Construction of Recombinant Adeno-Associated Virus Vectors Carrying the Combined Transcriptional Regulatory Sequences of α-Fetoprotein Enhancer and Albumin Promoter_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

ZHANG Mingman ¹ , LI Dehua ² , GOU Xinghua ² , YAN Lnan ¹ , ZHAO Yongheng ¹ , SU Zhi ² , HAN Lei ² , ZHAO Lanying. ²

Department of General Surgery， West China Hospital， Sichuan University, Chengdu 610041, China;

Corresponding author：

Keywords：

Adeno-associated virus Hepatocellular carcinoma Target Gene therapy

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【Abstract】ObjectiveTo construct a recombinant adeno-associated virus(rAAV2) vectors carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter for the purpose of targeted gene therapy for hepatocellular carcinoma (HCC).
MethodsThe fragment of combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was amplified through polymerase chain reaction (PCR) and cloned into the promoter site of pAAV-IRES-hrGFP instead of the CMV promotor in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-IRES-hrGFP-EP. Then the packaging cell lines (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-EP together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine.The recombinant adenoassociated virus vector(rAAV2-EP) carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was packaged and amplified in the HEK 293 cell. Then the viral titer was checked by GFP.
ResultsThe recombinant adeno-associated virus vector(rAAV2-EP) carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was constructed successfully, the b green fluorescence was observed in HEK 293 cells under fluorescence microscope. The viral titer was 1.2×105.
ConclusionConstruction of the recombinant adeno-associated virus vector rAAV2-EP driven by the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter would provide a sound basis and improved vector for targeted gene therapy for HCC.

Citation： ZHANG Mingman,LI Dehua,GOU Xinghua,YAN Lnan,ZHAO Yongheng,SU Zhi,HAN Lei,ZHAO Lanying.. Construction of Recombinant Adeno-Associated Virus Vectors Carrying the Combined Transcriptional Regulatory Sequences of α-Fetoprotein Enhancer and Albumin Promoter. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2005, 12(2): 142-146. doi: Copy

1.	Carroll NM, Tanabe KK. Gene therapy for liver tumors [Ｊ]. Surg Oncol Clin N Am， 1998; 7(3)617.
2.	贺平，叶胜龙，汤钊猷. 甲胎蛋白增强子白蛋白启动子联合转录调控序列的克隆 [Ｊ]. 中华肝脏病杂志，1998； 6（3）136.
3.	Watanabe K, Saito A, Tamaoki T. Cellspecific enhancer activity in a far upstream region of the human alphafetoprotein gene [Ｊ]. J Biol Chem, 1987; 262(10)4812.
4.	Urano Y, Watanabe K, Sakai M, et al. The human albumin gene. Characterization of the 5′ and 3′ flanking regions and the polymorphic gene transcripts [Ｊ]. J Biol Chem, 1986; 261(7)3244.
5.	Chen XP, Zhao H, Zhao XP. Alternation of AFPmRNA level detected in blood circulation during liver resection for HCC and its significance [Ｊ]. World J Gastroenterol, 2002; 8(5)818.
6.	Cao G, Kuriyama S, Gao J, et al. Gene therapy for hepatocellular carcinoma based on tumourselective suicide gene expression using the alphafetoprotein (AFP) enhancer and a housekeeping gene promoter [Ｊ]. Eur J Cancer, 2001; 37(1)140.
7.	Ishikawa H, Nakata K, Mawatari F, et al. Retrovirusmediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alphafetoprotein enhancer/promoter [Ｊ]. Biochem Biophys Res Commun, 2001; 287(4)1034.
8.	Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxiainducible enhancer linked to a human alphafetoprotein promoter [Ｊ]. Cancer Res, 2001; 61(7)3016.
9.	Jin JR, Wen P, Locker J. Enhancer sharing in a plasmid model containing the alphafetoprotein and albumin promoters [Ｊ]. DNA Cell Biol, 1995; 14(3)267.
10.	Monahan PE, Samulski RJ, Tazelaar J, et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia [Ｊ]. Gene Ther, 1998; 5(1)40.
11.	Rabinowitz JE, Samulski RJ. Building a better vector: the manipulation of AAV virions [Ｊ]. Virology, 2000; 278(2)301.
12.	Samulski RJ, Zhu X, Xiao X, et al. Targeted integration of adenoassociated virus (AAV) into human chromosome 19 [Ｊ]. EMBO J, 1991; 10(12)3941.
13.	Zolotukhin S, Potter M, Hauswirth WW, et al. A “humanized” green fluorescent protein cDNA adapted for highlevel expression in mammalian cells [Ｊ]. J Virol, 1996; 70(7)4646.

1. Carroll NM, Tanabe KK. Gene therapy for liver tumors [Ｊ]. Surg Oncol Clin N Am， 1998; 7(3)617.
2. 贺平，叶胜龙，汤钊猷. 甲胎蛋白增强子白蛋白启动子联合转录调控序列的克隆 [Ｊ]. 中华肝脏病杂志，1998； 6（3）136.
3. Watanabe K, Saito A, Tamaoki T. Cellspecific enhancer activity in a far upstream region of the human alphafetoprotein gene [Ｊ]. J Biol Chem, 1987; 262(10)4812.
4. Urano Y, Watanabe K, Sakai M, et al. The human albumin gene. Characterization of the 5′ and 3′ flanking regions and the polymorphic gene transcripts [Ｊ]. J Biol Chem, 1986; 261(7)3244.
5. Chen XP, Zhao H, Zhao XP. Alternation of AFPmRNA level detected in blood circulation during liver resection for HCC and its significance [Ｊ]. World J Gastroenterol, 2002; 8(5)818.
6. Cao G, Kuriyama S, Gao J, et al. Gene therapy for hepatocellular carcinoma based on tumourselective suicide gene expression using the alphafetoprotein (AFP) enhancer and a housekeeping gene promoter [Ｊ]. Eur J Cancer, 2001; 37(1)140.
7. Ishikawa H, Nakata K, Mawatari F, et al. Retrovirusmediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alphafetoprotein enhancer/promoter [Ｊ]. Biochem Biophys Res Commun, 2001; 287(4)1034.
8. Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxiainducible enhancer linked to a human alphafetoprotein promoter [Ｊ]. Cancer Res, 2001; 61(7)3016.
9. Jin JR, Wen P, Locker J. Enhancer sharing in a plasmid model containing the alphafetoprotein and albumin promoters [Ｊ]. DNA Cell Biol, 1995; 14(3)267.
10. Monahan PE, Samulski RJ, Tazelaar J, et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia [Ｊ]. Gene Ther, 1998; 5(1)40.
11. Rabinowitz JE, Samulski RJ. Building a better vector: the manipulation of AAV virions [Ｊ]. Virology, 2000; 278(2)301.
12. Samulski RJ, Zhu X, Xiao X, et al. Targeted integration of adenoassociated virus (AAV) into human chromosome 19 [Ｊ]. EMBO J, 1991; 10(12)3941.
13. Zolotukhin S, Potter M, Hauswirth WW, et al. A “humanized” green fluorescent protein cDNA adapted for highlevel expression in mammalian cells [Ｊ]. J Virol, 1996; 70(7)4646.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of Recombinant Adeno-Associated Virus Vectors Carrying the Combined Transcriptional Regulatory Sequences of α-Fetoprotein Enhancer and Albumin Promoter

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