| Purpose: To construct a capsid-mutant adeno-associated virus 2(AAV2) vector, which can display significantly increased transduction efficiency in the retina via intravitreal delivery; To assess its transduction in vitro and in vivo.Methods: Modified surface-exposed amino-acid residues on AAV2 capsids via Sitedirected Gene Mutagenesis Kit to construct AAV2 vectors containing four Y to F mutations and one T to V mutation(quard Y- F +T-V). In vitro, RGC-5 cells were infected with 0.1ml AAV2-Zs Green1, AAV8-Cherry and AAV2(quard Y-F+T-V)-Zs Green1 vectors respectively. FACS analysis was used to quantify the percentage of cells that were Zs Green positive and Cherry positive. In vivo, the right eyes of wildtype C57 BL /6 mice of three groups were administered 1ul AAV2-Zs Green1, 1ul AAV8-Cherry and 1ul AAV2(quard Y-F +T-V)-Zs Green1 via intravitreal delivery separately.Observed fluorescent protein expression in retinal sections by fluoresence microscope at 4 weeks after injection.Results: The results of FACS showed that the percent of cells among these groups: 2.31% in the AAV2- Zs Green1, 5.52% in the AAV8- Cherry, 4.77% in the AAV2(quard Y- F +T- V)-Zs Green1 and 1.80% in the control. The constructed AAV2(quard Y- F +T- V)-Zs Green1 vectors following intravitreal delivery were transducted to ganglion cell layer, inner plexiform lay, inner nuclear lay, outer plexiform lay and outer nuclear layer. While WT-AAV-based vectors were just transducted to ganglion cell layer. Results of the points of expression of fluorescent protein between AAV2(quard Y-F +T-V)-Zs Green1 group and WT-AAV-based group showed a significant difference. But there was no difference between two WT-AAV-based groups.Conclusions:There was a significantly increased transduction efficiency in the constructed capsid-mutant adeno-associated virus 2(AAV2) vectors following intraveal delivery, which will provide a more safety and efficient gene therapy to retinal disease. |
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