Figure 2.
Pseudotyping of rAAV Genomes with Capsids Derived from Four Additional Bocavirus Serotypes
(A) BoV helper plasmid (pCMVNS*Cap1) for chimeric rAAV/HBoV1 production and acceptor plasmid (pCMVNS*CapΔ) derived thereof for cloning of the different BoV cap ORFs. Each cap sequence was ordered as two gene blocks, assembled to a full-length cap ORF (capx, where x = HBoV2–4 or GBoV) and subsequently cloned into the acceptor plasmid using a Golden Gate reaction. BocaSR, BoV-transcribed small non-coding RNA. Numbers in brackets refer to the construct labels in Figure 1A. (B) Production and iodixanol purification of chimeric HBoV1-4 and GBoV vectors encoding Gluc. The amount of genome copies per milliliter was determined with TaqMan RT-PCR. Shown are averages (±SEM) of four independent productions. (C) Western blot analysis of the indicated iodixanol-purified BoV stocks. Detected are the three BoV capsid proteins VP1, VP2, and VP3. NEG, iodixanol gradient from untransfected cells. (D) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 2 × 104. Gluc activity in the medium was measured at 4 and 9 days post-transduction as arbitrary light units (ALU). Data are the mean Gluc expression (±SEM, n = 3). Numbers above the columns depict fold increases in expression. (E) Flow cytometry analysis of pHAEs transduced with scAAV-YFP/HBoV1 or scAAV-YFP/HBoV4. scAAV-Gluc/HBoV1 was used as a control (both at an MOI of 5 × 104, n = 4 independent transwells). Cells were co-stained for YFP and a cell type-specific marker: β-Tubulin IV (ciliated cells), MUC5AC (goblet cells), CC10 (club cells), or KRT5 (basal cells). Percentages of double-positive cells are shown in the upper right quarter. (F) Transduction of primary lung organoids with the indicated scAAV-Gluc/BoV variants. Several organoids per donor were either mechanically broken (br) and incubated with a total of 5 × 109 viral genomes, or they were individually microinjected (in) with 5 × 108–1 × 109 viral particles. Total Gluc activity in the medium was measured 3–12 days post-transduction and plotted on the y axis as ALU. Numbers above the columns depict fold increases in expression. (G) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 1 × 104 in the presence (+) or absence (−) of IVIg. Gluc activity in the medium was measured 5 days post-transduction as ALU. Shown is the mean Gluc activity (+SEM, n = 4, except for HB1 [–] n = 3). For pHAEs transduction, LLnL and Doxorubicin were added at concentrations of 40 and 5 μM, respectively. Transductions of primary lung organoids were performed in the presence of 1 μM Doxorubicin. For statistical analysis, one-way ANOVA was used. Significance at p < 0.001 is indicated by a triple asterisk. ns, non-significant.