The Construction And Application Of TALENs Targeting HPV E6E7Oncogenes

Objective:1. To construct8pairs of TALEN plasmids targeting HPV16E6/E7and8pairs of those targeting HPV18E6/E7, using SSA reporter system to select the TALENs with the best cleavage efficiency, with optimized FokI cleavage domain and TALEN architecture.2. Then confirm the intracellular effects of TALENs by detecting copies number of E6E7, E6E7oncoproteins expression, cell apoptosis and growth inhibition after various cell lines were treated with these TALENs.3. Then we construct mice intravaginal transfection system, detect the change of EGFP fluorescence in the EGFP transgenic mice after treated with TAL-EGFP which targeted EGFP gene. We hope these work could provide a novel strategy for the further intravaginal transfection of TALEN expression plasmids in the K14-HPV16transgenic mice.Methods:1. Using the Golden Gate Assembly method, we constructed a total of16pairs of TALENs, with4pairs targeting HPV16E6,4pairs targeting HPV16E7,4pairs targeting HPV18E6and4pairs targeting HPV18E7. All these TALENs were screened by SSA reporter system and optimized by combination of various mutation of wild type FokI cleavage domain and truncated architecture. Then pick out the best TALENs with most effective cleavage and the lowest toxicity.2. The candidate TALENs were transfected into SiHa, HeLa, C33A, S12and293T cell lines, then detect the DSBs in the cell by using T7endonuclease Ⅰ (T7E1) to reflect the cleavage efficiency of TALENs indirectly, test the copies number of HPV E6E7genes by using Fluorescent in situ hybridization (FISH) technology, check the protein expression of HPV16/18E6and E7oncoproteins and their targeting proteins, detect cell apoptosis by Flow cytometry and examine the cell proliferation.3. The BALB/c mice were directly transfected in the vagina with single or multiple administration of variant doses of polymer-complexed mRFP expression plasmids. Then monitor the transfection efficiency by detecting the fluorescence of mice vaginal exfoliated cells. Mice were killed at different time points with different doses, the mice vaginal and cervical tissues were separated, then observed the distribution of red fluorescence in the vaginal and cervical tissues frozen sections. According to these data, EGFP transgenic mice were intravaginaly administered FLAG marked TAL-EGFP, which targeted EGFP, in the same conditions. Then the fluorescence change of TALEN-treated EGFP were detected, tissue samples were fixed by4%paraformaldehyde and embedded in paraffin and cut into slices, using immunohistochemistry of FLAG tag which connected in the TALEN expression plasmids to reflect the distribution of expression in vivo. Local inflammation was tested by hematoxylin and eosin staining and immunohistochemistry of anti-mouse CD45polyclonal antibody.Results:1. We found all the16pairs of TALENs could effectively cleave the target, T27targeting HPV16E6, T512targeting HPV16E7, T34targeting HPV18E6and T519targeting HPV18E7were proved to be the most effective ones. TALENs with the combination of+63truncated architecture and S+KKR/ELD mutated FokI cleavage domain exhibited most effective cleavage effect.2. We found (1) T27-or T512-treated SiHa and S12cells and T34-or T519-treated HeLa cells were all exhibited positive results by using T7E1experiments.(2) The copies number of HPV16in T512-treated SiHa and S12cells were significantly decreased, the copies number of HPV18in T519-treated HeLa cells were also significantly reduced.(3) Results of Western Blotting suggested that T27and T34could specifically down-regulated the expression of corresponding HPV16and HPV18E6, and up-regulated the expression of p53, the target of E6oncogene. Similarly, T512and T519could specifically down-expressed HPV16and HPV18E7, respectively. And They up-expressed the expression of RB1, which is the target of E7oncogene.(4) By using Flow Cytometry, we observed significant apoptosis of T27-and T512-treated HPV16-positive SiHa and S12cell lines, while they did not induce significant apoptosis of HPV18-positive HeLa cells. Similarly, T34and T519specifically induced apoptosis of HeLa cells, while did not affect the growth of SiHa and S12cells. All of these four pairs of TALENs did not induced significant cell apoptosis of HPV-negative cervical cancer C33A cells and normal human embryonic kidney293T cells.(5) T27and T512specifically inhibited the growth of HPV16-positive SiHa and S12cells, while did not affect the growth of HeLa, C33A and293T cells; T34and T519arrested the growth of HeLa cells, while did not block SiHa, S12,C33A and293T cells.3. The red fluorescence can be detected in the cervical tissue48h later after intravaginal transfection, and the fluorescence signals lasted for at least6days. Most of the plasmids expressed around the vaginal and cervical epithelium. Change of EGFP fluorescence in the EGFP transgenic mice suggested that intravaginally transfected TAL-EGFP could effectively cleave the EGFP sequence in vivo, and consequently block the expression of EGFP proteins. H&E staining and IHC staining of CD45indicated that intravaginal administration of plasmids did not hurt local tissue and induce acute inflammatory reaction.Conclusions:1. We constructed8pairs of TALENs targeting HPV16E6E7and8pairs targeting HPV18E6E7, then4pairs with the best cleavage efficiency were selected, the TALEN architecture and FokI cleavage domain were optimized. 2. The selected T27, T512, T34and T519effectively cleaved the target HPV sequences of corresponding HPV subtype-positive cells, decreased the copies number of HPV, down-regulated E6and E7oncogenes and up-regulated their down-stream target proteins p53and RB1, respectively. And finally induced apoptosis and inhibited the cell growth of host cells.3. Intravaginal administration of TALEN expression plasmids could effective transfect mice cervical local tissue. TAELN could cleave the target sequences and block the proteins expression in cervical epithelium. Intravaginal transfection did not hurt local tissue and induce acute inflammatory reaction in vivo.