Generation of novel AAV variants by directed evolution for improved CFTR delivery to human ciliated airway epithelium

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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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 (ΔI_sc) 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.