Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

Abstract

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM +ve/let-7bdown-regulated), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM −ve/let-7bup-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM +ve/let-7bup-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat

“…For such diseases, RNAe holds a promise due to a likely tissue-specificity since it could enhance the target protein expression in a manner dependent on native mRNA distribution. RNAe also showed great simplicity compared with other tissue-specific methods ( 24 – 27 ).…”

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects

“…Danda et al . demonstrated that plasmid carrying the suicide gene was selectively expressed in the cancer cells (retinoblastoma) overexpressing Epithelial cell adhesion molecule (EpCAM) [ 51 ], and Dancer et al . demonstrated in vivo reduction of tumor volume and the number of blood vessels in the study targeting a suicide gene in the endothelium of transgenic mice [ 52 ].…”

Terapia génica en el manejo de las distrofias retinianas

“…En el retinoblastoma, la terapia génica ha producido reducción y detención del crecimiento de la masa por medio de los mecanismos antiapoptóticos del gen RB1, que no permite su crecimiento. Sin embargo, los efectos inflamatorios severos por el tipo de vector usado han hecho que los estudios se mantengan en fases experimentales (43,44).…”