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. 2016 Jun 22;11(6):e0158009. doi: 10.1371/journal.pone.0158009

Fig 3. Variations to ssAAV vector design.

Fig 3

Schematic illustration of modifications to the basic bicistronic ssAAV vector. (A) Variants of ssAAV vector comprise constructs with modifications to the Furin/F2A cleavage site and the 3' untranslated region (UTR); the changes include the addition of SGSG peptide and V5 peptide, deletion of furin peptide, and inclusion of a post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE). Abbreviations: ITR, inverted terminal repeat of AAV2; 5'ITRΔtrs, 5' inverted terminal repeat devoid of the terminal resolution site; Short CMV, a shortened variant of the immediate early CMV promoter; SV40 intron, an intron from simian virus 40; SP, signal peptide; VL, variable light domain; L, serine-glycine linker peptide; VH, variable heavy domain; H, hinge region; CH, constant heavy domain; CL, constant light domain; pA, polyadenylation signal; Furin, cleavage sequence for the cellular protease furin; F2A, 2A peptide from foot-and-mouth-disease virus. (B) Amino acid sequences of the Furin/F2A cleavage site in the different ssAAV vector constructs. Amino acids were colored to illustrate the range of specific sequences: encoded sequence of the heavy chain C-terminus (black), Furin peptide (red/orange), V5 peptide (gray), SGSG peptide (blue), F2A peptide (green) and the first amino acid (M, Methionine) of the light chain signal peptide (purple). Cleavage sites are indicated by arrows and are numbered in the order that cleavage is believed to occur: F2A self-cleavage (1.), Furin enzyme-mediated removal (2.), carboxypeptidase enzyme-mediated cleavage of basic amino acids (3.). The underlined amino acids represent the heavy chain C-terminus after the final cleavage within the secretory pathway. In B-cells, the heavy chain genes encode the amino acids Pro-Gly-Lys (PGK) at the C-terminus, however, the secreted IgG lacks the terminal Lys (K) due to removal by carboxypeptidases [31]. The ssAAV-4L6 IgG1 vector construct containing the appropriate Furin peptide was utilized in Fuchs et al. [17].