus treatment were used as reference control. PCR products obtained after viral (Ad TC) infections were sequenced. The positions of mutations due to DNA integration were detected through Sanger s sequencing.

3 B PC3 BP + Ad TC PC3 CMV + Ad TC DU145 BP + Ad TC DU145 CMV + Ad TC Cas9 D10A RFP 10 µm 10 µm 10 µm 10 µm EGFP tk 10 µm 10 µm 10 µm 10 µm Supplemental figure 2(B) Expression of Cas9 D10A and HSV1 tk in PC3 or DU145 tumors that contain TMEM135 CCDC67 breakpoint (PC3 BP and DU145 BP, respectively) and their control counterparts (PC3 CMV and DU145 CMV). The xenografted tumors were treated with pad5 Cas9 D10A grna TMEM135int13 grna CCDC67int9 and pad TMEM135 int13 EGFP tk CCDC67 int9 (Ad TC). Green arrows indicate unstained mouse stromal cells. Selected images were shown.

4 C HUH7 + Ad MF + G HEP3B + Ad MF + G HUH7 + Ad MF + PBS HUH7 + Ad TC + G Cas9 D10A RFP 10 µm 10 µm 10 µm 10 µm EGFP tk 10 µm 10 µm 10 µm 10 µm Supplemental figure 2C. Expression of Cas9 D10A and HSV1-tk in HUH7 or HEP3B tumors treated with Ad-TC or pad5-cas9 D10A -grna MAN2A1int13 -grna FERint14 /pad-man2a1 int13 -EGFP-tk-FER int14 (Ad-MF). Green arrows indicate unstained mouse stromal cells. Selected images were shown.

5 D Tumor: PC3 CMV DU145 CMV HUH7 Virus: Ad TC Ad TC Ad TC 10 µm 10 µm 10 µm Tumor: PC3 BP DU145 BP HUH7 Virus: Ad TC Ad TC Ad MF 10 µm 10 µm 10 µm Supplemental figure 2D. Genome therapy induced apoptosis of xenografted cancers that contain fusion gene breakpoints. Terminal deoxynucleotidyl transferase (TdT) dutp Nick-End Labeling (TUNEL) assays were performed on the PC3 BP, DU145 BP, PC3 CMV, DU145 CMV, or HUH7 xenografted cancers treated with either Ad-TC or Ad-MF.

6 Supplemental figure 3 A B TMEM135 CCDC67 GBP 1600 bp TMEM135 CCDC67 BP 500 bp MAN2A1 FER FER MAN2A1 FER 100 bp 113 Kd Genome Β actin PC3 BP DU145 BP PC3 CMV DU145 CMV 200 bp Β actin PC3 BP DU145 BP PC3 CMV DU145 CMV 100 bp Β actin HUH7 HEP3B 100 bp GAPDH HUH7 HEP3B 33 Kd Supplemental figure 3: Full view of figures 2B and 5B. (A) Full view of figure 2B. (B) Full view of figure 5B.

7 Supplemental Table 1. Chromosome breakpoint dependent cancer cell killing by Ganciclovir Samples Treatment % Apoptosis PC3 BP Ad-TC+Gan* 20.6%+1.4 PC3 CMV Ad-TC+Gan* 1.5%+0.2 DU145 BP Ad-TC+Gan* 19.8%+0.7 DU145 CMV Ad-TC+Gan* 1.2%+1.1 HUH7 Ad-MF+Gan** 26.9%+1.3 HUH7 Ad-TC+Gan* 2.6%+0.41 HEP3B Ad-MF-Gan** 2.8%+0.16 *- Treatment of Adeno+Gan includes AD5-Cas9 D10A -grna TMEM135int13 -grna CCDC67int9 and AD-TMEM135 int13 - EGFP-tk-CCDC67 int9 at 10 multiplicity of infection and Ganciclovir at 10µg/ml. **- Treatment of Adeno+Gan includes AD5-Cas9 D10A -grna MAN2A1int13 -grna FERint14 and AD-MAN2A1 int13 - EGFP-tk-FER int14 at 10 multiplicity of infection and Ganciclovir at 10µg/ml. BP=pCMV-TMEM135 int13 -CCDC67 int9. CMV=pCMVscript 1

8 Supplemental table 2. Primer sequences for PCR and RT-PCR Samples Forward primer/reverse primer Genome BP PCR GCCCATATATGGAGTTCCGCG/TCTGGCAAGCTATCTAACCCC RNA BP RT-PCR Genome -actin PCR RNA -actin RT-PCR Pre-integration 5 end Pre-integration 3 end CMV-EGFP PCR HSV1-tk-CMV PCR AGCACAGAGACCCAGAAGGTC/AGGAGGAGGAGGAGGAGAAAG TCTTTGCACTTTCTGCATGTCCCC/GTCCATCACGATGCCAGTGGTAC ATGATGATATCGCCGCGCTC/CACGATGGAGGGGAAGACG GCCCATATATGGAGTTCCGCG/AGGCAAAGAGCTCAGTGAGTG TGCCTCATTGGTAATGTTAGCTC/GGCGAATTGGGTACACTTACC ACTCACGGGGATTTCCAAGTC/AAGTCGTGCTGCTTCATGTGG TGTTCTAGCCAAGAGGCTGAG/GGCGAATTGGGTACACTTACC 2

9 Supplemental table 3 On- and off-target sequences Fusion gene grna target Sequences Chr. Position** TMEM135-CCDC67 On target CACTCACTGAGCTCTTTGCC Human Off-target 1 CACTGACTGAGCTCTCTGAC Human Off-target 2 CACTCACTGTCCTCTTTGCC Human Off-target 3 AACTCAGCGAGCTCTTTGCC Human Off-target 4 CACTCACTGAGATCTGTGCC Human Off-target 5 GACTCACTGAACTCTTTGGC Human Off-target 6 CCCTGAATGAGCTCTTTGCC Mouse Off-target 1 CACTGACTCAGTTCTTTGCC Mouse Off-target 2 CACTCCATCAGCTCTTTGCC Mouse Off-target 3 CACTCACTGGCCCCTTTGCC Mouse Off-target 4 TACCCACTGAGCTCTTTCCC Mouse Off-target 5 CACTCACTGAGCACTGTGTC Mouse Off-target 6 CACTGACTGAGTCCTTTGCC MAN2A1-FER On target TAGCATTAAGGGCCCCCTAA Human Off-target 1 TAGCACTGAAGGCCCCCTAA Human Off-target 2 TAGCATTAAGGGCCCACTTG Human Off-target 3 TAGCACTGAGGGCCCCCAAA Human Off-target 4 TAGTATTCAGGGCCCACTAA Human Off-target 5 TGGGATTAGGGGCCCCCTAA Mouse Off-target 1 TAGCTTTAAGTGCCTCCTAA Mouse Off-target 2 TACCATTAAGTGCCCCCAAA Mouse Off-target 3 TGGCATTAAGGGCCCATTAA Mouse Off-target 4 TTGCATTCAGGGTCCCCTAA Mouse Off-target 5 TAGCATTAAGTGCCCTCTTA ** Alignment to GRCh38.p7 primary assembly database for human genome or to GRCm38.p4 C57BL/6J for mouse genome. Chr Chromosome. 3

10 Supplemental table 4 On- and off-target sequencing primers for Illumina HiSeq2500 Fusion gene Genome sequencing primer Sequence Chr Position** TMEM135-CCDC67 On target CCCTGTTTTACATATGAGGAAAC Human Off-target 1 CCCACAAAAAGGGTACATGCC Human Off-target 2 TTTGGATTCAGCACAGTGGCC Human Off-target 3 AGGAGGACCATGCCATTTCCC Human Off-target 4 AGAGGCTCCAGACGCATTGTG Human Off-target 5a TCAGTGCCCTGCTCAAAACAG Human Off-target 5b CTCAGTCAGTATCCCTGCCAG Human Off-target 6a TGGGAAGGAATTGGAGGGAAG Human Off-target 6b GCTGTCATCTACAGCATCCTG Mouse Off-target 1 CAGGGAAGCTGCTTTAGAATG Mouse Off-target 2a TGAAATCCCTGACCCCCAATC Mouse Off-target 2b CAGACTGGACAAGGTGCTGCG Mouse Off-target 3a CACAGAGTATGCTAGGGGAAG Mouse Off-target 3b GTGAATGCCCTCTCTCTTTGC Mouse Off-target 4 AGGGTATTGTGGAGGTCACAG Mouse Off-target 5 CTTACTACTTAAGCCGCCCAG Mouse Off-target 6 ATCATCTAAGGGGAGTCTTGG EGFP-tk CAGCTCCTCGCCCTTGCTCAC N/A N/A MAN2A1-FER On target GACAGTCTGGCAGAGTTATGC Human Off-target 1a GAGCACGGGAGGCAAATAAAC Human Off-target 1b GTGAAGGGCACACTCTTCCAG Human Off-target 2 GGAGGAGGTATTGGAGGGTTG Human Off-target 3 AAGGCATCACTCACCACACTG Human Off-target 4a TCTATGATGTAGCCTCAGCTC Human Off-target 4b GTGGCATTTGCTTACCCTGGA Human Off-target 5a ATGGGGCTGTATGTGAAGAGG Human Off-target 5b CTGCATCTTCACAGGGTCGTC Mouse Off-target 1a AGAGCTTTCAGGTGTGCTGTC Mouse Off-target 1b TTGCCCACCTGCGACTAGAGA Mouse Off-target 2a TGGGTTGATCTAGCTGATGGAG Mouse Off-target 2b CTCAACCAACCTCTAATACTGTAC Mouse Off-target 3a GTCTGCTCCCTAAACGAGATG Mouse Off-target 3b CCAAACCAAAGAGTGGTGGAG Mouse Off-target 4a AAGGGATGCTACAGCCTGTCC Mouse Off-target 4b TGGGCACGGAGATAGGTTGTC Mouse Off-target 5a GCTTCTGTTAGGGCTTTCATGC Mouse Off-target 5b CACAGCATAGCCAAACTTATGG EGFP-tk CAGCTCCTCGCCCTTGCTCAC N/A N/A TMEM135-CCDC67 BP Primer 1 GCCTCATTGGTAATGTTAGCTC Primer 2 AGCATGGCACACTCAGTGAAC MAN2A1-FER BP Primer 1 CTCCTGACCCCGTGATCCACCT Primer 2 AAACCATAATCATGCTGACTG ** Alignment to GRCh38.p7 primary assembly database for human genome or to GRCm38.p4 C57BL/6J for mouse genome. Chr chromosome. N/A Not applicable; Chr Chromosome. 4

11 Supplemental table 5 Quantification of on and off targets reads of TMEM135 CCDC67 and MAN2A1 FER genome therapy in vitro and in vivo Therapy target Target cells total reads On target Off targets Off/On TMEM135 CCDC67 DU145 BP in vitro <0.1% PC3 BP in vitro <0.1% DU145 BP tumor <0.1% PC3 BP tumor <0.1% MAN2A1 FER HUH7 in vitro % HUH7 tumor % DU145 BP DU145 cell line harboring TMEM135 CCDC67 breakpoint. PC3 BP PC3 cell line harboring TMEM135 CCDC67 breakpoint. DU145 BP in vitro DU145 BP cell culture treated with Ad TC. PC3 BP in vitro PC3 BP cell culture treated with Ad TC. DU145 BP tumor DU145 BP xenografted tumor treated with Ad TC. PC3 BP tumor PC3 BP xenografted tumor treated with Ad TC. HUH7 in vitro HUH7 cell culture treated with Ad MF. HUH7 tumor HUH7 xenografted tumor treated with Ad MF. On target Pair end reads mapped to the correct on target genome sequence at one end and EGFP tk sequence at the paired end. Off target Pair end reads mapped to the off target genome sequence at one end and EGFP tk sequence at the paired end. Off/On: % Off target rate. Total reads Total number of reads including unmapped and mapped reads. 5

12 Supplemental table 6 Quantification of genomic EPGP tk reads in cells without fusion gene breakpoint Therapy viruses Target cells Total reads gegfp tk reads Mapped reads % Off Ad TC DU145 CMV in vitro % PC3 CMV in vitro % DU145 CMV tumor <0.1% PC3 CMV tumor <0.1% mliver (DU145 BP) <0.1% mliver (PC3 BP) <0.1% Ad MF HEP3B in vitro <0.1% HEP3B tumor % mliver (HUH7) <0.1% Ad TC AD5 Cas9 D10A grna TMEM135int13 grna CCDC67int9 and AD TMEM135 int13 EGFP tk CCDC67 int9. Ad MF AD5 Cas9 D10A grna MAN2A1int13 grna FERint14 and AD MAN2A1 int13 EGFP tk FER int14. gegfp tk reads Pair end reads mapped to correct on target or off target genome sequence at one end and EGFP tk sequence at the paired end. Mapped reads Pair end reads mapped to the correct genome sequence adjacent to the sequencing primers. Total reads Total number of reads including unmapped and mapped reads. mliver mouse liver cells from animals xenografted with cancer cells, and treated with recombinant adenoviruses. % Off gegfp tk/mapped reads DU145 CMV DU145 cells harboring pcmvscript. PC3 CMV PC3 cells harboring pcmvscript. DU145 CMV in vitro DU145 CMV cell culture treated with Ad TC. PC3 CMV in vitro PC3 CMV cell culture treated with Ad TC. DU145 CMV tumor DU145 CMV xenografted tumor treated with Ad TC. PC3 CMV tumor PC3 CMV xenografted tumor treated with Ad TC. mliver (DU145 BP) mliver from mice xenografted with DU145 BP cells and treated with Ad TC. mliver PC3 BP) mliver from mice xenografted with PC3 BP cells and treated with Ad TC. HEP3B in vitro HEP3B cell culture treated with Ad MF HEP3B tumor HEP3B xenografted tumor treated with Ad MF. mliver (HUH7) mliver from mice xenografted with HUH7 cells and treated with Ad MF. 6

13 Supplemental table 7 Integration rates of EGFP tk in genome therapy in vitro and in vivo Therapy target Samples genome EGFP tk reads BP reads Integration rates TMEM135 CCDC67 DU145 BP in vitro % DU145 BP tumor % PC3 BP in vitro % PC3 BP tumor % MAN2A FER HUH7 in vitro % HUH7 tumor % DU145 BP in vitro DU145 BP cell culture treated with Ad TC. DU145 BP tumor DU145 BP xenografted tumor treated with Ad TC. PC3 BP in vitro PC3 BP cell culture treated with Ad TC. PC3 BP tumor PC3 BP xenografted tumor treated with Ad TC. HUH7 in vitro HUH7 cell culture treated with Ad MF. HUH7 tumor HUH7 xenografted tumor treated with Ad MF. genome EGFP tk reads Pair end reads mapped to the correct on target genome sequence at one end and EGFP tk sequence at the paired end. BP reads Pair end reads mapped to the left side of the chromosome breakpoint at one end and the right side of the chromosome breakpoint at the paired end, or any mapped read containing the chromosomal breakpoint. Integration rate genome EGFP tk reads/bp reads 7