Figure 1.

Directed evolution of AAV5 capsid and candidate selection

(A) Screening of AAV5 capsid mutants with liver tropism. The AAV5 capsid NNK library plasmid was transfected into HEK293 cells together with AdHelper and 3-stop plasmids. Mosaic AAVs in HEK293 medium and lysate were purified by iodixanol gradient ultracentrifugation and used to inject mice via tail vein with a dose of 1 × 10¹³ vg/kg. Liver mRNAs were harvested 40 days post-injection, reverse-transcribed to cDNA, and subjected to NGS sequencing after 2 rounds of selection. CB, hybrid CB promoter with CMV enhancer and β-actin promoter elements; p40, p40 promoter region in the AAV2 Rep sequence; ITR, inverted terminal repeats. Representative oligopeptide inserts are shown in the table. (B) PDB construction of the 3-dimensional (3D) structure of AAVzk2 VP1 protein by Chimera 1.15 (University of California, San Francisco). The inserted oligopeptide was highlighted and magnified in the black rectangle frame. (C) Silver staining of the indicated AAV particles produced by 2 rounds of iodixanol gradient ultracentrifugation, with each AAV serotype in 3 lanes. (D) Table illustrating that the indicated AAVs packaged from 2 × 10⁸ cells (medium + lysates) were quantified by qPCR and silver staining. n = 3. vg, vector genome; vp, viral particle.