HBsAg阳性肝细胞的膜表面HBsAg抗原的检测 Hep3B HBsAg阳性肝细胞 91.3% 93.5% HepG2 2.3% Parvogen International

Map of AAV Genome Parvogen International

pHelper质粒的改建 pHelper Parvogen HBsAb Fd Cap E4 启动子 International Rep E2A 启动子 pHelper VAI Ori Parvogen International

A series of human cells infected by tropic AAV/AFPp-GFP virus （24 hours) HBsAg阳性 HBsAg阴性 Hep3B 肝细胞 肝癌细胞 HepG2 肝细胞 HBsAg阴性 PBMC Hela LNcap Hs578T H2125 Lovo Parvogen International

A series of human cells infected by tropic AAV/ALBp-GFP virus （24 hours) HBsAg阳性 HBsAg阴性 Hep3B 肝细胞 肝癌细胞 HepG2 肝细胞 HBsAg阴性 PBMC Hela LNcap Hs578T H2125 Lovo Parvogen International

Tropic AAV/ALBp-HLA-A2 virus infection （24 hours) HBsAg阳性肝细胞 HBsAg阴性肝细胞 未感染 感染 感染 5.6% 91.1% 1.4% Parvogen International

Tropic AAV/AFPp-HLA-A2 virus infection （24 hours) HBsAg阳性Hep3B细胞 HBsAg阴性HepG2细胞 未感染 感染 感染 87.8% 4.7% 10.7% Parvogen International

HLA-A2-expressed Primary Hepatic Carcinoma Cells Killed by the rAAV/AFP-infected-DC Pulsed CTL AAV/AFPp-HLA-A2- infected cells uninfected cells 90.1% 0.01% 0.01% 90.1% Uninfected cells AAV/HLA-2-infected cells uninfected cells % killing 50 40 30 20 10 Parvogen International

Section II Inhibit oncogene expression AAV-Rep78 function: interaction with HBV or AFP Inhibit oncogene expression Inhibit cervical cancer Inhibit Human papillomavirus p97 promoter Inhibit E6 & E7 oncogene Enhance recombinant AAV production Can Rep78 inhibit HBV or AFP expression? Parvogen International

Expression of HBsAg inhibited by tropic AAV/AFPp-Rep78 virus infection Parvogen International

Expression of HBeAg inhibited by tropic AAV/AFPp-Rep78 virus infection in HBV-positive PHC 感染前 24小时 48小时 72小时 96小时 68.9% 84.6% 53.9% 10.7% 32.9% Parvogen International

Expression of AFP inhibited by tropic AAV/ALBp-Rep78 virus infection Parvogen International

Elevation of albumin by tropic AAV/ALBp-Rep78 virus infection in Hep3B cells 未感染前 48小时 72小时 96小时 96.6 50.8% 22.3% 45.6% 12.6% Parvogen International

AAV Rep78与HBV基因的相互作用具有剂量依赖效应 Enh I X基因启动子 Enh II 前C/C启动子 Rep78 (μg): 0 0.2 0.5 0 0.2 0.5 0 0.2 0.5 0 0.2 0.5 Parvogen International

Inhibition of HBV-C and –X Transcription by Rep78 C+Rep78 C X+Rep78 X+GST X 1.0ug 1.0ug Parvogen International

Section III Functions of expressed interferon in rAAV-transfeted hepatic cells 甲胎蛋白启动子 Parvogen International

构建携带干扰素基因的嗜肝性重组腺相关病毒 ALBp TR Poly A IFN-α基因 rAAV/Albp-IFN-α ALBp TR Poly A IFN-γ基因 rAAV/Albp-IFN-γ AFPp TR Poly A IFN-γ基因 rAAV/Albp-IFN-γ Parvogen International

rAAV在肝细胞中表达IFN-α抑制HBsAg和HBeAg的表达 rAAV在肝细胞中表达IFN-a对HBeAg表达的影响 0小时 48小时 72小时 96小时 77.2% 12.1% 52.9 52.9% 25.7% Parvogen International

rAAV在感染的黑猩猩中表达人IFN-a AAV/Albp-IFN-α Virus, 1011 copies Control AAV Parvogen International

Expressed IFN-γ promote expression of MHC class I on rAAV-infected Hep3B cells IFN-γ MHC class I 2.5% \ % 感染前 感染AAV/AFPp -IFN-γ病毒后 52.3% 33.7% 12 hr 78.0% 48.5% 48 hr 85.2% 76.0% 96 hr Parvogen International

rAAV在肝细胞中表达的IFN-γ增强抗原特异性，MHC class I限制性CTL的杀伤活性 70 60 50 40 30 20 10 CTL杀伤率 A B C D E F G H HBsAg+肝细胞：B: HepG2 C: +anti-HLA I D:+INF-r Hep3B: F: HBsAg+肝细胞 G:+anti-HLA I H:+INF-r Parvogen International

Conclusions Generate rAAV with liver cell-specific promoters. Gernerate liver-tropic rAAV. Recover MHC class I expression by transfection of liver-tropic rAAV. AAV Rep78 may become new anti-HBV infection or hepatic cancer drug. Expressed INF-αcan inhibit HBV expression in the rAAV-transfected liver cells. The liver-tropic AAV carrying IFN-Αcan express in animal models. Expressed IFN-γcan promote expression of the HLA class I antigen in the rAAV-transfected liver cells. Parvogen International

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