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 bond to transcription activator TRE, which will activate downstream--miRNA sponge expression. MiRNA sponge will absorb the miRNA targeting MAP4K4 to block RNAi. When Tet or Dox is present, Dox will bond to tTA. The conformation of tTA will change and leave away from TRE. Thus the downstream genes can’t express (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.

HULC, Long non-coding RNA (lncRNA), highly up-regulated in liver cancer, is one of the most up-regulated genes in hepatocellular carcinoma.

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.

When Tet or Dox is present, Dox will bond to Tet. The conformation of Tet will change and leave away from TRE. Thus the downstream genes can’t express. Tet-off regulating 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 to the human body.

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, Tet 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 NS5BNLS

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)

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