Click me!

A gene therapy strategy to target hepatocellular carcinoma based on conditional RNA interference

1.Description

As described in the Background page, we know spatial and/or temporal regulation of RNAi is of significant importance for basic research as well as practical applications of RNAi. For this reason, we design a gene therapy strategy to target hepatocellular carcinoma based on conditional RNA interference.

1.1 Key procedures

As shown in the following figure, there are three key procedures in our conditional RNAi gene therapy:

A. Drosha can’t cleavage pri-miRNA (green) which is bind with the inhibitory strand (yellow) in this stage, with no RNAi (Figure A).

B. SLD3, a short template competent for RNA synthesis by NS5B, is located in the 3’end of the inhibitory strand. NS5B, as the RNA dependent RNA polymerase, can interact with the 2’-OH and 3’-OH of the two base cytosines in the 3’end of the SLD3. And then, RNA synthesis, inhibitory strand as template, will happen. NS5B separates the inhibitory strand from the pri-miRNA and the latter will be cleavaged by Drosha and Dicer successively (Figure B).

C. For Tet-off system, when tetracycline (Tc) or Doxycycline (Dox) is absence, transcription factor tTA will bind to transcription activator TRE, which will activate downstream--miRNA sponge expression. MiRNA sponge will absorb the miRNA targeting MAP4K4 to block RNAi. When Tc or Dox is present, they will bind to tTA which leads to conformation exchange of this protein, thus will not bind to TRE, which result in non-coding of downstream genes (Figure C).

1.2 Features

1.2.1 Cancer-specific promoters

Our gene therapy strategy utilize two cancer-specific promoters (one HCC-specific) to open an AND-gated system to target HCC, the selectivity supposed to be extremely high.

HTERT is the core promoter of human telomerase reverse transcriptase (hTERT) gene. It’s one of the most known cancer-specific promoters. A number of factors, like c-Myc, HIF-1, regulate the hTERT promoter, most of which comprise tumor suppressor gene products.

Long non-coding RNA (lncRNA), highly up-regulated in liver cancer(Hulc) plays an important role in tumorigenesis,is one of the most up-regulated genes in hepatocellular carcinoma.Hulc promote is acutually the core promoter of this gene.

Hence, Only tumor cells (malignant ones especially) can express NS5B and “open” the AND gate, releasing miRNA that damage themselves.

1.2.2 Controlled by small molecule

Our gene therapy strategy is controlled by a small molecule.

As we mentioned above, tet-off system adjust the coding of miRNA sponge. This system is highly specific, the level of gene expression induced by it is relative to the dosage of the inducer and time, which can make adjusting the design accurately come true and is safety when we apply it in human.

1.2.3 Apply on other disease

Our gene therapy strategy has the flexibility to be adapted to target any mRNA and, if there are disease-specific promoters, other diseases.

Remarkably, such disease-specific approach would be suitable other diseases. Not only the target can be any messenger RNA. But also the promoters (“keys” to the AND gate) here can be replaced with other disease-specific promoters, in gene therapy for the corresponding diseases.

1.3 Plasmids construction

PlasmidⅠincluding hTERT promoter activate transactivation protein tTA for TRE promoter and NS5B to separate pri-miRNA and inhibitory strand. We load a nuclear location sequence (NLS) to the N terminal of NS5B (NS5B^NLS) to transport the RdRP into the nucleus to duplicate the inhibitory strand and release pri-miRNA.

PlasmidⅡcontains HULC promoter, pri-miRNA to silence MAP4K4 and the inhibitory strand of pri-miRNA. If the parts work, the pri-miRNA will not processed further because the combination of inhibitory strand.

tTA, TRE with downstream sponge are located in these two plasmids separately. Under the control of Tc or Dox, the two prats can regulate the expression of sponge. Sponge will regulate the miRNA targeting MAP4K4 to influence the level of MAP4K4 translation.

2.Results

To engineer our parts, we chose pIRES and pcDNA3.1 as backbones for the plasmidⅠandⅡvectors, respectively. And we constructed the plasmids containing partial parts to detect the function of system. Here the plasmids we used involed p1 (pIRES-hTERT-tTA-NS5B^NLS), p-H (pcDNA3.1-HULC-pri-miRNA(MAP4K4)), p-H* (pcDNA3.1-HULC- hulc-pri-miRNA), p-H-U6 (pcDNA3.1-HULC-pri-miRNA(MAP4K4)-U6-inhSi-pri-miRNA), p-H*-U6 (pcDNA3.1-HULC-pri-miRNA-U6-inhSi-pri-miRNA) and p2 (pcDNA3.1-HULC-pri-miRNA(MAP4K4)-U6-inhSi-pri-miRNA-tre-sponge).

2.1 Determine the activity of the promoters

To improve the selectivity of our therapy system, we utilize hTERT, the cancer-specific promoter, and HULC, the HCC-specific promoter, to open an AND-gated system to target HCC. pGL3-Basic vector is a promoterless vector for measuring the activity of promoter with a luciferase assay. We determined the activity of the two promoters by it.

Figure1. The activity of the promoters

2.2 Function verifiction of NS5B

We applied indirect immunofluorescence to prove the expression (Figure2 A) and nuclear translocation (Figure2 B) of NS5B^NLS.

Figure2. The expression and nuclear translocation of NS5B^NLS

2.3 Conditional RNA interference

To test the kill ability of this conditional RNA interference system, MTT assay was involved. The effect was obvious when introduced two plasmids to the cells under the control of Tetracycline (Tc) (Figure3 A).

Figure3. MTT Assay

To analysis the quantity of miRNA and its silencing effect to MAP4K4 mRNA, Real-time Quantitative Polymerase Chain Reaction (QPCR) was preformed. When the inhibitory strand worked, the expression of miRNA(MAP4K4) was reduced (Figure4 A) and MAP4K4 mRNA was increased (Figure4 B). As expected, when the two plasmids worked, MAP4K4 mRNA was increased (Figure4 B) because sponge could absorb the miRNA(MAP4K4).

Figure4. The expression of miRNA(MAP4K4) (A) and MAP4K4 mRNA (B)

[1] Cai, X., Hagedorn, C. H., & Cullen, B. R. (2004). Human micrornas are processed from capped, polyadenylated transcripts that can also function as mrnas. RNA, 10(12), 1957.

[2] Amuthan, G. (2004). The microprocessor complex mediates the genesis of micrornas. Nature, 432(7014), 235-40.

[3] Han, J., Lee, Y., Yeom, K. H., Nam, J. W., Heo, I., & Rhee, J. K., et al. (2006). Molecular basis for the recognition of primary micrornas by the drosha-dgcr8 complex. Cell, 125(5), 887-901.

[4] Castanotto, D., & Rossi, J. J. (2009). The promises and pitfalls of rna-interference-based therapeutics. Nature, 457(7228), 426-433.

[5] Zeng, Y., & Cullen, B. R. (2005). Efficient processing of primary microrna hairpins by drosha requires flanking nonstructured rna sequences. Journal of Biological Chemistry, 280(30), 27595-603.

[6] Beisel, C. L., Chen, Y. Y., Culler, S. J., Hoff, K. G., & Smolke, C. D. (2011). Design of small molecule-responsive micrornas based on structural requirements for drosha processing. Nucleic Acids Research,39(7), 2981-2994.

[7] Kumar, D., An, C. I., & Yokobayashi, Y. (2009). Conditional rna interference mediated by allosteric ribozyme. Journal of the American Chemical Society, 131(39), 13906-13907.

[8] Cheng, H., Zhang, Y., Wang, H., Sun, N., Liu, M., & Chen, H., et al. (2016). Regulation of map4k4 gene expression by rna interference through an engineered theophylline-dependent hepatitis delta virus ribozyme switch. Molecular Biosystems, 12(11), 3370-3376.

[9] Zhang, Y., Wang, J., Cheng, H., Sun, N., Liu, M., & Wu, Z., et al. (2017). Inducible bcl-2 gene rna interference mediated by aptamer-integrated hdv ribozyme switch. Integrative Biology Quantitative Biosciences from Nano to Macro, 9(7), 619.

[10] Moradpour, Darius, Volker, Gosert, Rainer, & Wölk, et al. (2002). Hepatitis c: molecular virology and antiviral targets. Trends in Molecular Medicine, 8(10), 476-482.

[11] Brass, V., Gouttenoire, J., Wahl, A., Pal, Z., Blum, H. E., & Penin, F., et al. (2010). Hepatitis c virus rna replication requires a conserved structural motif within the transmembrane domain of the ns5b rna-dependent rna polymerase. Journal of Virology, 84(21), 11580.

[12] Vo, N. V., Tuler, J. R., & Lai, M. M. (2004). Enzymatic characterization of the full-length and c-terminally truncated hepatitis c virus rna polymerases: function of the last 21 amino acids of the c terminus in template binding and rna synthesis. Biochemistry, 43(32), 10579.

[13] Lee, K. J., Choi, J., Ou, J. H., & Lai, M. M. (2004). The c-terminal transmembrane domain of hepatitis c virus (hcv) rna polymerase is essential for hcv replication in vivo. Journal of Virology, 78(7), 3797.

[14] Lohmann, V., Körner, F., Herian, U., & Bartenschlager, R. (1997). Biochemical properties of hepatitis c virus ns5b rna-dependent rna polymerase and identification of amino acid sequence motifs essential for enzymatic activity. Journal of Virology, 71(11), 8416-8428.

[15] http://parts.igem.org/Part:BBa_K1442100

[16] Kao, C. C., Yang, X., Kline, A., Wang, Q. M., Barket, D., & Heinz, B. A. (2000). Template requirements for rna synthesis by a recombinant hepatitis c virus rna-dependent rna polymerase. Journal of Virology,74(23), 11121.

[17] http://parts.igem.org/wiki/index.php?title=Part:BBa_K1442304

[18] O'Farrell, D., Trowbridge, R., Rowlands, D., & Jager, J. (2003). Substrate complexes of hepatitis c virus rna polymerase (hc-j4): structural evidence for nucleotide import and de-novo initiation. Journal of Molecular Biology,326(4), 1025-1035.

[19] Rhyu, M. S. (1995). Telomeres, telomerase, and immortality. J Natl Cancer Inst, 87(12), 884-894.

[20] Buseman, C. M., Wright, W. E., & Shay, J. W. (2012). Is telomerase a viable target in cancer?. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 730(1-2), 90-97.

[21] Nakayama, J., Tahara, H., Tahara, E., Saito, M., Ito, K., & Nakamura, H., et al. (1998). Telomerase activation by htrt in human normal fibroblasts and hepatocellular carcinomas. Nature Genetics, 18(1), 65-68.

[22] Poole, J. C., Andrews, L. G., & Tollefsbol, T. O. (2001). Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene, 1-12.

[23] Kyo, S., Kanaya, T., Takakura, M., Tanaka, M., & Inoue, M. (1999). Human telomerase reverse transcriptase as a critical determinant of telomerase activity in normal and malignant endometrial tissues. International Journal of Cancer, 80(1), 60-63.

[24] Aisner, D. L., Wright, W. E., & Shay, J. W. (2002). Telomerase regulation: not just flipping the switch. Current Opinion in Genetics & Development,12(1), 80-85.

[25] Nakamura, T. M., & Cech, T. R. (1997). Telomerase catalytic subunit homologs from fission yeast and human. Science, 277(5328), 955.

[26] Meyerson, M., Counter, C. M., Eaton, E. N., Ellisen, L. W., Steiner, P., & Caddle, S. D., et al. (1997). Hest2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell, 90(4), 785-795.

[27] Zhou, X. U., Lu, J., & Zhu, H. (2016). Correlation between the expression of htert gene and the clinicopathological characteristics of hepatocellular carcinoma. Oncology Letters, 11(1), 111.

[28] Kyo, S., Takakura, M., Fujiwara, T., & Inoue, M. (2010). Understanding and exploiting htert promoter regulation for diagnosis and treatment of human cancers. Cancer Science, 99(8), 1528-1538.

[29] Takakura, M., Kyo, S., Kanaya, T., Hirano, H., Takeda, J., & Yutsudo, M., et al. (1999). Cloning of human telomerase catalytic subunit (htert) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells. Cancer Research, 59(3), 551-557.

[30] http://parts.igem.org/Part:BBa_K1722002

[31] http://parts.igem.org/wiki/index.php?title=Part:BBa_K1922001

[32] http://parts.igem.org/Part:BBa_K1699001

[33] http://parts.igem.org/Part:BBa_K1722001

[34] Siegel, R. L., Miller, K. D., & Jemal, A. (2015). Cancer statistics, 2015. CA: A Cancer Journal for Clinicians, 65(1), 5-29.

[35] Riordan, S. M., & Williams, R. (2017). Medical management of hepatocellular carcinoma. Journal of Oncology Practice, 13(6), 356.

[36] Panzitt, K., Tschernatsch, M. M., Guelly, C., Moustafa, T., Stradner, M., & Strohmaier, H. M., et al. (2007). Characterization of hulc, a novel gene with striking up-regulation in hepatocellular carcinoma, as noncoding rna. Gastroenterology, 132(1), 330-342.

[37] Wang, J., Liu, X., Wu, H., Ni, P., Gu, Z., & Qiao, Y., et al. (2010). Creb up-regulates long non-coding rna, hulc expression through interaction with microrna-372 in liver cancer. Nucleic Acids Research, 38(16), 5366-5383.

[38] Collins, C. S., Hong, J., Sapinoso, L., Zhou, Y., Liu, Z., & Micklash, K., et al. (2006). A small interfering rna screen for modulators of tumor cell motility identifies map4k4 as a promigratory kinase. Proc Natl Acad Sci U S A, 103(10), 3775-3780.

[39] Han, S. X., Zhu, Q., Ma, J. L., Zhao, J., Huang, C., & Jia, X., et al. (2010). Lowered hgk expression inhibits cell invasion and adhesion in hepatocellular carcinoma cell line hepg2. World Journal of Gastroenterology, 16(36), 4541-4548.

[40] Gao, X., Gao, C., Liu, G., & Hu, J. (2016). Map4k4: an emerging therapeutic target in cancer. Cell & Bioscience, 6(1), 56.

[41] Das, A. T., Tenenbaum, L., & Berkhout, B. (2016). Tet-on systems for doxycycline-inducible gene expression. Current Gene Therapy, 16(3),

[42] Stieger, K., Belbellaa, B., Guiner, C. L., Moullier, P., & Rolling, F. (2009). In vivo, gene regulation using tetracycline-regulatable systems ☆. Advanced Drug Delivery Reviews, 61(7), 527-541.

[43] Gu, J., Zhang, L., Huang, X., Lin, T., Yin, M., & Xu, K., et al. (2002). A novel single tetracycline-regulative adenoviral vector for tumor-specific bax gene expression and cell killing in vitro and in vivo. Oncogene,21(31), 4757-4764.

[44] https://2013.igem.org/Team:SYSU-China/Project/Design