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. 2009 Jul 14;17(12):2067–2077. doi: 10.1038/mt.2009.155

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Wuping Li 1,*, Liqun Zhang 2, Jarrod S Johnson 1,3, Wu Zhijian 1, Joshua C Grieger 1, Xiao Ping-Jie 1, Lauren M Drouin 4, Mavis Agbandje-McKenna 4, Raymond J Pickles 1,2,5, R Jude Samulski 1,3
PMCID: PMC2801879  NIHMSID: NIHMS161684  PMID: 19603002

Abstract

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

Introduction

Cystic fibrosis (CF) is the most common recessive lethal genetic disorder in Caucasian populations resulting from a defect in a single gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate–activated chloride ion channel. The pulmonary manifestations of CF account for over 90% of the morbidity and mortality.1 In the airway epithelium, mutations in CFTR affect the normal regulation of ion transport, leading to a reduced volume of airway surface liquid, mucus dehydration, decreased mucus transport, and mucus plugging of the airways. These events result in an inability to prevent or eradicate bacterial infections that, over several decades, lead to a progressive decline of lung function.

Restoration of CFTR function to the airway epithelium of CF patients is a major goal for alleviating CF lung disease. One therapeutic approach has been focused on delivering a normal copy of the CFTR gene to the airway epithelium using viral and nonviral-based gene delivery vectors. Two decades of intense preclinical and clinical research have identified diverse vector systems that hold promise for CFTR gene delivery to the lung. However, to date, the currently available vectors have failed to result in efficacious CFTR gene delivery to the ciliated airway epithelium of CF patients.

Adeno-associated virus (AAV)–derived vectors hold considerable promise for CFTR gene delivery due to advances in vector production, the relatively stable expression of transgenes and the low incidence of inflammation induced by these vectors in vivo. AAV-2 vectors carrying the complete human CFTR complementary DNA (tgAAVCF) have been delivered to the nasal epithelium, the sinuses, and lungs of subjects with CF. Although these studies indicated that AAV delivery was safe and well tolerated, they did not demonstrate expression of functional CFTR or a statistically significant improvement in lung function.2 These shortcomings likely resulted from a low efficiency of CFTR gene transfer to cells that enable corrective CFTR function in the CF airways. The efficiency of CFTR delivery to airway epithelium in vivo using viral vectors may be limited by multiple cellular barriers, such as lack of relevant receptors in the airway lumen that reduce receptor binding, internalization, and subcellular processing of vectors. Strategies to circumvent these barriers have mainly focused on identification and development of different AAV serotypes or by re-engineering existing serotypes for improved gene transfer efficiency.3,4

Although AAV-2-based vectors were originally proposed for CFTR delivery to CF airways, several groups have now shown that other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a variety of models of the lung epithelium.5,6,7,8,9 AAV-1 has been demonstrated to be ~100-fold more efficient than AAV-2 and AAV-5 at transducing human airway epithelial cells in vitro,5 although AAV-1 transduced murine tracheal airway epithelia in vivo with an efficiency equal to that of AAV-5.6 Other studies have shown that AAV-5 is 50-fold more efficient than AAV-2 at gene delivery to human airway epithelium (HAE) in vitro and significantly more efficient in the mouse lung airway epithelium in vivo.7 AAV-6 has also been shown to be more efficient than AAV-2 in human airway epithelial cells in vitro and murine airways in vivo.8 The more recent isolate, AAV-9, was shown to display greater gene transfer efficiency than AAV-5 in murine nasal and alveolar epithelia in vivo with gene expression detected for over 9 months suggesting AAV may enable long-term gene expression in vivo, a desirable property for a CFTR gene delivery vector. Furthermore, it was demonstrated that AAV-9 could be readministered to the murine lung with no loss of CFTR expression and minimal immune consequences.9

DNA shuffling combined with directed evolution has been previously used to generate novel AAV vectors that display improved transduction efficiency in specific cell types.10,11 Recently, it has been demonstrated that directed evolution of the AAV capsid can select viral variants with enhanced infection of HAE in vitro.12 Clearly, the cellular model used for AAV evolution will dictate the usefulness of this approach and, therefore, the cellular model should represent the appropriate in vivo characteristics of the target tissue, i.e., the human ciliated airway epithelium. We have now applied a directed evolution strategy to select AAV variants that exhibit increased transduction efficiency for an in vitro model of human ciliated airway epithelium in an attempt to generate more efficient gene delivery vectors for CFTR. In our approach, a chimeric AAV plasmid library was generated by DNA shuffling a series of genes encoding the capsid sequences of eight different AAV serotypes (1–9 with the exception of AAV-7).10 Using a plasmid library of chimeric AAV capsids we performed directed evolution of AAV on HAE. After five successive rounds of screenings, two novel AAV variants were identified with greater transduction efficiency for HAE than the parental serotypes AAV-1 and AAV-6. We next inserted the shortened CFTR construct (CFTRΔR)13 into the two evolved AAV variants and the parental AAV-6 and used these vectors to show that our novel variants improved CFTR gene transfer efficiency sufficiently to partially restore the CF bioelectric defect characteristic of HAE derived from CF patients (CF HAE). Our data demonstrate that directed molecular evolution of AAV using DNA-shuffling can be exploited to identify novel virions with improved efficiency of gene delivery to human ciliated airway epithelium and suggest that further development of AAV for the treatment of CF lung disease should be explored.

Results

Directed evolution of AAV variants by DNA shuffling and screening on cultures of human ciliated airway epithelium

Because traditional AAV-derived vectors transduce human ciliated airway epithelium in vitro and in vivo at levels unlikely to provide sufficient CFTR delivery for correction of the CF airways phenotype, we sought to generate novel AAV variants with improved transduction efficiency for human ciliated airway epithelium by directed evolution on HAE. We cycled our AAV capsid library as previously described10 in HAE using co-infection with wild-type adenovirus (AdV, dl309) to amplify infectious AAV variants (Figure 1a)14 Because HAE are relatively resistant to AdV infection after lumenal inoculation of HAE unless epithelial tight junctions are disrupted to reveal basolateral AdV receptors,15,16 the apical surfaces of HAE were first exposed to sodium caprate to transiently open tight junctions immediately before AdV inoculation.17 A schematic representation of our strategy is shown in Figure 1a. After five cycles of screening on HAE derived from different donors, DNA extracted from cell lysates obtained after the fifth cycle served as a template for PCR and was cloned into the AAV helper plasmid pXR. Ten individual clones were picked and subjected to sequence analysis. Two chimeric AAV variants were present in the population: a chimera of AAV-1 and AAV-6 capsids and a chimera of AAV-1, AAV-6, and AAV-9 capsids. These chimeras are referred to as HAE-1 and HAE-2, respectively and are represented schematically in Figure 1b. HAE-1, a chimera of AAV-1 and AAV-6 with only amino acid residues 583–641 derived from AAV-6, results in only two amino acid changes, F584L and A598V compared to the parent AAV-1. For HAE-2, the chimera was more complex, with amino acid residues 1–30, 105–193 derived from AAV-9 and, amino acid residues 31–104, 194–641 from AAV-6. The carboxyl-terminal amino acid residues 642–737 represented those of AAV-1 (Figure 1b).

Figure 1.

Figure 1

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Selection of novel AAV variants tropic to human ciliated airway epithelium in vitro. (a) The strategy for enrichment of HAE tropic AAV variants by repetitive screenings of combinatorial AAV shuffling library on HAE (see Materials and Methods for details). A DNA-shuffling AAV library was inoculated onto the apical surface of HAE, followed by sodium caprate treatment/wild-type adenovirus infection at 24 hours, and cell lysis at 5 days to recover selected AAV viruses. The harvested AAV pool was then used for subsequent screening, and the process was repeated four more times. The recovered AAV viruses at the end of screenings were subjected to sequencing analysis. (b) Primary structures of two dominant AAV variants selected from successive screening on HAE, named as chimeric HAE-1, chimeric HAE-2, respectively. Sequence analysis and alignment with parental serotype sequences revealed the chimeric capsid subunit (VP1) are derived from AAV-1, AAV-6, and AAV-9. (c) Surface contour representations of the VP3 three-dimensional models of the HAE chimeric viruses. The front and back views of a trimer are shown for HAE-1 (top left and right) and HAE-2 (bottom left and right) which the surface colored according to the sequence contribution from the parental virus (red for AAV-1, blue for AAV-6) as depicted in b. The five amino acids that differ between AAV-1 and AAV-6 in the modeled region are colored black if contributed from AAV-1 or yellow if contributed from AAV-6 and are labeled. These images were generated in the program PyMol. AAV, adeno-associated virus; HAE, human airway epithelium.

Amino acid differences between the chimeric HAE-1 and HAE-2 and their parental viruses are localized around the icosahedral threefold axes

Three-dimensional models were generated for the chimeric HAE-1 and HAE-2 viruses and compared to the structures of the parental AAV-1 and AAV-6 viruses with respect to the role of capsid amino acid contributions to their enhanced transduction phenotypes. Mapping of the amino acid contributions from the parental AAV-1 and AAV-6 viruses onto a surface contour representation of these models showed a difference in parental origin of the five amino acids which differ between AAV-1/AAV-6, all located either on the interior or exterior capsid surface of these new viruses (Figure 1c). Of the five amino acid differences, AAV-1 contributes one outer surface residue (E531) and two inner surface residues (E418 and N642) to HAE-1 and AAV-6 contributes two outer surface residues (L584 and A598). For chimeric HAE-2, all outer capsid surface localized amino acid differences between AAV-1 and AAV-6 are contributed from AAV-6 (K531, L584, and V598). The two inner capsid surface amino acid differences are contributed from both AAV-1 (N642) and AAV-6 (D418). The contribution of the AAV-9 amino acids to the chimeric HAE-2 virus could not be modeled because these are located on the N-terminal region of the AAV viral protein (VP) that is disordered in all the structures that have been determined to date by X-ray crystallography and cryo-electron microscopy.

Enhanced transduction of human ciliated airway epithelium by novel AAV variants

We determined the transduction efficiency of the chimeric AAV variants, HAE-1 and HAE-2, on our in vitro model of HAE by packaging all recombinant AAV vectors with double-stranded green fluorescent protein (GFP) or luciferase expression cassettes. Both novel AAV capsids were competent in packaging recombinant AAV vectors to titers similar to the parental serotype AAV-1/AAV-6. To assess the numbers of cells transduced after inoculation of the apical surface of HAE with equal titers of chimeric capsids HAE-1, HAE-2, or the parental serotypes, AAV-1, AAV-5, AAV-6, and AAV-9 (multiplicity of infection (MOI) ~105), we monitored GFP transgene expression en face over time. The number of GFP-positive cells was found to be maximal at 1 week postinoculation (p.i.) and representative epifluorescence images are shown in Figure 2a. These data show that the parental serotypes, AAV-1, AAV-5, and AAV-9, transduced HAE poorly although AAV-6 transduction was repeatedly the best of the parental strains. Both chimeric capsids HAE-1 and HAE-2 transduced significantly more cells than either AAV-1, AAV-5, AAV-6, or AAV-9, with HAE-2 transduction being the most efficient of the two variants. Thus, based on this assay, we find that our directed evolution variants HAE-1 and HAE-2 are the most efficient vectors in HAE cells, outperforming all other AAV serotypes tested in parallel (AAV-1, AAV-5, AAV-6, and AAV-9).

Figure 2.

Figure 2

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Improved transduction efficiencies by the novel AAV variants. (a) HAE were inoculated with equal titers (MOI = 100,000) of AAV-1, AAV-5, AAV-6, AAV-9, HAE-1, and HAE-2, all of which contain a double strand GFP reporter gene. Representative en face fluorescence photomicrographs showing GFP-positive cells in HAE cells were obtained at 14 days postinoculation. (b) Quantitative comparisons of transduction efficiency using luciferase expressing vectors. HAE were inoculated with luciferase expressing AAV vectors (MOI = 1,000), cell lysates were harvested at 2 weeks postinoculation, and luciferase activities measured. *P < 0.01. AAV, adeno-associated virus; GFP, green fluorescent protein; MOI, multiplicity of infection.

Although monitoring GFP-positive cells in real-time enabled quantitation of the numbers of cells transduced, to further assess improvements in gene transfer efficiency with our novel AAV variants, we also performed experiments using AAV expressing a luciferase transgene. Luciferase enzyme activity in HAE was assessed 2 weeks p.i. with our novel AAV variants and the two best parent vectors (AAV-1 and AAV-6) (MOI ~103) (Figure 2b). Consistent with the findings for GFP-expressing vectors, AAV-6 expressed more luciferase activity in HAE than AAV-1, whereas the AAV variants, HAE-1 and HAE-2, produced two- to threefold more luciferase activity than AAV-1 and AAV-6, likely due to the increased numbers of cells targeted by these novel variants. Virus binding assay (see Supplementary Figure S1) supports a step downstream of initial binding as likely enhancement for these chimeric capsid transduction on HAE cells. Thus, two novel AAV variants have been generated by our molecular evolution strategy that have improved transduction efficiency for HAE compared to the parental AAV serotypes, AAV-1, AAV-6, and AAV-9. In addition, the two novel variants have improved transduction efficiencies over AAV-5 that has been previously reported to efficiently transduce HAE cells in vitro.7

Transduction efficiency of chimeric capsids HAE-1 and HAE-2 in human ciliated airway epithelia can be enhanced using pharmacological methods

It has been previously shown that AAV transduction of HAE can be enhanced by coadministration at the time of inoculation with proteasome-modulating agents, such as tripeptidyl aldehyde [N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL)], and chemotherapeutic agents, such as doxorubicin (Dox).18,19 To test whether these agents improved the transduction efficiency of chimeric capsids HAE-1 and HAE-2 in HAE cells, the proteosome inhibitor LLnL (40 µmol/l) and/or the DNA disrupting agent Dox (5 µmol/l) were co-inoculated onto the apical surfaces of HAE with AAV-6, chimeric capsids HAE-1, and HAE-2 expressing GFP. These agents (and AAV) were maintained on the apical surface of HAE for a further 10 hours before removal. GFP transgene expression assessed over time indicated that the maximal numbers of transduced cells was reached at 1 week p.i. In the absence of pharmacological reagents, the percentage of GFP-positive cells was greater for chimeric capsids HAE-1 and HAE-2 compared to AAV-6 confirming our earlier data (Figure 3a,b). For all vectors, both the numbers of AAV transduced cells and the intensity of GFP fluorescence increased after co-incubation with LLnL or Dox or both drugs together (Figure 3a,b). Both LLnL and Dox increased AAV transduction independently with the highest level of transduction efficiency produced by co-incubation of AAV with both drugs in combination. These data are consistent with previous reports that proteasome inhibitors LLnL and Dox can be additive.19 The highest transduction efficiencies were obtained with chimeric capsids HAE-1 or HAE-2 when coadministered with both LLnL and Dox with 6–7% of the surface epithelial cells being transduced (Figure 3b). Under these conditions, HAE-1 and HAE-2 vectors provided a twofold increase in transduction efficiency compared to AAV-6 when coadministered with LLnL and Dox. Because it has been reported that AAV transduction efficiency in HAE increased after disruption of epithelial cell tight junctions,20 we determined whether the addition of these reagents affected tight junction permeability at the time of AAV inoculation. Transepithelial resistance was measured immediately after inoculation of chimeric HAE-2 in the absence and presence of LLnL and Dox. Figure 3c shows that neither AAV vector nor LLnL/Dox affected transepithelial resistance indicating that enhancement of AAV-mediated transduction did not occur by exposing basolateral membranes to vectors. These results suggest a downstream step to virus binding is influenced by chimeric capsid (e.g., endosome escape, nuclear entry, etc.). To identify the cell types targeted by AAV vectors +/− LLnL/Dox, we used a ciliated cell-specific immunomarker (β-tubulin IV antibody) and determined colocalization of immunofluorescence with that of GFP by optical XZ confocal microscopy. These analyses revealed that AAV-6, HAE-1, and HAE-2 vectors only transduced columnar, lumen-facing epithelial cells and that both ciliated and nonciliated cells were transduced (Figure 3d). Although the numbers of cells transduced were variable dependent on the AAV vector used the cellular tropism was not obviously different for variants HAE-1 or HAE-2 versus AAV-6 with and without reagent co-incubations.

Figure 3.

Figure 3

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Effects of pharmacological modulation on AAV transduction of HAE. HAE cultures were inoculated apically with AAV variants expressing GFP (MOI = 10,000), in the presence or absence of the proteasome inhibitor, LLnL (40 µmol/l), and/or the anthracycline, Dox (5 µmol/l), as outlined in Materials and Methods. (a) At 2 weeks postinoculation, en face epifluorescence images of GFP expression were obtained, with representative images. (b) The relative abundance of GFP-positive cells was quantitated and compared. (c) After inoculation of HAE-2 in the absence and presence of LLnL and Dox, TER was measured. (d) Optical x-z confocal fluorescent images showed both ciliated and nonciliated columnar epithelial cells were transduced by AAV. Cilia were immunolabeled red as described in Materials and Methods. *P < 0.05. AAV, adeno-associated virus; Dox, doxorubicin; GFP, green fluorescent protein; LLnL, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; TER, transepithelial resistance.

Packaging of CFTR into AAV and determination of expression efficiencies

To test whether chimeric capsids HAE-1 or HAE-2 were capable of delivering CFTR to sufficient cells to provide correction of the CF bioelectric Cl− defect we engineered our novel variants to express CFTR. However, the open reading frame size of full-length CFTR (4.2 kb) poses a significant challenge for producing AAV-CFTR vectors as additions of regulatory elements including promoter/enhancer and poly-adenylation (poly-A) sequences would exceed the normal packaging capacity of AAV (~5 kb). We have attempted to circumvent this problem by testing three different CFTR constructs to enable efficient packaging of AAV-CFTR. These constructs are shown schematically in Figure 4a. AAV-CFTR-1 contains a full-length cytomegalovirus promoter (CMV), full-length CFTR, and a poly-A sequence. With these elements the genome size (5.7 kb) exceeds the typical 5 kb packaging capacity of AAV. AAV-CFTR-2 contains full-length CFTR and a poly-A sequence with no exogenous promoter producing a 5.1-kb genome with CFTR expression driven by the AAV terminal repeat sequence. AAV-CFTR-3 contains a shortened CMV promoter, an R-domain-deleted CFTR and shortened poly-A tail, with a total genomic size of 4.9 kb. To verify whether these constructs could be packaged by AAV vectors, we generated viruses in 293T cells using AAV-6 capsids and quantitated viral particles by dot-blot. Viral DNA isolated from equal numbers of viral particles (genome copy number tittered by dot-blot) was analyzed by Southern blot to determine the genomic sizes and genome integrity. Compared to a 5 kb plasmid marker (Figure 4b, lane 1), AAV-CFTR-1 produced a smear with no discrete band at 5.7 kb (Figure 4b, lane 2), suggesting either inefficient packaging or truncated fragments were incorporated by the capsid. AAV-CFTR-2 and AAV-CFTR-3, however, each displayed one discrete band at their respective predicted sizes (Figure 4b, lanes 3 and 4) indicating efficient packaging of these smaller constructs.

Figure 4.

Figure 4

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Analysis of CFTR expressing constructs. (a) The three CFTR expressing constructs that were tested. Construct #1 (AAV-CFTR-1) contains a CMV promoter, a full-length CFTR open reading frame, and a poly-adenylation signal. Construct #2 (AAV-CFTR-2) contains no exogenous promoter sequence with CFTR expression driven by AAV ITR. Construct #3 (AAV-CFTR-3) contains a truncated minimal CMV promoter, an R-domain-deleted CFTR, and a truncated poly-A signal. The length of each construct is indicated. TR, terminal repeats; CMV, CMV promoter; CFTR, cystic fibrosis transmembrane conductance regulator; PA, poly-adenylation signal sequence; mCMV, minimal CMV promoter; CFTRΔR, R-domain-deleted CFTR; mPA, minimal poly-A sequence. (b) Packaging efficiency is dependent on vector size. Vector DNA isolated directly from AAV virions was run on an alkaline agarose gel, followed by Southern-blot analysis probed with CFTR-specific fragment. The AAV-CFTR vector fragment was a control plasmid of known size (5.0 kb) (lane 1), AAV-CFTR-1 (lane 2), AAV-CFTR-2 (lane 3), and AAV-CFTR-3 (lane 4). (c). Western-blot analysis of CFTR transgene expression in 293T cells transfected with AAV-CFTR plasmids. 293T cells were transfected with constructs #1 and #2 at 3 µg/10-cm plate, and cell lysates were prepared at 48 hours postinoculation, and subjected to western-blot analysis for CFTR expression. Calu-3 cells were used as positive control (lane 1). Lane 2 was mock transduced. Lanes 3 and 4 were from AAV-CFTR-1 and AAV-CFTR-2, respectively. Arrow indicates the fully glycosylated mature form of CFTR. (d). Western-blot analyses CFTR transgene expression in HeLa cell 48 hours postinoculation. HeLa cells were infected with different AAV serotypes packaged with AAV-CFTR-3 at MOI of 10,000 and co-infected with dl309 (MOI of 5). Positive control (lane 1), mock infection control (lane 2), transfection of plasmid AAV-CFTR-3 (lane 3), lanes 4–7 are infected from AAV-1, AAV-6, HAE-1, HAE-2. (e) CF HAE were inoculated with AAV-CFTR-3 vectors packaged with different capsids (MOI = 10,000), super-infected with wild-type adenovirus (MOI = 5) following caprate treatment 24 hours after infection, and at 1 week after AAV inoculations, analyzed for CFTR expression by western blot. Lane 1, mock; 2, AAV-1; 3, AAV-6; 4, HAE-1; 5, HAE-2, respectively. AAV, adeno-associated virus; CFTR, cystic fibrosis transmembrane conductance regulator; CMV, cytomegalovirus; MOI, multiplicity of infection.

To determine the relative transcriptional activities of the different constructs in directing CFTR expression, 293T/HeLa cells were transfected with these three plasmid constructs, and CFTR present in cell lysates analyzed by western blot. Using human Calu-3 cell lysate as a positive control for human fully glycosylated CFTR (Figures 4c,d, lane 1), transfection of 293T cells with the genomes of AAV-CFTR-1 (Figure 4c, lane 3) and AAV-CFTR-3 (Figure 4d, lane 3) resulted in robust CFTR expression suggesting the shortened CMV/poly-A sequences achieve a high level of CFTR expression. On the contrary, no CFTR expression was observed from cells transfected with AAV-CFTR-2 genomes (Figure 4c, lane 4), indicating the atypical AAV terminal repeat promoter in AAV-CFTR-2 has a much lower transcriptional activity than CFTR-1 or CFTR-3. In summary, AAV-CFTR-1 was found to be too large to be efficiently packaged in AAV, and AAV-CFTR-2 did not express significant amounts of CFTR. Therefore, only AAV-CFTR-3 was used in subsequent experiments.

Expression of CFTRΔR in human CF ciliated airway epithelial cells

To test the efficiency of AAV-mediated CFTR expression in cells, AAV-CFTR-3 was packaged by capsids of AAV-6, HAE-1 and HAE-2 variants, and inoculated on to HeLa cells and HAE derived from CF patients (CF HAE), with co-inoculation with wild-type AdV (MOI = 5). CFTR expression in HeLa and CF HAE was analyzed by western blot at 2 and 5 days p.i., respectively. For HeLa cells (Figure 4d), AAV6-CFTR-3 expressed the largest amount of CFTR protein (Figure 4d, lane 5) when compared to that expressed by chimeric HAE-1 (Figure 4d, lane 6) and HAE-2 (Figure 4d, lane 7). However, for CF HAE cells (Figure 4e), chimeric HAE-1-CFTR-3 (Figure 4e, lane 4) and HAE-2-CFTR-3 (Figure 4e, lane 5) showed a greater level of CFTR expression compared to AAV1-CFTR3 (Figure 4e, lane 2) and AAV6-CFTR3 (Figure 4e, lane 3). These data suggest that the novel AAV variants are adapted for transduction and replication in HAE cells unlike the parental AAV that transduce HeLa cells more efficiently.

Novel AAV variants deliver CFTRΔR to CF HAE and partially correct the CF bioelectric chloride ion transport defect

To test whether AAV capsid variants were capable of restoring sufficient CFTR function to CF HAE to generate cyclic adenosine monophosphate–activated chloride ion transport, CF HAE cells were inoculated with AAV-CFTR-3 packaged by AAV-6, variants HAE-1, HAE-2 capsids. Vectors were inoculated onto CF HAE cells at an MOI of 4 × 105 in the presence of LLnL and Dox as described earlier. Non-CF HAE and CF HAE cells inoculated with AAV6-GFP or mock-inoculated with vector vehicle alone were included as controls and assays performed 2 weeks p.i.

First, we determined by quantitative reverse transcription–PCR the relative expression levels of CFTR mRNA in CF HAE cells that was produced by AAV vectors compared to endogenous CFTR mRNA levels. Chimeric HAE-1 and HAE-2, but not AAV6 CFTR or AAV6-GFP, produced significantly increased levels of CFTR mRNA over endogenous CFTR mRNA levels (Figure 5a). Variant HAE-1 and HAE-2 likely expressed CFTR mRNA at greater levels than AAV6-CFTR because more cells were transduced by these chimeric capsids confirming our earlier transduction efficiency data (Figure 2).

Figure 5.

Figure 5

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Novel AAV variants mediate CFTR gene delivery to CF HAE and partially restore forskolin-sensitive chloride ion transport. CF HAE were inoculated with AAV-CFTR-3 vectors packaged with the capsids indicated at MOI of 4 × 105 vector genomes/cell in the presence of LLnL and Dox (see Materials and Methods for details). (a) CFTR mRNA levels in CF HAE 14 days after inoculation with AAVGFP or AAV-CFTR and relative to mRNA levels in mock-transduced CF HAE (n = 2). (b) Representative traces of transepithelial PD measurements (PD spikes in response to a current pulse for resistance calculation were removed) performed in Ussing chambers at 14 days postinoculation showing responses to sequentially added amiloride (Amil), forskolin (Fskl), and CFTR172. A representative PD response by a non-CF HAE is shown for comparison. A small Fskl response was seen in CF HAE inoculated with AAVGFP or mock consistent with the residual activity of CFTR 621 + 1G (T allele). (c) Summary data for Fskl-activated changes in calculated short-circuit current (ΔIsc) in CF HAE at 14 days postinoculation (mean (SD)). Fskl responses for non-CF HAE were included for comparison. *P < 0.01. AAV, adeno-associated virus; CFTR, cystic fibrosis transmembrane conductance regulator; LLnL, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; PD, potential difference.

Next, we performed Ussing chamber studies to determine the presence of functional CFTR ion channel activity by maximally activating CFTR with forskolin (a cell permeable activator of adenylate cyclase to increase intracellular cyclic adenosine monophosphate). Significantly increased forskolin-inducible short-circuit currents (Isc) were observed in CF HAE cells inoculated with chimeric capsids HAE-1 (2.95 ± 0.82 µA/cm2, n = 5, P < 0.01) and HAE-2 (3.92 ± 2.0 µA/cm2, n = 5, P < 0.01) when compared to those in CF HAE cells inoculated with AAV6-CFTR, AAV6-GFP, or mock-inoculated controls. Representative traces and calculated mean changes in forskolin-mediated short-circuit currents are shown in Figure 5b,c. No significant differences in forskolin-induced Isc between AAV6-CFTR and AAV6-GFP or mock inoculations were observed. By comparison to forskolin-mediated CFTR activation in non-CF HAE cells performed in parallel, we conclude that chimeric HAE-1 and HAE-2 expressed sufficient CFTR in enough cells to restore 25 and 31% of the normal CFTR response to forskolin, respectively. These data demonstrate that the novel AAV variants partially restore the CFTR bioelectric defect that is a characteristic of the CF airway epithelia phenotype. As the most likely reason that these novel variants restore 20–30% CFTR-mediated ion transport to that of normal levels was the relatively low numbers of cells (~6%) transduced by chimeric HAE-1 and HAE-2 capsids, it is not clear if continued further evolution of these vectors for increased transduction efficiency in HAE will likely improve the ability to correct the bioelectric defect in CF HAE cells. Although further development and evolution of AAV-based vectors holds significant promise for the potential use of AAV-derived vectors as a therapeutic strategy to reverse CF lung disease, success may require additional steps to remove physical barriers (mucus layer, etc.) in order to reach full potential.

Discussion

AAV vectors remain promising candidates for gene therapy applications, due to low pathogenicity, broad tissue and cell tropism, lack of cellular immune responses, and the ability to mediate long-term gene expression. However, to date, some cell types and tissues are refractory to the commonly available serotypes of AAV vectors.

As CF lung disease is due to the genetic absence of functional CFTR in the airway epithelium, this tissue is a major target for AAV-based vector gene delivery strategies. Until recently, attempts using commonly available AAV vectors for gene delivery to human ciliated airway epithelium in vitro and in vivo have resulted in little or no evidence that efficacious CFTR delivery was obtained, a result attributed to the low transduction efficiency of the commonly available AAV vector serotypes.

One strategy to increase the transduction efficiency of AAV vectors is to identify new AAV variants with improved transduction efficiencies for ciliated airway epithelium. In addition to naturally evolved AAV isolates, several strategies to develop hybrid AAV serotype vectors have been formulated in recent years. The generation of mosaic or chimeric vectors through rational design,21 transcapsidation,22 and marker-rescue/domain-swapping approaches,23 respectively, have advanced progress in this regard. More recently, combinatorial strategies for engineering AAV vectors using error-prone PCR, DNA shuffling, and other molecular cloning techniques have been established.10,11,12,24,25,26 The latter library-based approaches can serve as powerful tools in the generation of novel AAV capsids with altered retargeted tropism, immunogenicity, and improved efficacy as gene delivery vectors.

In this study, we subjected a chimeric AAV capsid library, generated by DNA-shuffling serotypes 1–9 (excluding serotype 7), to five cycles of screening after inoculation of the apical surface of an in vitro model of HAE. Using these methods, we identified two novel chimeric AAV isolates, HAE-1 and HAE-2, with improved transduction efficiency, from which the majority of their capsid sequences were derived from serotypes AAV-1 and AAV-6 (Figure 1b). Clear differences in transduction efficiency are observed in HAE between AAV-1, AAV-5, AAV-6, and AAV-9 when compared to the chimeric HAE viruses with a rank order of efficiency as follows: AAV-1/AAV-5/AAV-9 < AAV-6 < HAE-1 < HAE-2. The improved transduction in HAE observed for AAV-6 compared to AAV-1 and that of chimeric HAE-1 compared to AAV-6 suggests that some of the six amino acids that differ between AAV-1 and AAV-6 confer an advantage to AAV-6. We have initiated efforts to map critical residues using both AAV-1 and AAV-6 single amino acid exchanged these residues between capsid backbone as previously described in our lab. For HAE-2 which showed the best overall transduction efficiency in HAE, all the surface amino acids are derived from AAV-6 with an interior residue at 418 also from AAV-6. These observations suggest that the different AAV-1 and AAV-6 variable amino acids combine to confer improved infection of the chimeric viruses for HAE. Such molecular events could improve cell binding, internalization, and/or trafficking of the novel variants. Mutation of these variable amino acids in the parental viruses and the characterization of the mutant viruses by biochemical and cell-based assays, such as ability of the viruses to bind to cells, is expected to provide further insight into the mechanism(s) underlying the increased airway epithelial cell transduction of these AAV variants. As a first step toward this analysis, we performed binding assays with our parental and variant AAVs on HAE that showed no significant differences in the ability of these vectors to bind to the apical surfaces of HAE (see Supplementary Figure S1). Given these data, we predict that increased transduction efficiencies observed with our novel variants are likely due to alteration of events that occur post-attachment of AAV to the airway epithelial cells.

The initiating cause of CF lung disease is the dysfunction of mutant CFTR in epithelial cells that line the human airways. Although it is not established which cell types or airway regions (i.e., airway surface epithelium versus submucosal gland epithelium) will require correction of CFTR function, the accessibility of the surface airway epithelium to intralumenally delivered vectors and the presence of CFTR in epithelial cells that line the surface of the conducting airways suggest that targeting CFTR delivery to the airway surface epithelium will provide therapeutic benefit to CF patients. Reintroduction of CFTR to airway epithelial cells using gene delivery vectors remains a rational strategy toward developing a treatment for this disease. However, preclinical studies and clinical trials in CF patients using viral and nonviral vector systems have demonstrated negligible efficacy of CFTR delivery. Previously, it was shown that delivery of CFTR to as few as 6–10% of human CF airway epithelial cells in vitro could restore normal levels of chloride ion transport to levels of that measured in non-CF cells. However, these experiments performed in homogenous epithelial cell types that over expressed CFTR using a retroviral-based vector were polarized but undifferentiated thus did not represent the morphological characteristics of the human ciliated airway epithelium in vivo.27 More recently, using a novel, recombinant virus vector that targets ciliated cells it has been shown that restoration of airway surface liquid volume and mucus transport to CF HAE requires CFTR expression in ~25% of surface epithelial cells.28 In particular, this study also demonstrated that if CFTR expression in individual cells exceeded endogenous levels of CFTR expression, then the ability to restore fluid and mucus transport to CF HAE was directly related to the numbers of cells expressing CFTR. In this report, we demonstrate that ~3% of surface epithelial cells are transduced after apical inoculation of CF HAE with our novel AAV variants (Figure 3a,b). Coadministration of LLnL and Dox, further increased this transduction efficiency to ~6–7%, although the augmentation of our novel AAV variants by these reagents were much less than reported previously for AAV-2 and AAV-5.5 We assume that the transduction ability of these novel serotypes in HAE differs from that of AAV-2 and AAV-5 by virtue of altered subcellular trafficking profiles or distinct ubiquitin/proteasome sensitivities. Therefore, our novel AAV variants although greatly improved over the parental AAV serotypes, still fall short of the transduction efficiencies likely required restoring fluid and mucus transport to CF HAE. Further evolution of AAV vectors for increased transduction efficiency in HAE may enable efficacious transduction efficiencies to be obtained.

In addition to identifying novel AAV capsid variants that exhibit improved HAE transduction, in this report we have applied a strategy to overcome the small packaging capacity of AAV. Due to the relatively large size of full-length CFTR, addition of a CMV promoter and a poly-A sequence exceeds the standard AAV packaging capacity (~5 kb) (Figure 4a), resulting in a five to tenfold decrease in viral titer (data not shown) and the packaging of transgene fragments under 5 kb (Figure 4b). As mentioned earlier, several approaches have been reported to overcome the packaging limitations of AAV, including the use of split genome systems,29,30 the construction of CFTR minigenes,13,31,32 the use of minimal promoter elements,33 and the use AAV vectors to deliver targeted trans-splicing signals to repair the endogenous CFTR mRNA.34 In this study, we tested several options that were available to us to generate a useful construct, AAV-CFTR-3, which contained a shortened CMV promoter (173 bp), an R-domain-deleted CFTR and 49 bp poly-A tail, flanked by TR2 with a total size of 4.9 kb. We determined this construct could be packaged by AAV with no impact on vector production (data not shown) or infectivity (Figure 4d,e).

Several groups are developing AAV vectors for CF gene transfer purposes and contradictory data exist as to which AAV serotype is most efficient at transducing HAE. Previously, it has been shown that AAV-5 expressing a CFTR minigene was able to fully correct the chloride ion transport defect in CF epithelial cells.13 In our hands, we would predict based on comparison of AAV-5 and AAV-6 transduction efficiency in HAE that AAV-5 would not target sufficient numbers of epithelial cells in our model of human CF ciliated airway epithelium to display any significant correction of the CF bioelectric defect. More recently, a novel AAV serotype generated by directed evolution by methods similar to those described here was shown to fully correct the CF bioelectric defect to non-CF levels.12 Another previous report indicated that AAV-2 vectors expressing a similar CFTR construct and coadministered with pharmacological reagents as we used here dramatically enhanced CFTR gene delivery and the ability to correct CFTR-mediated ion transport.35 In our hands, using our model of human ciliated airway epithelium, AAV-1 and AAV-6 were consistently more efficient (>100 times) than AAV-25 or AAV-5 after apical inoculation. The discrepancies between these studies with AAV serotypes from different laboratories may be due to several variables such as the vector purification methods, the MOI used and/or the differentiation state of the HAE models used for screening/transduction studies. We note that one difference between our studies and those of others is that AAV library selection and transduction studies were performed on human airway cultures 2 weeks after cell seeding, whereas we routinely used cultures 4–6 weeks after seeding.12 In our hands, this extended cell culture time is required for full differentiation of airway epithelial cells into ciliated/nonciliated cells. After only 2 weeks of culture, our model of epithelial cells represents a polarized, but not fully differentiated, airway epithelium (see Supplementary Figure S2). To test whether transduction efficiency of AAV would be affected by the time of culture of our airway model, we inoculated the apical surfaces of cultures derived from the same patient grown for 2 or 4 weeks with equal amounts of AAV6-GFP and monitored GFP-positive cells over time. Transduction efficiency in 2-week old cultures was significantly better than for 4-week old cultures (see Supplementary Figure S3), suggesting an increased ability of AAV vectors to transduce younger, less differentiated airway epithelial cells. Although differences in culture techniques between laboratories may also account for the differences in transduction efficiencies of AAV variants, further testing of genetically identical AAV vectors on fully differentiated cells derived from single patient sources will be required to resolve some of these differences. It should be noted that full correction of in vitro HAE may not be a complete predictor of human lung but is the gold standard at the moment.

In conclusion, our data presented here provides evidence that novel AAV variants generated through directed evolution on a specific target tissue, i.e., human ciliated airway epithelium are capable of transducing the target tissue more efficiently than the parental serotypes included in the library. When these novel AAV variants expressing CFTR are coadministered with proteasome inhibitors and anthracycline compounds, they are capable of partially correcting the bioelectric defect of CF HAE. It remains to be determined whether this partial level of correction would be beneficial to CF airway disease. Thus, we suggest that directed in vitro evolution of AAV through screening a DNA-shuffled virus library on relevant target cells is useful in generating novel AAV variants with improved tropism. The two novel AAV variants HAE-1 and HAE-2 identified in this study warrant further studies in vitro and in vivo.

Materials and Methods

Cell lines and viruses. 293T cells and HeLa cells were maintained in Dulbecco's modified Eagle's medium (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum and penicillin–streptomycin (100 U/ml) with 5% CO2 at 37 °C. Wild-type AdV dl309 has been described previously.14

AAV production and titration. Recombinant AAV vectors containing GFP, luciferase, or CFTR, were produced using the triple plasmid transfection protocol as described and virus titers were determined by dot-blot analysis.36

Generation of ciliated HAE in vitro. Fresh human tracheobronchial epithelial cells were isolated from excess surgical specimens resected at lung transplantation by the University of North Carolina Cystic Fibrosis Center Tissue Culture Core under University of North Carolina institutional review board–approved protocols. Briefly, primary cells derived from single patient sources were expanded on plastic to generate passage 1 cells, which were plated at a density of 250,000 cells/cm2 on permeable membrane support (Transwell-Col, 12 or 24-mm diameter, Corning; Millicells, 12-mm diameter, Millipore, Corning, NY), and maintained in a specialty media.37 Differentiation was induced under air–liquid interface; and the cultures were considered “matured” (in ~4–8 weeks) when significantly ciliated (>50% ciliated cells). Only fully differentiated ciliated cultures were used in all experiments.

HAE-1 and HAE-2 model building. To enable three-dimensional visualization of the amino acid differences between the HAE chimeric viruses and the parental AAV-1 and AAV-6, a homologous VP3 structural model (residues 217–736, VP1 numbering) was first generated for AAV-6 based on the crystal structure of AAV-1 VP3 monomer (L. Govindasamy and M. Agbandje-McKenna, unpublished results) as a template using the SWISS-MODEL model building program.38 The first 217 N-terminal residues of AAV-1 are disordered in the crystal structure and thus this region could not be modeled in AAV-6. The three-dimensional models for HAE-1, and HAE-2 were then created by “cutting and pasting” the structural regions from the parental virus followed by regularization of the model geometry in the program Coot.39 Due to the structural localization of the amino acids that differ between AAV-1 and AAV-6 in a VP3 monomer around the icosahedral threefold axes, a trimer was generated for each chimeric HAE virus model by matrix multiplication for further visualization of the amino acid contributions from each parental serotype. The position of these amino acids (five in the ordered VP3 region) were then colored (black for AAV-1, yellow for AAV-6) on a surface contour representation of the chimeric virus models using the program PyMol.40

Construction of the chimeric plasmid library and selection on HAE. The construction of the chimeric AAV library by capsid DNA shuffling of eight natural AAV serotypes, i.e., serotypes 1–9 except 7, has been previously described.10 To select for AAV variants with enhanced tropism toward airway epithelium, the AAV library was screened with well-ciliated HAE cultures, illustrated in Figure 1a. Briefly, 100 µl of the AAV library (MOI of 1,000) were inoculated onto the apical surface of four 24-mm HAE cultures, incubated for 4 hours at 37 °C before removal of the library and washing of the apical surface of HAE cultures three times with phosphate buffered saline. At 24 hours after AAV inoculation, the HAE cultures were infected with wild-type AdV dl309 (MOI of 20) on treatment with sodium caprate.17 AAV virus particles were harvested at 4 days after AdV infection by lysing the cells with repeated freezing and thawing, followed by heat inactivation of wild-type Adv. The harvested AAV pool was then used for the next round of screening as carried out in the first round, and so on. At the end of each round of screening, viral genomic DNA was purified from 50 µl aliquots of crude cell lysates using the DNeasy kit (Qiagen, Germantown, MD) for the determination of viral genome titer by dot-blot hybridization. After five successive rounds of screening, individual AAV clones were picked and subjected for DNA sequencing analysis. Representative AAV variants were cloned into the pXR2 backbone and used as AAV helper plasmids to produce recombinant AAV viruses.

Apical transduction of HAE with recombinant AAV vectors. CF and non-CF HAE cultures were inoculated on the apical surface with 100 µl of AAV vectors for 2 hours. The MOI varied from 1 × 103 to 4 × 105 vector genomes/cell, depending on virus concentration and purposes of the experiments. Equal MOI was used within each experiment. The proteasome-modulating agent, LLnL (40 µmol/l), or the chemotherapeutic anthracycline, Dox (5 µmol/l),19,34 were applied when noted. Photomicrographs of transduced GFP-positive cells were acquired using a Leica Leitz DMIRB inverted fluorescence microscope equipped with a cooled-color charge-coupled device digital camera (Retiga 1300; Q-Imaging, Burnaby, British Columbia, Canada). Images of five random fields for each HAE culture were captured and quantified using ImageJ software (National Institutes of Health, Bethesda, MD). To identify AAV transduced cell types, ciliated cells were immunolabeled with a β-tubulin IV antibody (Sigma-Aldrich, St Louis, MO), followed by AlexaFluor594 conjugated goat anti-mouse antibody (Invitrogen, Carlsbad, CA) as described previously.41 Optical x-z fluorescent confocal images were obtained using a Zeiss 510 Meta Laser Scanning Confocal Microscope (Carl Zeiss Micro Imaging, Thornwood, NY). In HAE inoculated with luciferase expressing AAV vectors, luciferase activities were measured using a luminometer (Victor2 1420 Multilabel Counter; Perkin Elmer Life Sciences, Canton, MA).

Western-blot analyses of CFTR expression. HAE/293T/HeLa cells were lysed in M-PER buffer (Pierce, Waltham, MA), and equal amounts of total protein were separated with a NuPAGE 3–8% Tris–Acetate Gel (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride membranes. The membranes were then probed with a CFTR-specific antibody (#596, a gift of Dr Jack Riordan) followed by HRP conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA); and CFTR visualized with SuperSignal West Dura Substrate (Pierce, Waltham, MA).

Ion transport measurements. HAE were mounted in Ussing chambers for measurement of transepithelial resistance and transepithelial potential difference. The cultures were studied under open circuit conditions using a voltage clamp (Physiologic Instruments, San Diego, CA). The electrical potential difference across the tissue was continually recorded and a constant current pulse (2–10 µA) applied across the tissue at 1-minute intervals to calculate tissue resistance. From these measurements, the equivalent short-circuit current (Isc) was calculated. All other details of Ussing chamber techniques have been previously published.42 HAE were bathed in Krebs bicarbonate ringer solution on the serosal side, and high K+ low Cl− (HKLC) buffer on the lumenal side. Both solutions were gassed with 95% O2, 5% CO2. Drugs [amiloride (10−4 mol/l), forskolin (10−5 mol/l), and CFTR172 (10–5 mol/l)] were added from concentrated stock solutions to either lumenal and/or serosal surfaces (all obtained from Sigma-Aldrich). CFTR functional activities (forskolin ΔIsc) were measured in AAV inoculated CF HAE at 14 days p.i.

Statistics. All data are expressed as means ± SEM. Unpaired Student's t-test was used to assess the difference between groups. P value of <0.05 was considered significant.

SUPPLEMENTARY MATERIALFigure S1. The binding of AAV to the apical surface of HAE is not significantly different among HAE-1, HAE-2, AAV-1, and AAV-6. The experiments were carried out by incubating serotypes HAE-1, HAE-2, AAV1, and AAV6 at doses of 5× 1010 and 1× 109 vg per HAE for 1 hour at 4 °C, followed by three rinses with PBS. Viral DNA from cell-associated AAV was extracted and analyzed by dot-blot assay.Figure S2. Histological analysis of HAE at 2 and 4 weeks post ALI. HAE were fixed with 4% PFA, paraffin-embedded, thin-sectioned, and stained with hematoxylin and eosin (H&E). As opposed to the pseudostratified ciliated cell abundant morphology of 4-week-old cultures, 2-week-old HAE displayed thin, non-differentiated, non-ciliated epithelial cell morphology.Figure S3. AAV transduction was more efficient in nondifferentiated than fully differentiated HAE. HAE grown for 2 or 4 weeks post ALI were inoculated with equal titers (MOI=105 vg/cell) of AAV-6-GFP. Representative en face fluorescence photomicrographs show abundant GFP-positive cells in 2 week old but not 4 week old HAE cells at 7 days pi.

Supplementary Material

Figure S1.

The binding of AAV to the apical surface of HAE is not significantly different among HAE-1, HAE-2, AAV-1, and AAV-6. The experiments were carried out by incubating serotypes HAE-1, HAE-2, AAV1, and AAV6 at doses of 5× 1010 and 1× 109 vg per HAE for 1 hour at 4 °C, followed by three rinses with PBS. Viral DNA from cell-associated AAV was extracted and analyzed by dot-blot assay.

Click here for supplemental data (3.8MB, tiff)
Figure S2.

Histological analysis of HAE at 2 and 4 weeks post ALI. HAE were fixed with 4% PFA, paraffin-embedded, thin-sectioned, and stained with hematoxylin and eosin (H&E). As opposed to the pseudostratified ciliated cell abundant morphology of 4-week-old cultures, 2-week-old HAE displayed thin, non-differentiated, non-ciliated epithelial cell morphology.

Click here for supplemental data (1.8MB, tiff)
Figure S3.

AAV transduction was more efficient in nondifferentiated than fully differentiated HAE. HAE grown for 2 or 4 weeks post ALI were inoculated with equal titers (MOI=105 vg/cell) of AAV-6-GFP. Representative en face fluorescence photomicrographs show abundant GFP-positive cells in 2 week old but not 4 week old HAE cells at 7 days pi.

Click here for supplemental data (1.4MB, tiff)

Acknowledgments

We thank the Directors and Teams of the UNC Cystic Fibrosis Center Tissue Culture Core and Dr Barbara Grubb of the UNC CF Center Correction Core. We also thank Dr Michael J. Welsh at University of Iowa for providing plasmid pTR5-173-CFTR-R6. The National Institutes of Health (NIH) and Cystic Fibrosis Foundation (CFF) supported these studies: NIH R01 –AI 072176; NIH R01 HL77844-1; NIH P01 HL051818-15; NIH Molecular Therapy Core Center P30 DK065988-01.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

The binding of AAV to the apical surface of HAE is not significantly different among HAE-1, HAE-2, AAV-1, and AAV-6. The experiments were carried out by incubating serotypes HAE-1, HAE-2, AAV1, and AAV6 at doses of 5× 1010 and 1× 109 vg per HAE for 1 hour at 4 °C, followed by three rinses with PBS. Viral DNA from cell-associated AAV was extracted and analyzed by dot-blot assay.

Click here for supplemental data (3.8MB, tiff)
Figure S2.

Histological analysis of HAE at 2 and 4 weeks post ALI. HAE were fixed with 4% PFA, paraffin-embedded, thin-sectioned, and stained with hematoxylin and eosin (H&E). As opposed to the pseudostratified ciliated cell abundant morphology of 4-week-old cultures, 2-week-old HAE displayed thin, non-differentiated, non-ciliated epithelial cell morphology.

Click here for supplemental data (1.8MB, tiff)
Figure S3.

AAV transduction was more efficient in nondifferentiated than fully differentiated HAE. HAE grown for 2 or 4 weeks post ALI were inoculated with equal titers (MOI=105 vg/cell) of AAV-6-GFP. Representative en face fluorescence photomicrographs show abundant GFP-positive cells in 2 week old but not 4 week old HAE cells at 7 days pi.

Click here for supplemental data (1.4MB, tiff)

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