1. Volume 21, Issue 12, Pages 2181-2194 (December 2013)
A Novel Chimeric Adenoassociated Virus 2/Human Bocavirus 1 Parvovirus Vector Efficiently Transduces Human Airway Epithelia 
Ziying Yan, Nicholas W Keiser, Yi Song, Xuefeng Deng, Fang Cheng, Jianming Qiu, John F Engelhardt 
Molecular Therapy 
Volume 21, Issue 12, Pages (December 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
rHBoV1 vector production and infection of primary polarized HAE. (a) Schematic structure of the HBoV1 genome and the proviral plasmids used in this study. pHBoV1KUm630 is the helper plasmid for trans-complementation of HBoV1 viral proteins, pIHBoV1 is the infectious clone of the HBoV1 complete genome, and prHBoV1-CBAluc is the rHBoV1 cis transfer proviral plasmid. Critical restriction enzyme cutting sites used for cloning are also indicated and small deletions within NS and VP genes are marked (Δ). (b) Replication complementation assay of the rHBoV1 proviral plasmid in HEK293 cells. pIHBoV1 (lane 1), prHBoV1-CBAluc (lane 2), or prHBoV1-CBAluc + pHBoV1KUm630 (lane 3) plasmids were transfected to HEK293 cells. Hirt DNA was extracted at 48 hours after transfection and digested by DpnI before resolving on an agarose gel. HBoV1 replication intermediates (indicated by arrows) were visualized by Southern blotting with a 32P-labeled HBoV1 probe. Hirt DNA from cells transfected with the HBoV1 infectious clone (pIHBoV1, lane 1) was used as positive control. Short and long exposures are shown on the left and right of the panel, respectively. (c) DNase I–digested cell lysates from HEK293 cotransfected with prHBoV1-CBAluc and pHBoV1KUm630 were fractionated by CsCl equilibrium ultracentrifugation. The plot shows the distribution of rHBoV1.CBAluc (solid dots) genomes against the observed density of the gradient (open dots). The genome copies in each fraction (~750 µl) were determined by TagMan PCR. (d) Transduction assay following rHBoV1.CBAluc infection of HEK293 cells, IB3 cells, undifferentiated (UD) CuFi8 cell monolayers, polarized CuFi8 cells in ALI cultures, and primary HAE ALI cultures. Data represent the mean (±SEM) relative luciferase activity per well at 2 days after infection (n = 4). (e) Transgene expression from rHBoV1.CBAluc infected HAE ALI cultures at different time points after infection. Data represent the mean (±SEM) relative luciferase activity per well (n = 3). ALI, air–liquid interface; HAE, human-polarized airway epithelia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Pseudopackaging rAAV2 genomes in HBoV1 capsid. (a) DNase I–digested cell lysates from the indicated HEK293 cell plasmid transfections were fractionated by CsCl equilibrium ultracentrifugation. The number of viral genomes in each fraction was determined by TaqMan PCR. (b) HEK293 cells were transfected with the indicated combinations of plasmids (M: Molecular weight marker; lane 1: pAV2-F5tg83luc + pAV-Rep2; lane 2: pAV2-F5tg83luc + pAVRC2.3; lane 3: pAV2-CF5tg83luc + pAV-Rep2; land 4: pAV2-CF5tg83 + pAV-Rep2 + pHBoV1KUm630) together with the Ad helper pAd4.1. Low molecular weight (Hirt) DNA was extracted from transfected cells after 48 hours and digested with Dpn I, followed by Southern blotting using a 32P-labeled luciferase probe. The 4.8 kb and 5.4 kb replicative form (RF) DNA of the rAV2.F5tg83Luc and rAV2.CF5tg83Luc genomes are indicated by arrows. (c) Negatively stained transmission electron micrographs of the chimeric vector rAV2/HBc.F5tg83luc, (bar = 100 nm in the 15,000× image and 50 nm for the 50,000× image). The virus-like particle with incompletely packaged viral DNA (<1% of total virions) is marked by a white arrow in the inset. (d) A two-color western blot (red: AAV; green: HBoV1) was performed on the indicated viral preparations using an Infrared Image System. Converted single channel images are also shown with dark arrows pointing to the AAV2 and HBoV1 VP proteins in the left and right panels, respectively. Gray arrows and white arrows mark cleaved protein fragments from HBoV1 VP1 and VP2 proteins.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Package polarity and capacity of rHBoV1 and rAAV2/HBoV1 vectors. (a) Viruses AV2/2.F5tg83luc, AV2/HBc.F5tg83luc, and rHBoV1.CBAluc were loaded on nylon membrane by slot blotting and visualized with 32P-labeled 32-mer oligonucleotide probes against the minus and plus strand of the luciferase gene (left panels). The percentages of the minus and plus strands in each viral preparation was calculated based on the signal density quantitated with NIH ImageJ software (right panel). (b) 2 × 108 DRP of rAAV vector AV2/2.F5tg83luc, chimeric viruses AV2/HBc.F5tg83luc, and AV2/HBc.CF5tg83luc were heated in alkaline gel loading buffer at 95 °C for 10 minutes and then resolved in a 0.9% alkaline agarose gel. Following transferred to Nylon membrane, Southern blotting was performed with 32P-labeled luciferase probe. Black and white arrows mark the shorter rAV2.F5tg83luc (4.8 kb) and longer rAV2.CF5tg83luc (5.4 kb) genomes, respectively. (c) Left panels depict slot blots of AV2/HBc.F5tg83luc and rHBoV1.CBAluc viral preparations (~109 DRP based on TaqMan PCR for the luciferase transgene) probed with 32P-labeled fragments recognizing the luciferase gene (1.7 kb) or the HBoV1 genome region unique to the helper plasmid (a 2.64 kb HindIII/BglII fragment covering the NP1 coding region). Right panel depicts the relative copies of luciferase or NP1 gene fragments based on the signal intensity relative to the plasmid standards. NIH ImageJ software was used to quantify the mean (±range) signal density for rAAV2/HBoV1 and rHBoV1 viral preparations shown. DRP, DNase I–resistant particles.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Transduction comparisons between rAAV2/HBoV1 and rAAV vectors. (a) Luciferase expression at 2 days following infection of HEK293 cells with AV2/2.F5tg83luc (MOI = 2,500 DRP/cell) or AV2/HBc.F5tg83luc (MOI = 50,000 DRP/cell). Results show the mean (±SEM, N = 4) relative luciferase activities per well of a 24-well plate. (b) Primary HAE ALI cultures were infected with AV2/2.F5tg83luc, AV2/1.F5tg83luc, or AV2/HBc.F5tg83luc from the apical or basolateral surface. The vector amount in the inoculum was 1010 DRP for each Millicell insert, roughly 5,000 to 10,000 DRP/cell. Data represent the mean (±SEM) relative luciferase activities measured at 7 days after infection (RLU/well) for N = 6 independent infections of HAE ALI cultures derived from three donors. (c,d) Virion internalization and subcellular distribution analyses were performed at 18 hours after primary HAE ALI cultures were apically infected with rAAV2/1, rAAV2/2, and rAAV2/HBoV1 vectors of 1010 DRP per Millicell insert. Viral genomes in the cytoplasmic and nuclear fractions were quantified by TaqMan PCR. The total viral genomes detected in each culture is presented in (c) with the black bars representing the nuclear fraction, whereas bars representing the cytoplasmic fraction. The percentage of viral genomes in each fraction is presented in (d). Data represent the mean (±SEM) viral genome copies (per well) for N = 3 independent infections. ALI, air–liquid interface; DRP, DNase I–resistant particles; HAE, human-polarized airway epithelia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Effect of proteasome inhibitors on rAAV2/HBoV1 transduction in polarized and non-polarized cultures of human airway epithelial cells. (a,b) Primary HAE ALI cultures were apically infected with 1010 DRP per Millicell insert with (a) AV2/2.F5tg83luc or (b) AV2/HBc.F5tg83luc for a period of 16 hours. When indicated, proteasome inhibitors (PI) LLnL (40 nmol/l) and doxorubicin (5 µmol/l) were applied only during the infection period. Luciferase expression was monitored over 11 days by biophotonic imaging of live cells using the Xenogen 200 IVIS (Xenogen). Data represent the mean (±SEM, n = 6) relative luciferase activity per well at three time points of 3, 7, and 11 days after infection. (c) CuFi8 cells cultured as a polarized epithelium at an ALI (CuFi ALI; a: apical infection, b: basolateral infection) or non-polarized undifferentiated monolayers on plastic (CuFi-UD), and HEK293 cells, were incubated with 1.5 × 109 DRP of AV2/HBc.F5tg83luc at 37 °C for 4 hours. All cultures contained ~5 × 105 cells at the time of infection. Following infection, unbound virus was washed off and cells were either detached from the culture supports with trypsin and lysed for TaqMan PCR quantification of viral genomes, or returned to the incubator for luciferase expression assays at 24 hours after infection using cell lysates. When indicated (+PI), CuFi8 cells were treated with proteasome inhibitors doxorubicin (1 µmol/l) and LLnL (8 nmol/l) during the 4 hours infection period. Data represent the mean (±SEM) total vector genomes (n = 4) at 4 hours after infection and relative luciferase activity (n = 3) at 24 hours after infection. ALI, air–liquid interface; DRP, DNase I–resistant particles; HAE, human-polarized airway epithelia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Partial correction of CFTR-dependent chloride transport by primary CF HAE ALI cultures following infection with AV2/HBc.CBAhCFTR. CF HAE ALI cultures derived from two CF patient donors (genotypes: ΔF508/ΔF508 homozygous) were infected with AV2/HBc.CF5tg83luc or AV2/HBc.CBAhCFTR at 1010 DRP per Millicell insert (MOI of 5,000–10,000 DRP/cell) in the presence of proteasome inhibitors LLnL (40 nmol/l) and doxorubicin (5 µmol/l). Uninfected non-CF HAE were also cultured for electrophysiologic comparisons and experimental cultures were evaluated at 10 days after infection. (a) Representative traces of transepithelial short-circuit current (Isc) of CF HAE following the sequential addition of various inhibitors and agonists as indicated. Amiloride and DIDS were used to block ENaC-mediated sodium currents and non-CFTR chloride channels before cAMP agonists (forskolin and IBMX) induction and GlyH101 inhibition of CFTR currents. ΔIsc(cAMP) reflects the activation of CFTR-mediated chloride currents following cAMP agonist induction and ΔIsc(glyH) reflects the inhibition of CFTR-mediated chloride currents following addition of GlyH101. (b) Summary data of the ΔIsc(cAMP) and ΔIsc(glyH) (mean ± SEM, n = 6 independent transwells) for both CF infected cultures and non-CF controls. (c) Immunofluorescent detection of CFTR expression (green) in CF HAE following infection with AV2/HBc.CBAhCFTR (left panels) or AV2/HBc.CF5tg83luc (right panels). ALI, air–liquid interface; DRP, DNase I–resistant particles; HAE, human-polarized airway epithelia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
One potential model for how polarization of human airway epithelia cells influences HBoV1 virion infection and transduction. (a) Polarized HAE may contain multiple binding and/or coreceptors for HBoV1. In this illustrated scenario, a single binding receptor exists on the apical membrane and is significantly reduced or absent on the basolateral membrane. Two different coreceptors exist including an efficient coreceptor-1 on the apical membrane and a more abundant inefficient coreceptor-2 on the basolateral membrane. Endocytosis through coreceptor-1 leads to functionally efficient (from a transduction standpoint) virion processing that is highly influences by activity of the proteasome, whereas internalization through coreceptor-2 is ineffective at processing the virion and not influenced by proteasome function. This model is consistent with significantly less viral uptake and transduction from the basolateral surface, as compared with the apical membrane. Other models not shown might include a second type of binding receptor on the basolateral surface that is inefficiently endocytosed with coreceptor-1 or coreceptor-2. (b) In non-polarized human airway cells, the primary binding receptor, coreceptor-1, and coreceptor-2 exist in the same membrane. Both coreceptors can interact with the same binding receptor, however, coreceptor-2 is in greater abundance than coreceptor-1. Thus, endocytosis of HBoV1 virions through coreceptor-2 predominates, and since this pathway inefficiently processes HBoV1 virions for productive transduction, transgene expression is low. These findings are consistent with high-level HBoV1 virion endocytosis, but poor transduction and weak proteasome inhibitor responsiveness, in non-polarized human airway cells. HAE, human-polarized airway epithelia.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions