FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080778399> ?p ?o ?g. }

• W2080778399 endingPage "2077" @default.
• W2080778399 startingPage "2067" @default.
• W2080778399 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. 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. 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.1Koch C Høiby N Pathogenesis of cystic fibrosis.Lancet. 1993; 341: 1065-1069Abstract PubMed Scopus (364) Google Scholar 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.2Moss RB Milla C Colombo J Accurso F Zeitlin PL Clancy JP et al.Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial.Hum Gene Ther. 2007; 18: 726-732Crossref PubMed Scopus (214) Google Scholar 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.3Flotte TR Recent developments in recombinant AAV-mediated gene therapy for lung diseases.Curr Gene Ther. 2005; 5: 361-366Crossref PubMed Scopus (49) Google Scholar,4Duan D Yue Y Yan Z Engelhardt JF A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation.Nat Med. 2000; 6: 595-598Crossref PubMed Scopus (161) Google Scholar 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.5Yan Z Lei-Butters DC Liu X Zhang Y Zhang L Luo M et al.Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia.J Biol Chem. 2006; 281: 29684-29692Crossref PubMed Scopus (38) Google Scholar,6Virella-Lowell I Zusman B Foust K Loiler S Conlon T Song S et al.Enhancing rAAV vector expression in the lung.J Gene Med. 2005; 7: 842-850Crossref PubMed Scopus (56) Google Scholar,7Zabner J Seiler M Walters R Kotin RM Fulgeras W Davidson BL et al.Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer.J Virol. 2000; 74: 3852-3858Crossref PubMed Scopus (276) Google Scholar,8Halbert CL Allen JM Miller AD Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors.J Virol. 2001; 75: 6615-6624Crossref PubMed Scopus (246) Google Scholar,9Limberis MP Wilson JM Adeno-associated virus serotype 9 vectors transduce murine alveolar and nasal epithelia and can be readministered.Proc Natl Acad Sci USA. 2006; 103: 12993-12998Crossref PubMed Scopus (128) Google Scholar 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,5Yan Z Lei-Butters DC Liu X Zhang Y Zhang L Luo M et al.Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia.J Biol Chem. 2006; 281: 29684-29692Crossref PubMed Scopus (38) Google Scholar although AAV-1 transduced murine tracheal airway epithelia in vivo with an efficiency equal to that of AAV-5.6Virella-Lowell I Zusman B Foust K Loiler S Conlon T Song S et al.Enhancing rAAV vector expression in the lung.J Gene Med. 2005; 7: 842-850Crossref PubMed Scopus (56) Google Scholar 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.7Zabner J Seiler M Walters R Kotin RM Fulgeras W Davidson BL et al.Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer.J Virol. 2000; 74: 3852-3858Crossref PubMed Scopus (276) Google Scholar 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.8Halbert CL Allen JM Miller AD Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors.J Virol. 2001; 75: 6615-6624Crossref PubMed Scopus (246) Google Scholar 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.9Limberis MP Wilson JM Adeno-associated virus serotype 9 vectors transduce murine alveolar and nasal epithelia and can be readministered.Proc Natl Acad Sci USA. 2006; 103: 12993-12998Crossref PubMed Scopus (128) Google Scholar DNA shuffling combined with directed evolution has been previously used to generate novel AAV vectors that display improved transduction efficiency in specific cell types.10Li W Asokan A Wu Z Van Dyke T DiPrimio N Johnson JS et al.Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.Mol Ther. 2008; 16: 1252-1260Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar,11Grimm D Lee JS Wang L Desai T Akache B Storm TA et al.In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.J Virol. 2008; 82: 5887-5911Crossref PubMed Scopus (443) Google Scholar Recently, it has been demonstrated that directed evolution of the AAV capsid can select viral variants with enhanced infection of HAE in vitro.12Excoffon KJ Koerber JT Dickey DD Murtha M Keshavjee S Kaspar BK et al.Directed evolution of adeno-associated virus to an infectious respiratory virus.Proc Natl Acad Sci USA. 2009; 106: 3865-3870Crossref PubMed Scopus (119) Google Scholar 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).10Li W Asokan A Wu Z Van Dyke T DiPrimio N Johnson JS et al.Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.Mol Ther. 2008; 16: 1252-1260Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar 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)13Ostedgaard LS Rokhlina T Karp PH Lashmit P Afione S Schmidt M et al.A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia.Proc Natl Acad Sci USA. 2005; 102: 2952-2957Crossref PubMed Scopus (75) Google Scholar 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. 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 described10Li W Asokan A Wu Z Van Dyke T DiPrimio N Johnson JS et al.Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.Mol Ther. 2008; 16: 1252-1260Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar in HAE using co-infection with wild-type adenovirus (AdV, dl309) to amplify infectious AAV variants (Figure 1a)14Jones N Shenk T Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.Cell. 1979; 17: 683-689Abstract Full Text PDF PubMed Scopus (476) Google Scholar Because HAE are relatively resistant to AdV infection after lumenal inoculation of HAE unless epithelial tight junctions are disrupted to reveal basolateral AdV receptors,15Pickles RJ McCarty D Matsui H Hart PJ Randell SH Boucher RC Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer.J Virol. 1998; 72: 6014-6023Crossref PubMed Google Scholar,16Walters RW Grunst T Bergelson JM Finberg RW Welsh MJ Zabner J Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.J Biol Chem. 1999; 274: 10219-10226Crossref PubMed Scopus (312) Google Scholar the apical surfaces of HAE were first exposed to sodium caprate to transiently open tight junctions immediately before AdV inoculation.17Coyne CB Kelly MM Boucher RC Johnson LG Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate.Am J Respir Cell Mol Biol. 2000; 23: 602-609Crossref PubMed Scopus (98) Google Scholar 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). 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. 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). 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.7Zabner J Seiler M Walters R Kotin RM Fulgeras W Davidson BL et al.Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer.J Virol. 2000; 74: 3852-3858Crossref PubMed Scopus (276) Google Scholar 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).18Duan D Yue Y Yan Z Yang J Engelhardt JF Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus.J Clin Invest. 2000; 105: 1573-1587Crossref PubMed Scopus (311) Google Scholar,19Yan Z Zak R Zhang Y Ding W Godwin S Munson K et al.Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.J Virol. 2004; 78: 2863-2874Crossref PubMed Scopus (101) Google Scholar 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.19Yan Z Zak R Zhang Y Ding W Godwin S Munson K et al.Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.J Virol. 2004; 78: 2863-2874Crossref PubMed Scopus (101) Google Scholar 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,20Bals R Xiao W Sang N Weiner DJ Meegalla RL Wilson JM Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.J Virol. 1999; 73: 6085-6088PubMed Google Scholar 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. 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. 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. 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. 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). 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 inocul" @default.
• W2080778399 created "2016-06-24" @default.
• W2080778399 creator A5009651009 @default.
• W2080778399 creator A5020247252 @default.
• W2080778399 creator A5037092076 @default.
• W2080778399 creator A5044065218 @default.
• W2080778399 creator A5050422690 @default.
• W2080778399 creator A5054521114 @default.
• W2080778399 creator A5055991434 @default.
• W2080778399 creator A5059305777 @default.
• W2080778399 creator A5085482867 @default.
• W2080778399 creator A5087559484 @default.
• W2080778399 date "2009-12-01" @default.
• W2080778399 modified "2023-10-02" @default.
• W2080778399 title "Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium" @default.
• W2080778399 cites W1582366477 @default.
• W2080778399 cites W1963623233 @default.
• W2080778399 cites W1970138286 @default.
• W2080778399 cites W1978515356 @default.
• W2080778399 cites W1982346550 @default.
• W2080778399 cites W1999723571 @default.
• W2080778399 cites W2004288362 @default.
• W2080778399 cites W2008036455 @default.
• W2080778399 cites W2012104351 @default.
• W2080778399 cites W2013083986 @default.
• W2080778399 cites W2017558860 @default.
• W2080778399 cites W2026370952 @default.
• W2080778399 cites W2029513032 @default.
• W2080778399 cites W2033689077 @default.
• W2080778399 cites W2042805311 @default.
• W2080778399 cites W2053222398 @default.
• W2080778399 cites W2053706844 @default.
• W2080778399 cites W2060809301 @default.
• W2080778399 cites W2061752094 @default.
• W2080778399 cites W2064736759 @default.
• W2080778399 cites W2082274611 @default.
• W2080778399 cites W2083457187 @default.
• W2080778399 cites W2084711412 @default.
• W2080778399 cites W2087358560 @default.
• W2080778399 cites W2090943591 @default.
• W2080778399 cites W2096753000 @default.
• W2080778399 cites W2102111681 @default.
• W2080778399 cites W2113082692 @default.
• W2080778399 cites W2116951270 @default.
• W2080778399 cites W2125375279 @default.
• W2080778399 cites W2126829401 @default.
• W2080778399 cites W2137130539 @default.
• W2080778399 cites W2143864438 @default.
• W2080778399 cites W2147193337 @default.
• W2080778399 cites W2148223197 @default.
• W2080778399 cites W2151534641 @default.
• W2080778399 cites W2159953474 @default.
• W2080778399 cites W2327242592 @default.
• W2080778399 doi "https://doi.org/10.1038/mt.2009.155" @default.
• W2080778399 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2801879" @default.
• W2080778399 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19603002" @default.
• W2080778399 hasPublicationYear "2009" @default.
• W2080778399 type Work @default.
• W2080778399 sameAs 2080778399 @default.
• W2080778399 citedByCount "79" @default.
• W2080778399 countsByYear W20807783992012 @default.
• W2080778399 countsByYear W20807783992013 @default.
• W2080778399 countsByYear W20807783992014 @default.
• W2080778399 countsByYear W20807783992015 @default.
• W2080778399 countsByYear W20807783992016 @default.
• W2080778399 countsByYear W20807783992018 @default.
• W2080778399 countsByYear W20807783992019 @default.
• W2080778399 countsByYear W20807783992020 @default.
• W2080778399 countsByYear W20807783992021 @default.
• W2080778399 countsByYear W20807783992022 @default.
• W2080778399 countsByYear W20807783992023 @default.
• W2080778399 crossrefType "journal-article" @default.
• W2080778399 hasAuthorship W2080778399A5009651009 @default.
• W2080778399 hasAuthorship W2080778399A5020247252 @default.
• W2080778399 hasAuthorship W2080778399A5037092076 @default.
• W2080778399 hasAuthorship W2080778399A5044065218 @default.
• W2080778399 hasAuthorship W2080778399A5050422690 @default.
• W2080778399 hasAuthorship W2080778399A5054521114 @default.
• W2080778399 hasAuthorship W2080778399A5055991434 @default.
• W2080778399 hasAuthorship W2080778399A5059305777 @default.
• W2080778399 hasAuthorship W2080778399A5085482867 @default.
• W2080778399 hasAuthorship W2080778399A5087559484 @default.
• W2080778399 hasBestOaLocation W20807783991 @default.
• W2080778399 hasConcept C105922876 @default.
• W2080778399 hasConcept C141071460 @default.
• W2080778399 hasConcept C159047783 @default.
• W2080778399 hasConcept C2776938444 @default.
• W2080778399 hasConcept C529295009 @default.
• W2080778399 hasConcept C54355233 @default.
• W2080778399 hasConcept C71924100 @default.
• W2080778399 hasConcept C86803240 @default.
• W2080778399 hasConcept C87568996 @default.
• W2080778399 hasConcept C95444343 @default.
• W2080778399 hasConceptScore W2080778399C105922876 @default.
• W2080778399 hasConceptScore W2080778399C141071460 @default.
• W2080778399 hasConceptScore W2080778399C159047783 @default.
• W2080778399 hasConceptScore W2080778399C2776938444 @default.
• W2080778399 hasConceptScore W2080778399C529295009 @default.

• next