The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy / 中西医结合学报

OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.@*RESULTS@#The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Inhibition effect of anti-epidermal growth factor receptor antibody mediated by recombinant adeno-associated virus vector on pancreatic cancer cell lines / 中华消化外科杂志

Objective To construct a recombinant adeno-associated virus(rAAV)vector containing a human anti-epidermal growth factor receptor(anti-EGFR)single-chain variable fragment antibody gene,and observe its inhibitory effects on pancreatic cancer cell lines.Methods Human anti-EGFR single-chain variable fragment antibody gene was inserted into the Kpn I and Bgl Ⅱ sites to construct a rAAV-anti EGFR vector,and then rAAV1-EGFP group and rAAV1-anti EGFR group were established.The expression of anti-EGFR antibody was observed.Antibody expression was detected by Western blot,and the inhibition and apoptosis rates of human pancreatic cancer cell lines(PCT-3,SW1990,Capan-1,ASPC-1,MiaPaCa-2 and PANC-1 cells)were detected by CCK-8 assay and flow cytometry,respectively.All data were analyzed using the t test.Results The results of Western blot assay demonstrated that anti-EGFR antibody was expressed in 6 pancreatic cancer cell lines.The inhibition rates of rAAV1-EGFP and rAAVl-anti EGFR on pancreatic ASPC-1 cells were 1.1%± 2.4% and 15.1%±3.5%,respectively,with a significant difference between the 2 groups(t =6.598,P ＜0.05).The apoptosis rates of PANC-1 cells were 7.0% ± 3.0% in the rAAV1-EGFP group and 1 1.4% ± 2.5% in the rAAV1-anti EGFR group,with no significant difference between the 2 grouvs(t = 1.952,P ＞0.05).The apoptosis rates of SW1990,ASPC-1,Capan-1,PCT-3,MiaPaCa-2 cells were 1.1% ± 0.8%,1.5% ± 0.7%,1.7% ± 1.2%,1.1%±0.7% and 2.2% ± 1.1% in the rAAV1-EGFP group,and 17.6% ± 2.2%,46.9% ± 3.9%,20.0% ±2.8%,12.1% ± 1.6% and 31.1% ±2.5% in the rAAV1-anti EGFR group,respectively,with significant differences between the 2 groups(t = 12.208,19.846,10.405,10.909,18.327,P ＜0.05).Conclusions A rAAV-anti EGFR vector with human anti-EGFR single-chain variable fragment antibody gene was constructed.Anti-EGFR antibody has obvious inhibition effects on pancreatic cancer cell lines.

Recombinant-adeno-associated viral vector-mediated gene therapy for cardiovascular diseases / 中南大学学报(医学版)

Adeno-associated virus is a kind of DNA defective parvovirus which is non-pathogenic. Recombinant-adeno-associated virus vector comes from wild-type non-pathogenic adeno-associated virus and is highly secure, and it also has the advantages of broad host range. Recombinant-adeno-associated virus vector has become a hot spot for gene therapy and is widely used in gene therapy for cardiovascular diseases, especially for hypertension, heart failure, arteriosclerosis, and myocardial infarction.