Skeletal muscle targeting AAVT42 vector showed high tropism to the central nervous system in the adult mice and monkeys (IMAGE)
Caption
(A) Generation of the novel AAVT42 vector by directed evolution and in vivo screening. The cap genes of AAV1, AAV2, AAV3B, AAV4, and AAV6-9 were mixed, after digestion, denaturation, and reannealing, the shuffled novel AAV cap genes were generated, which were then inserted behind the rep gene and established a library of AAV viral particles. These viruses were injected intravenously in mice, after skeletal muscle isolation, AAV variants displaying tissue tropism were collected, and the corresponding cap genes were recovered by polymerase chain reaction, which were re-cloned into infectious plasmids, forming the viral particles. Then in vivo screening in the skeletal muscle was conducted again for further enrichment, characterization, and the sequencing validation of the novel variant. .
(B) The schematic of stereotactic AAV injection in mice.
(C) Representative confocal microscope images of GFP expression in the mice dorsal hippocampus, and the cell nuclei were marked with DAPI (blue). DG, dentate gyrus; CA1, cornu ammonis 1; CA3, cornu ammonis 3. Yellow arrows indicated GFP-positive dendrites.
(D) Quantification analysis of GFP fluorescent intensity in the dorsal hippocampus. Both the left and right half of the dorsal hippocampus of each mouse were quantified (dorsal hippocampus: AAV9-GFP = 1342 ± 359, n = 5 mice; AAVT42-GFP = 2916 ± 354, n = 5 mice, with two injection sites per mouse, AAV9-GFP versus AAVT42-GFP, ∗∗P = 6.8 × 10−3).
(E) The schematic of stereotactic AAV injection in monkey brain.
(F) Confocal images showed GFP-positive cells, which colocalized with neuron marker (NeuN, red) in the hippocampus of monkeys after AAVT42-GFP injection.
(G) Representative confocal microscope images of antibodies to GFP, NeuN, astrocyte marker GFAP, and microglia marker Iba1 in monkey hippocampus. AAV, adeno-associated virus; GFP, green fluorescent protein.
Credit
Siqi Tang, Wenshu Luo, Shihao Wu, Meng Yuan, Jiashuo Wen, Guoshen Zhong, Leshan Shen, Wei Jiang, Cheng Cheng, Xia Wu, Xiao Xiao
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