[Optimized AAV package and experimental application of recombinant AAV8/hFâ
§ for gene therapy on hemophilia A mice].
Zhonghua Xue Ye Xue Za Zhi
; 41(1): 34-39, 2020 Jan 14.
Article
em Zh
| MEDLINE
| ID: mdl-32023752
ABSTRACT
Objective:
To evaluate the effects of adeno-associated virus (AAV) carrying hFâ § by serotype 8 (AAV8/hFâ §) on hemophilia A (HA) mice by gene therapy strategy.Methods:
pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFâ §, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFâ § in HEK-293 cells using the optimized package condition. The purified AAV8/hFâ § were intravenously injected into HA mice and the effects of gene therapy were estimated.Results:
The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 µg plasmids, 20 cm-dish to 20 µg, 30 µg and 40 µg plasmids were employed, and the condition of 20 cm-dish to transfect 20 µg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFâ § was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFâ § was detected by Western blot. Fractions of AAV8/hFâ § at the dose of 8×10(12) vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of Fâ § was measured by aPTT assay. Results showed that the activity of Fâ § maintained at the therapeutic level and lasted up to 12 weeks after injection.Conclusion:
The purified AAV8/hFâ § based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFâ § were observed in vivo.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dependovirus
/
Hemofilia A
Limite:
Animals
/
Humans
Idioma:
Zh
Ano de publicação:
2020
Tipo de documento:
Article