Analysis of the epigenetic factors for the transduction efficiency of hepatoma cell lines in gene therapy with recombinant adeno-associated virus

Abstract

[한글]유전자치료에서 gene delivery vehicle로써 rAAV(recombinant Adeno-associated virus)를 이용하는데, 이는 염증반응이나 면역반응이 거의 없고 neuron, myocyte, hepatocyte와 같은 non-dividing cell의 transduction에 효과가 있다. AAV는 다양한 장기에서 발현을 보이는데 각각의 장기마다 단백질의 발현이 다르게 나타나기에 유전자 치료를 함에 있어 단백질의 발현기관으로 간 (liver)을 대상으로 하여 단백질의 발현을 증가시킬 수 있다면 좀더 효과적인 유전자 치료가 되리라 예측된다. 일부의 연구에서 DNA-damaged agent인 irradiation이나 heparin, topoisomerse inhibitor(camptothecin)등이 간의 transduction efficiency을 높여준다는 보고가 있다 3-5. 따라서 본 연구에서는 rAAV를 여러 물리적 화학적 방법을 사용하여 transduction에 차이가 있는지를 살펴 보았고 heparin, chloroquine, butyrate, hydroxyurea 혹은 chemotherapeutic agents를 rAAV와 함께 처리했을 때 hepatoma cell에서 transduction에 어떠한 영향이 있는지를 살펴보았다. 또한 γ-irradiation을 hepatoma cell에 expose시켜서 rAAV를 infection하면 transduction efficiency에 영향을 주는지를 살펴보았다. γ-irradiation과 함께 여러 화학적 agent를 처리하여 synergistic effect가 있는지를 살펴보고, hepatoma cell에서 효과가 있는지를 AAV-mediated β-galactosidase gene인 pAAV Lac Z를 이용한 β-galactosidase의 발현여부와 activity를 통한 transduction efficiency를 평가하였다. cell type에 따라 각각 다르게 나타났으나 chemotherapeutic agents인 camptothecin, etoposide를 rAAV와 함께 처리했을 때 transduction에 효과가 있었다. 또한 γ-irradiation만을 cell에 expose하여 rAAV를 infection해도 permissive environment가 되었다. 이를 좀더 세분화시켜 γ-irradiation과 chemotherapeutic agents인 camptothecin이나 5-FU를 combination했을 때 효과가 있었다. 이상의 결과를 토대로 rAAV를 이용한 permissive environment를 위한 적정 농도와 적정 조건이 향후 유전자 치료의 protocol을 결정하는 자료가 되리라 여겨진다. 이를 통해 앞으로 in vivo실험에서 좀더 체계적인 유전자 치료를 하는데 좋은 평가가 되리라 여겨진다.

[영문]Gene therapy is one of the promising treatment modalities to overcome incurable diseases. However, there are many problems to be solved, such as transient expression or not sufficient level of therapeutic protein. rAAV has been widely used as a gene delivery vehicle in gene therapy, not only because it is less likely to induce inflammatory or immune response which result in prolonged expression, but because it is effective at transducing non-dividing cells such as neurons, myocytes, and hepatocytes. The expression by AAV varies in different tissues and organs. The liver is the good factory for protein synthesis, so liver-targeted gene therapy would be more effective and rAAV can be used as a vehicle to transfer the target gene to the liver. Still, it is difficult to reach therapeutic range of target protein in liver-targeted gene therapy with rAAV. /It has been reported that DNA-damaging agents like UV irradiation, heparin, or topoisomerase inhibitor facilitate the transduction of organ with rAAV. These prompted us to study the epigenetic factors that affect the transduction efficiency of liver in rAAV gene therapy. We generated rAAV by transfecting 293 cells with three plasmids namely, pAAV-LacZ, which has ITR of wild type2 upstream of CMV promoter and LacZ gene, pAAV-RC, which allows the expression of cap gene, and pHelper, which is required for AAV replication. We then examined the possible effects of several chemicals, i.e., heparin, chloroquine, butyrate, hydroxyurea, 5-FU, etoposide, cisplatin or camptothecin on the transduction of hepatoma cell lines after rAAV infection. Also, we investigated whether hepatoma cells infected with rAAV, after being exposed to gamma-irradiation, affects their transduction efficiency, and if there is effect when cells are treated with gamma-irradiation and various chemical agents. /We found that pre-treatment of hepatoma cell lines with permissive growth medium or 4mM of hydroxyurea increased the transduction efficiencies of rAAV significantly (p<0.05). There was no decrease in the viability of cells in the concentration of 4mM of hydroxyurea or permissive medium. However, heparin, sodium butyrate and chloroquine did not affect the transduction efficiency of hepa1c1c7 cell lines with rAAV. Still more, high dose of hydroxyurea (100mM) or chloroquine (50μM) showed the cytotoxicity and induced the downregulation of Lac-Z expression. Pretreatment of hepatoma cell lines with camptothecin, etoposide, 5-FU or cisplatin increased the transduction efficiency of rAAV-LacZ and etoposide, one of topoisomerase inhibitor, was the most effective for increasing the transduction of hepatoma cell lines with rAAV. When the cells were exposed with γ-irradiation and infected with AAV-LacZ alone, the effect was similar to that obtained with permissive growth medium. The results of combinations with chemotherapeutic agents (i.e., 1μM of etoposide, 3μM of 5-FU, 1μM of cisplatin, 30μM of camptothecin) and γ-irradiation were confusing. Combinations of chemotherapeutic agents(1μM of etoposide, 3μM of 5-FU, 1μM of cisplatin, 30μM of camptothecin) and γ-irradiation increased the transduction efficiency of H4IIE cells significantly. (p<0.05) /In contrast, combination of permissive medium and γ-irradiation or 3μM of etoposide and γ-irradiation in hepa1c1c7 cells, and combination of permissive medium and γ-irradiation in H4IIE cells showed the downregulation of LacZ expression with cytotoxicity.(p<0.05) Taken together, the preteatment of hepatoma cell lines with some chemicals such as hydroxyurea, chemotherapeutic drugs such as camptothecin, etoposide, 5-FU, cisplatin, and γ-irradiation resulted in higher transduction efficiencies in hepatoma cell lines compared to no pretreatment. Some combinations of γ-irradiation and chemotherapeutic drugs increased transduction efficiency in hepatoma cell lines by rAAV-LacZ. However, most combinations of chemotherapeutic agent and γ-irradiation induced the downregulation of LacZ expression. /These suggested us that chemotherapeutic agents or γ-irradiation might increase the effect of rAAV gene therapy, but the circumstances that show the cell cytotoxicity induce the down regulation of target gene expression in hepatoma cell lines with rAAV gene therapy.