Expression of PTEN-long mediated by CRISPR/Cas9 can repress U87 cell proliferation
Na Fang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Jiangsu Superbio Co.,Ltd, Nanjing, China
Search for more papers by this authorTingxuan Gu
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorYahui Wang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorShuzhen Wang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorFengling Wang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorYang An
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorWenqiang Wei
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorWeijuan Zhang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorXiangqian Guo
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorAdil J Nazarali
College of Pharmacy and Nutrition and Neuroscience Research Cluster, University of Saskatchewan, Saskatchewan, Canada
Search for more papers by this authorCorresponding Author
Shaoping Ji
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Department of Oncology, The First Affiliated Hospital of Henan University, Kaifeng, China
College of Pharmacy and Nutrition and Neuroscience Research Cluster, University of Saskatchewan, Saskatchewan, Canada
Jiangsu Superbio Co.,Ltd, Nanjing, China
Correspondence to: Shaoping JI
E-mail: [email protected]
Search for more papers by this authorNa Fang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Jiangsu Superbio Co.,Ltd, Nanjing, China
Search for more papers by this authorTingxuan Gu
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorYahui Wang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorShuzhen Wang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorFengling Wang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorYang An
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorWenqiang Wei
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorWeijuan Zhang
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorXiangqian Guo
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Search for more papers by this authorAdil J Nazarali
College of Pharmacy and Nutrition and Neuroscience Research Cluster, University of Saskatchewan, Saskatchewan, Canada
Search for more papers by this authorCorresponding Author
Shaoping Ji
Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, Henan Province, China
Department of Oncology, The First Affiliated Hospital of Henan University, Kaifeng, China
College of Pharmacy and Nutrition and Neuroscience Research Cluster, University of Saskatchewan, Saskatchewan, Canada
Jiangsu Superbio Co.,Ltd, Nanjing, China
Correspondence to: Shaoping JI
E-mail: [email protected]
Search for more papers by this authorAbstract
PTEN is a tumour suppressor that is frequently mutated in a variety of cancers. Hence, PTEN has significant potential as a therapeutic molecule. PTEN-long is an alternative translation variant, with an additional 173 amino acids added to the N-terminal of the canonical PTEN when CUG of the mRNA is utilized as the start codon. PTEN-long is secreted into serum and can re-enter cells throughout the body. One of the major barriers for gene therapy is to efficiently and specifically deliver DNA or RNA material to target cells. As an alternative approach, if a therapeutic protein can be directly delivered to target cell of interest, it should theoretically function well within the cells, particularly for genes that are deficiently expressed in vivo. Most therapeutic proteins are incapable of efficiently permeating the cell membrane. In this study, we have employed CRISPR/Cas9 gene editing tool combined with single-stranded template to edit CTG of PTEN-long to ATG in the genome. Two guide RNAs close to CTG site were found to have similar efficiency in driving PTEN-long expression. Furthermore, we detected PTEN-long expression in transfected whole-cell lysate and in concentrated culture media in Western blot. Interestingly, the culture media of PTEN-long expression can reduce Akt phosphorylation level and repress U87 cell proliferation compared to wild-type U87 or control media. Taken together, PTEN-long driven by CRISPR/Cas9 imports and exports cells and represses nearby cell proliferation, indicating the PTEN-long generated by CRISPR/Cas9 has potential to be an alternative strategy for PTEN gene therapy.
References
- 1Zhang R, Miner JJ, Gorman MJ, et al. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature. 2016; 535: 164–8.
- 2Zhou J, Wang C, Zhang K, et al. Generation of human embryonic stem cell line expressing zsGreen in cholinergic neurons using CRISPR/Cas9 system. Neurochem Res. 2016; 41: 2065–74.
- 3Yin H, Song CQ, Dorkin JR, et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. 2016; 34: 328–33.
- 4Zuckermann M, Kawauchi D, Gronych J. Applications of the CRISPR/Cas9 system in murine cancer modeling. Brief Funct Genomics. 2016; 16: 25–33.
- 5Gagnon KT, Corey DR. Stepping toward therapeutic CRISPR. Proc Natl Acad Sci USA. 2015; 112: 15536–7.
- 6High K, Gregory PD, Gersbach C. CRISPR technology for gene therapy. Nat Med. 2014; 20: 476–7.
- 7Tang H, Shrager JB. CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer: a personalized molecular surgical therapy. EMBO Mol Med. 2016; 8: 83–5.
- 8Nelson CE, Hakim CH, Ousterout DG, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016; 351: 403–7.
- 9Tabebordbar M, Zhu K, Cheng JK, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016; 351: 407–11.
- 10Long C, Amoasii L, Mireault AA, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016; 351: 400–3.
- 11Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013; 8: 2281–308.
- 12Chu VT, Weber T, Wefers B, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015; 33: 543–8.
- 13Wang H, Yang H, Shivalila C, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013; 153: 910–8.
- 14Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013; 154: 1380–9.
- 15Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016; 353: aaf5573.
- 16Sun H, Lesche R, Li DM, et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci USA. 1999; 96: 6199–204.
- 17Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 1997; 275: 1943–7.
- 18Hopkins BD, Fine B, Steinbach N, et al. A secreted PTEN phosphatase that enters cells to alter signaling and survival. Science. 2013; 341: 399–402.
- 19Legg K. PTEN goes paracrine. Nat Cell Biol. 2013; 15: 894.
- 20Zhang H, Zhou X, Xu C, et al. Synergistic tumor suppression by adenovirus-mediated ING4/PTEN double gene therapy for gastric cancer. Cancer Gene Ther. 2016; 23: 13–23.
- 21Li D, Zhang Y, Xie Y, et al. Enhanced tumor suppression by adenoviral PTEN gene therapy combined with cisplatin chemotherapy in small-cell lung cancer. Cancer Gene Ther. 2013; 20: 251–9.
- 22Wang D, Mou H, Li S, et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum Gene Ther. 2015; 26: 432–42.
- 23Gerashchenko MV, Su D, Gladyshev VN. CUG start codon generates thioredoxin/glutathione reductase isoforms in mouse testes. J Biol Chem. 2010; 285: 4595–602.
- 24Starck SR, Jiang V, Pavon-Eternod M, et al. Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I. Science. 2012; 336: 1719–23.
- 25Shalygin A, Skopin A, Kalinina V, et al. STIM1 and STIM2 proteins differently regulate endogenous store-operated channels in HEK293 cells. J Biol Chem. 2015; 290: 4717–27.
- 26Maruyama T, Dougan SK, Truttmann MC, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015; 33: 538–42.
- 27Ambulos NP Jr, Smith T, Mulbry W, et al. CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis. Gene. 1990; 94: 125–8.
- 28Wang H, Zhang P, Lin C, et al. Relevance and therapeutic possibility of PTEN-long in renal cell carcinoma. PLoS ONE. 2015; 10: e114250.
- 29Cucchiarini M. Human gene therapy: novel approaches to improve the current gene delivery systems. Discov Med. 2016; 21: 495–506.
- 30Ibraheem D, Elaissari A, Fessi H. Gene therapy and DNA delivery systems. Int J Pharm. 2014; 459: 70–83.
- 31Amer MH. Gene therapy for cancer: present status and future perspective. Mol Cell Ther. 2014; 2: 27.
- 32Moussa EM, Panchal JP, Moorthy BS, et al. Immunogenicity of therapeutic protein aggregates. J Pharm Sci. 2016; 105: 417–30.
- 33Yin L, Chen X, Tiwari A, et al. The role of aggregates of therapeutic protein products in immunogenicity: an evaluation by mathematical modeling. J Immunol Res. 2015; 2015: 401956.
- 34Lu Y, Sun W, Gu Z. Stimuli-responsive nanomaterials for therapeutic protein delivery. J Control Release. 2014; 194: 1–19.
- 35Sachdeva M, Sachdeva N, Pal M, et al. CRISPR/Cas9: molecular tool for gene therapy to target genome and epigenome in the treatment of lung cancer. Cancer Gene Ther. 2015; 22: 509–17.
- 36Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339: 819–23.
- 37Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339: 823–6.
- 38He Z, Kee K. Generating a genome editing nuclease for targeted mutagenesis in human cells. Methods Mol Biol. 2017; 1498: 153–62.
- 39Gaj T, Gersbach CA, Barbas CF 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013; 31: 397–405.
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