Original Article

Gene Therapy (2017) 24, 290–297; doi:10.1038/gt.2017.19; published online 13 April 2017

Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract

F Kurosaki1,2, R Uchibori1,3, N Mato2, Y Sehara1, Y Saga1,4, M Urabe1, H Mizukami1, Y Sugiyama2 and A Kume5

  1. 1Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Japan
  2. 2Division of Pulmonary Medicine, Department of Medicine, Jichi Medical University, Shimotsuke, Japan
  3. 3Division of Immuno-Gene and Cell Therapy (Takara Bio), Jichi Medical University, Shimotsuke, Japan
  4. 4Department of Obstetrics and Gynecology, Jichi Medical University, Shimotsuke, Japan
  5. 5Support Center for Clinical Investigation, Jichi Medical University, Shimotsuke, Japan

Correspondence: Professor A Kume, Support Center for Clinical Investigation, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke 329-0498, Japan. E-mail: kume@jichi.ac.jp

Received 31 August 2016; Revised 16 March 2017; Accepted 17 March 2017
Accepted article preview online 27 March 2017; Advance online publication 13 April 2017

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Abstract

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1–9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken β-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

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Introduction

Intractable respiratory diseases such as cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) are devastating illnesses. Curative therapies are not available, and current management is limited to symptomatic therapy and the prevention of severe infections. Therefore, patients have a poor prognosis; the median life expectancy of CF patients is 39.3 years,1 while the median survival time from the diagnosis of IPF is only 3 to 5 years.2 Extensive investigations have identified several genes related to intractable respiratory diseases. For example, mutations in the CF transmembrane conductance regulator (CFTR) gene lead to CF,3 and the mucin-5B (MUC5B) gene is related to IPF.4 These findings suggest that gene therapy has potential as a curative modality to treat CF and IPF by correcting pathological cellular functions and ameliorating disease phenotypes.

To deliver therapeutic genes to the respiratory tract, adeno-associated virus (AAV) vectors have emerged as promising tools in gene therapy due to their safety profiles, long-term gene expression and broad tissue specificity.5, 6 The efficacy of AAV vectors has so far been demonstrated in clinical trials for hemophilia B,7 Parkinson’s disease8, 9 and congenital ocular amaurosis.10, 11 In contrast, AAV vectors have failed to successfully treat CF patients, with weak or negligible transgene expression being observed in the respiratory epithelium.12, 13, 14 Several reasons have been suggested for the low transduction efficiency in earlier studies; the AAV serotype 2 capsid may not be optimal for respiratory cells, the promoter activities of the previous vectors may be weak and administration with a jet nebulizer may not deliver AAV vectors to the appropriate target tissue.

Vectors with AAV1, 5, 6 and 9 capsids were recently found to transduce airway epithelial cells more efficiently than AAV2.15, 16, 17, 18, 19 Transgene expression in the lung may be enhanced by stronger promoters, including the cytomegalovirus (CMV) promoter,20 Rous sarcoma virus promoter21 and chicken β-actin promoter with the CMV immediate-early enhancer (CAG).17, 22, 23 Furthermore, an appropriate administration device needs to be selected based on the polarity of airway epithelial cells because the transduction pattern may vary depending on the approach (apical or basolateral infection) and AAV serotype.18, 24, 25

In the present study, we investigated several parameters affecting the efficiency of AAV-mediated gene transfer to the respiratory tract. Based on in vitro screening, an efficient AAV vector was constructed and examined for its in vivo transduction properties in the murine respiratory system.

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Results

Construction of an optimal AAV vector for respiratory cell transduction

Previous studies have suggested the advantage of several AAV serotypes, including AAV1, AAV5, AAV6 and AAV9, in respiratory cell transduction,15, 16, 17, 18, 19 and prompted us to study AAV pseudotyping for the lung in a comprehensive and detailed way. To screen AAV serotypes for efficient respiratory cell transduction, we constructed β-galactosidase (β-gal)-expressing AAV vectors pseudotyped with AAV1–9 capsids (AAVn-CMV-LacZ; N=1, 2, 3, 4, 5, 6, 7, 8 and 9). These vectors were challenged for β-gal expression in five respiratory cell lines (human bronchial epithelial cells (BEAS-2B), murine alveolar epithelial cells (MLE12), human fetal lung fibroblast cells (MRC-5), murine alveolar macrophages (MH-S) and normal human primary bronchial epithelial cells (NHBE)). As shown in Figure 1, the AAV6 vector achieved the strongest transgene expression in all the examined cell lines (BEAS-2B, MLE12, MRC-5, MH-S and NHBE), whereas other AAV vectors resulted in very weak expression. When transgene expression was corrected by cell numbers, the results were unaffected, that is, the AAV6 vector transduced these cells most efficiently (Supplementary Figure 1). The result that the AAV6 yielded by far the highest expression in murine and human respiratory cells was consistent with a report by Limberis et al.,18 which suggested that the serotype may be the most promising one to treat lung diseases. Therefore, we designed the subsequent experiments centering upon AAV6.

Figure 1.
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In vitro transduction efficiency of AAV serotypes 1–9. (ac and e) BEAS-2B, MLE12, MRC-5 and NHBE cells were transduced with 3 × 105 viral genomes (vg) per cell of the AAV1–9-CMV-LacZ vector. β-Galactosidase was assayed 72h after transduction. (d) MH-S was transduced with 1 × 106 vg per cell of the AAV1–9-CMV-LacZ vector. β-Galactosidase was assayed 48h after transduction. Data are presented as the mean±s.e.m. (N=4 for each group). *P<0.05, **P<0.01, ***P<0.001 versus all lung values (one-way analysis of variance (ANOVA) with Tukey’s correction or Games-Howell’s correction).

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We then compared promoter activity in the respiratory cell lines. Two luciferase-expressing AAV6 vectors were constructed with CMV and CAG promoters (AAV6-CMV-Luc and AAV6-CAG-Luc) and administered to the five respiratory cell lines. In all the cell lines, the CAG promoter drove the transgene stronger than the CMV promoter (Figure 2). Based on these results, we used a combination of the AAV6 capsid and CAG promoter in subsequent experiments to investigate in vivo transduction to the lung.

Figure 2.
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In vitro comparison of CMV and CAG promoters. The respiratory cell lines were transduced with AAV6-CAG (or CMV)-Luc at 3 × 105 vg per cell, except for MH-S cells (1 × 106 vg per cell). Luciferase activity was assayed 48h after transduction. Data are presented as the mean±s.e.m. (N=4 for each group). RLU, relative light units. **P<0.01, ***P<0.001 versus CMV (Welch’s t-test, two-sided).

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Appropriate route of administration for lung transduction

To elucidate how the route of delivery influences biodistribution, the AAV6-CAG-Luc vector (1 × 109 viral genomes (vg) per mouse) was administered to mice by an intravenous, intranasal or intratracheal injection. In vivo imaging 1 month after the injection demonstrated that the route of administration affects AAV6 gene transfer in the major target organ. Luciferase expression was localized to the nasal cavity and liver following intranasal and intravenous administration routes, respectively. An intratracheal administration led to a robust luciferase expression in the lung, whereas intravenous and intranasal instillation resulted in negligible signals in the chest (Figure 3). Our failure in lung transduction via transnasal delivery appeared to differentiate from several previous reports showing successful intranasal administration.18, 23 To explore the reason for such a difference, we administered 1 × 1011 vg of AAV6-CAG-Luc intranasally, the same dose that Limberis et al.18 used. With this dose (that is, 100-fold greater dose of intratracheal administration), 5 out of 16 mice showed moderate-to-strong expression in the chest area (Supplementary Figure 2). Thus, the apparent inefficiency of our intranasal administration may partly result from difficulty in this technique,26 whereas we successfully delivered 1 × 109 vg via trachea in every mouse (Supplementary Figure 2). As for intravenous administration, we assayed in vivo luciferase expression from 5 days to 6 months post injection, considering the possibility of distinct time course and biodistribution from local administration. Throughout the observation period, intravenously given AAV6-CAG-Luc expressed only in the liver (Supplementary Figure 3).

Figure 3.
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Noninvasive in vivo imaging after AAV6-luciferase vectors. The AAV6-CAG-Luc vector (1 × 109 vg per mouse) was administered to mice via three routes: intravenous, intranasal and intratracheal. For comparison, the AAV6-CMV-Luc vector (1 × 109 vg per mouse) was administered intratracheally to another cohort of mice. The expression range was 10000–300000 photonss−1cm−2. Images were taken 30 days post injection in a ventral view. Three representative images from each group (N=11–16) are shown.

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Notably, the superiority of intratracheal administration for lung transduction was emphasized when combined with AAV6 vectors. When the AAV1, 2 and 9 vectors (AAV1, 2, 9-CAG-Luc; expression in cell lines are shown in Supplementary Figure 4) were intratracheally administered to mice (1 × 109 vg per mouse), AAV1 and AAV2 vectors did not produce significant signals; only very weak signals were observed in the lung by AAV1. The expression of AAV9-CAG-Luc was modest in the liver and weak in the lung (Supplementary Figure 5a). An analysis of luminescence in the region of interest (average radiance; photonss−1cm−2 per steradian) demonstrated that maximum expression of the recombinant AAV (1, 2, 6, 9)-CAG-Luc vector occurred 1 to 2 months after the injection. Although the strength of the signals gradually decreased, the expression of AAV6-CAG-Luc in the lung was sustained for at least 6 months (Supplementary Figure 5b). These results confirmed that the AAV6 vector was the most appropriate for lung transduction.

To elucidate whether the CAG promoter functions better than the CMV promoter in vivo, mice were also intratracheally injected with an AAV6-CMV vector (1 × 109 vg per mouse). In vivo imaging showed that the CAG promoter was stronger than the CMV promoter (Figure 3), and the durability of luciferase expression was significantly greater with the CAG promoter than with the CMV promoter during the observation period (up to 12 months; Figure 4a). Although gene expression with the CAG promoter decreased over time, luminescence remained 6.7-fold greater than the baseline, whereas expression with the CMV promoter groups returned to the baseline in 8 months.

Figure 4.
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Differences in the in vivo efficacy of AAV serotypes and promoters. (a) AAV6-CAG-Luc and AAV6-CMV-Luc vectors (1 × 109 vg per mouse) were administered intratracheally to mice. Bars show the average radiance (photonss−1cm−2 per steradian) of luciferase bioluminescence emitted from the chest area at different time points after gene delivery. In each group, average radiance was measured for each animal at each time point. Values were obtained from ventral images and expressed as the mean±s.e.m. (AAV6-CAG-Luc or AAV6-CMV-Luc; N=6 for each group at each time point). *P<0.05, **P<0.01 versus AAV6-CMV-Luc (Welch’s t-test, two-sided). (bd) Vector distribution and transgene expression in lung tissue 2 months after the intratracheal injection of AAV1, 2, 6, 9-CAG-Luc or AAV6-CMV-Luc (1 × 109 vg per mouse). (b) Vector distribution in the lung. Genomic DNA was isolated from lung tissue and the copy number of the luciferase gene in 300ng DNA was measured by qPCR. To normalize each sample, copy number of the mouse Rplp1 gene was also quantified as an internal control. *P<0.05 versus all lung values, except for AAV6-CAG (or AAV6-CMV). NS, not significant (one-way analysis of variance (ANOVA) with Games-Howell’s correction). (c) mRNA expression was assessed by reverse transcriptase directed-qPCR. Luciferase mRNA levels were measured using the comparative Ct method, and the GAPDH mRNA expression was used as a control. *P<0.05 versus all lung values (one-way ANOVA with Games-Howell’s correction). (d) Luciferase enzyme quantity was expressed as ng luciferase per mg of total protein. Values are shown as the mean±s.e.m. (AAV1, 2, 9-CAG-Luc; N=4, AAV6-CAG or AAV6-CMV-Luc; N=7 for each group). ***P<0.001 versus all lung values (one-way ANOVA with Games-Howell’s correction).

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Tissue distribution and transgene expression

To further analyze the transduction efficiency of the different AAVs, vector distribution and expression in dissected tissues were investigated 2 months after administration when the expression of luciferase was maximum in vivo (Figure 4a and Supplementary Figure 5b). Vector genome copy numbers in the lung and liver were measured using a real-time quantitative polymerase chain reaction (qPCR). The intratracheal administration of the AAV1, 2 and 9-CAG-Luc vectors was associated with low copy numbers in the lung (Figure 4b). Very low copy numbers in the lung after the intravenous and intranasal administration of AAV6 vectors indicated that few vectors reached the lung via these delivery routes (data not shown). In contrast, the intratracheal administration of AAV6-CAG-Luc and AAV6-CMV-Luc resulted in higher copy numbers in the lung, with no significant difference between the two promoter cohorts (Figure 4b). Vector DNA was not detected in the liver following the intratracheal administration of any vector (AAV1, 2, 6, 9-CAG-Luc or AAV6-CMV-Luc), intravenous injection of AAV6-CAG-Luc or intranasal dosing of AAV6-CAG-Luc (data not shown).

Luciferase messenger RNA (mRNA) was then examined in the dissected tissues. In the lung, the intratracheal administration of AAV6-CAG-Luc resulted in the highest mRNA level, whereas the other AAV vectors produced very small amounts of luciferase mRNA when dosed via the same route of administration (Figure 4c). The mRNA levels derived from AAV6-CAG-Luc were sevenfold higher than those from AAV6-CMV-Luc. Taken together with vector copy numbers being similar in mice given AAV6-CAG-Luc and AAV6-CMV-Luc, more efficient transcription from the CAG promoter was confirmed in the lung tissue. As expected from the negligible amounts of vector DNA, mRNA was not detected in the lung after the intravenous or intranasal administration of AAV6-CAG-Luc. Similarly, mRNA was not found in the liver for any vectors or delivery routes (data not shown).

Luciferase enzyme was also measured in the dissected tissues. In the lung, the intratracheal administration of the AAV6-CAG-Luc vector produced the highest enzyme expression. Only weak or no luciferase activity was associated with the other vectors administered intratracheally, which parallels with the results obtained for mRNA levels (Figure 4d). A 14.4-fold difference was observed between AAV6-CAG-Luc and AAV6-CMV-Luc. The intravenous and intranasal administration of AAV6-CAG-Luc resulted in undetectable luciferase activity (data not shown). Enzymatic activity was not found in the liver, except for very weak signals following the intravenous administration of AAV6-CAG-Luc and intratracheal administration of AAV9-CAG-Luc (data not shown). This may reflect some remnant luciferase protein in the liver as a result of transient transduction.

Pulmonary cell types transduced by the AAV6-CAG vector

The results of the present study suggest that the AAV6-CAG vector is optimal for transduction to respiratory tract cells in vitro and in vivo. To identify which cell types in the lung are preferentially transduced by this combination, a humanized Renilla reniformis green fluorescent protein (hrGFP)-expressing AAV6 vector was constructed with the CAG promoter (AAV6-CAG-hrGFP) and administered to mice (1 × 1011 vg per mouse) intratracheally. Lung cryosections were obtained 1 month after the intratracheal injection, and hrGFP expression was examined together with cell type-specific immunostaining. Based on the number of hrGFP-positive cells in cryosections, the overall transduction efficiency of the lung was estimated to be 14.5%. Fluorescent microscopy confirmed a comparable prevalence of hrGFP-positive cells in the bronchial epithelium (12.8%; Figure 5a). In the lung periphery, cytokeratin-positive alveolar epithelial cells (Figure 5b) and F4/80-positive macrophages (Figure 5c) expressed hrGFP infrequently (6.3% and 1.5%, respectively). Transgene expression was mostly observed within the alveolar septa and co-localized with alpha smooth muscle actin (αSMA)-positive cells (67.3%; Figure 5d), suggesting that the AAV6 vector preferentially transduced pericytes.

Figure 5.
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Immunostaining of the lung 1 month after the intratracheal injection of AAV6-CAG-hrGFP (1 × 1011 vg per mouse, N=4). Three lung sections were made from each mouse, and representative images are shown. (a) hrGFP expression was assessed directly by fluorescent microscopy of the bronchial epithelium. (bd) Lung sections were immunostained with an anti-pan Cytokeratin antibody (AE1/AE3) for the detection of alveolar epithelial cells (b), an anti-F4/80 antibody for macrophages (c) and an anti-αSMA antibody for mesenchymal cells (d), followed by the appropriate fluorescence-labeled secondary antibodies (red) for each. Nuclei were stained with DAPI (blue). White arrowheads represent transduced cells. Original magnification, × 200.

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Discussion

In the present study, we investigated the optimization of AAV vector-mediated gene transfer to the respiratory tract. The most promising AAV vector was composed of the serotype 6 capsid and CAG promoter, and intratracheal administration was the most efficient delivery route for the transduction of respiratory tract cells. Here, the intratracheal injection of the AAV6-CAG vector achieved a robust and sustained transgene expression in the lung, and our histological examination suggested that most transduced cells were lung pericytes.

Previous studies have suggested that several serotypes, including AAV1, 5, 6 and 9, are effective for pulmonary gene therapy.15, 16, 17, 18, 19 To more directly clarify the optimal serotype for gene transduction to the respiratory tract, we herein examined the transduction efficiencies of different AAV serotypes in vitro and in vivo. Among the AAV serotypes screened with several representative respiratory cell lines, AAV6 produced the strongest transgene expression. Noninvasive bioimaging confirmed that AAV6 was the most appropriate for transduction to the lung, particularly when administered intratracheally. The mechanisms underlying the enhanced transduction of AAV6 to the lung have not yet been elucidated in detail. AAV6 uses α-2,3 and α-2,6 sialic acids on N-linked glycoproteins and heparan sulfate proteoglycans for infection.27, 28 These sialic acids are abundantly expressed on the apical side of airway epithelial cells,29 while heparan sulfate proteoglycans are expressed on the basolateral side.24 Adequate expression of the receptor molecules is a prerequisite for efficient transduction, but receptor biodistribution may considerably vary among species,19, 30 and pathological conditions may modulate transducibility even in the same species. Studies with nonhuman primates will be required and ultimately, clinical trials should prove the safety and the efficacy of AAV6 vectors.

In addition to more suitable AAV serotypes, gene expression levels may be enhanced by stronger promoters. The CMV promoter used in this study contains an immediate-early enhancer that provides a strong and constitutive expression.31, 32 It is, however, prone to be silenced over time in certain cell types, which is often observed with viral promoters.33 Incorporation of cellular cis-elements (for example, the β-actin promoter and a β-actin/β-globin fusion intron in the CAG promoter) can prevent such downregulation.17, 22, 23, 33 Indeed, the CAG promoter resulted in more robust and durable in vivo expression than the CMV promoter. We observed a significant transgene expression in the mouse lung for at least 12 months, the longest period reported thus far. A limitation of the CAG promoter is its relatively large size, particularly when packaged in a vector with restricted capacity such as AAV. To overcome such a packaging limitation, several approaches have been developed. First, trimming of promoter (for example, a shortened CAG) and cDNA (for example, CFTR) have been pursued.34, 35, 36, 37 Second, split vector systems have been created taking advantage of homologous recombination or trans-splicing.38 With the latter strategy, Song et al.39 successfully reconstituted full-length and functional CFTR in human CF airway epithelial cells. Fragment AAV vectors can be used as well, where annealing of overlapped complementary AAV genomes will trigger second-strand DNA synthesis to generate a large expression cassette.40

Target-specific vector delivery is a major issue in in vivo gene therapy as it promises superior efficacy and less adverse effects. Intranasal injection has been a preferred method for mouse lung transduction, because of its noninvasiveness and easiness. However, a considerable part of vector solution may be trapped in the upper airway and even misdirected to gastrointestinal tract, resulting in inconsistent transgene expression in the lower airway as has been experienced by several researchers.26 Similarly, intranasal AAV administration gave us a very little, if any, expression with the same vector dose as reported elsewhere, and a moderate expression with the 100-fold larger dose. On the other hand, intratracheal injection has been a dependable method of vector delivery to the lung in our present study. Although it is an invasive procedure for mice,41 bronchoscopy is a routine medical intervention and allows easy access to airway and alveolar epithelial cells. Therefore, it is a translatable route of AAV administration to an actual clinical application demanding high efficiency. In in vivo gene therapy, cell polarity also needs to be considered at the site of an injection; transduction efficiency may differ when the same AAV vector is administered from the apical or basolateral side. The AAV2 vector used in CF clinical trials preferentially transduces airway epithelial cells from the basolateral membrane,24 whereas the AAV6 vector favors the apical membrane.18 This may be one of the reasons for the efficient transduction of airway cells by AAV6 vectors when administered intratracheally.

Feasibility of repeated administration is another important issue of consideration in gene therapy. Preexisting neutralizing antibodies may prevent vector re-administration,42 and the prevalence of seropositive adults is higher for AAV2 than for AAV5 or AAV6.43 In addition, several investigators have reported that re-administration of AAV5 and AAV6 to the airway was feasible and even boosting,44, 45 whereas that was not the case with AAV2.46 As the feasibility of repeated administration depends on multiple parameters such as the host immune status,47 the route of delivery and the nature of AAV serotype, this issue awaits further extensive study. Of note, a long-term transgene expression reduces the number of dosing and intensity of immune suppression. Therefore, a year-long in vivo transgene expression, shown in this study, is an encouraging result to further refine the vector and the strategy to treat respiratory diseases.

Regarding transduced cell types in pulmonary gene transfer, only a few histological studies have been conducted on lung tissues, and cells other than bronchial ones have not yet been extensively examined.18, 23 We found that not only bronchial epithelial cells, but also many αSMA-positive cells were transduced. In the adult mouse lung parenchyma, αSMA is expressed in alveolar ring cells and pericytes as well as vascular and airway smooth muscle cells. Alveolar ring cells are mostly found at the corners of alveolar ducts, whereas pericytes are located around capillaries in the alveolar septa. In our lung sections, hrGFP/αSMA co-localization was not detected in the alveolar ducts, but was present within the alveolar septa, suggesting that most hrGFP-positive cells are pericytes. Pericytes are mural cells of a mesenchymal origin that attach to capillaries and venules,48 and have been suggested to control the morphology and function of lung parenchyma by altering microvascular blood flow.49 The mechanisms by which AAV6 reaches pericytes currently remain unclear. AAV6 may penetrate the alveolar epithelium by transcytosis or translocate through tight junction.50, 51 It has been also suggested that pericytes constitute a source of myofibroblast progenitors.52 This is particularly interesting because the sustained activation and proliferation of myofibroblasts may have a pivotal role in lung fibrosis by producing an excessive fibrillary extracellular matrix,53 and thus, may be an appropriate therapeutic target.

The present study demonstrated that the AAV6-CAG vector successfully transduced respiratory cells in vitro and in vivo. Gene transfer may be targeted to bronchial cells and pericytes by intratracheal administration, and this strategy will contribute to the development of successful gene therapy for lung diseases such as CF and IPF.

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Materials and methods

Plasmids and recombinant AAV vector production

Recombinant AAVs for β-gal screening were prepared with an AAV vector plasmid pW1, in which the LacZ gene cassette encoding an Escherichia coli β-gal with the CMV promoter, the first intron of the human growth hormone gene, and the SV40 early polyadenylation sequence are flanked by inverted terminal repeats. The luciferase AAV vector plasmid, pAAV-CMV-Luc was generated by inserting the luciferase gene from the pGL3-Control Vector (Promega, Madison, WI, USA) into the pAAV-MCS vector containing the CMV promoter and a β-globin intron (Agilent Technologies, Palo Alto, CA, USA). To construct pAAV-CAG-Luc, the CMV promoter and the β-globin intron in pAAV-CMV-Luc was replaced with the CAG promoter derived from pCAGGS.22 The GFP AAV vector plasmid pAAV-CAG-hrGFP was based on the pAAV-hrGFP vector (Agilent Technologies) and the promoter was replaced as described above. Recombinant AAV-CMV-LacZ (serotypes 1–9), AAV-CAG (or CMV)-Luc (serotypes 1, 2, 6 and 9) and AAV-CAG-hrGFP vectors (serotype 6) were prepared as previously described.54 Briefly, 60% confluent human embryonic kidney cells (HEK293; Agilent Technologies) were incubated in large culture vessels and co-transfected with an AAV vector plasmid, adenoviral helper plasmid pHelper (Agilent Technologies), and one of the following AAV1–9 chimeric helper plasmids: pAAV2 Rep/AAV1 Cap for AAV1,55 pAAV-RC (Agilent Technologies) for AAV2, pAAV2 Rep/AAV3 Cap for AAV3,56 pAAV2 Rep/AAV4 Cap for AAV4,57 pAAV5 Rep/AAV5 Cap for AAV5,58 pAAV2 Rep/AAV6 Cap for AAV6,59 pAAV2 Rep/AAV7 Cap for AAV7,60 pAAV2 Rep/AAV8 Cap for AAV8 (ref. 60) and pAAV2 Rep/AAV9 Cap for AAV9.16 Crude viral lysates were purified twice on a CsCl two-tier centrifugation gradient. Viral titers were measured by qPCR, and the viral stock was dissolved in HN buffer (50mmoll−1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 150mmoll−1 NaCl) before the injection. The genome and infectious titers of the vector stocks were tested along with the reference standard materials.61, 62

AAV transduction of respiratory cell lines

Five respiratory cell lines were used; human bronchial epithelial cells (BEAS-2B), murine alveolar epithelial cells (MLE12), human fetal lung fibroblast cells (MRC-5), murine alveolar macrophages (MH-S; all purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA), and normal human primary bronchial epithelial cells (NHBE; Lonza, Walkersville, MD, USA). The BEAS-2B and NHBE cells were maintained in serum-free bronchial epithelial cell growth medium (BEGM; Lonza) supplemented with a bullet kit containing bovine pituitary extract, insulin, hydrocortisone, gentamicin/amphotericin, retinoic acid, transferrin, epinephrine and human epithelial growth factor (Lonza). The MLE12 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100Uml−1 penicillin, and 100mgml−1 streptomycin (Pen Strep; Life Technologies). MH-S cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum, 0.05mm 2-mercaptoethanol, and Pen Strep. The MRC-5 cells were cultured in Eagle’s Minimum Essential Medium (ATCC) supplemented with 10% heat-inactivated fetal bovine serum and Pen Strep. The HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium/F-12 medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum and Pen Strep. All the cultures were kept in an incubator at 37°C with 5% CO2.

In the initial screening, BEAS-2B, MLE12, MRC-5, MH-S and NHBE cells were seeded on 96-well plates at densities of 2 × 104, 4 × 104, 3 × 104, 5 × 104 and 1 × 104 per well, respectively. After 24h, AAV1–9-CMV-LacZ was added at a concentration of 3 × 105 vg per cell for all cell types, except for MH-S cells (1 × 106 vg per cell). After 48h for MH-S cells and 72h for other cell types, β-gal expression was evaluated using a β-Gal Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The total protein concentration of the supernatant was measured by the Bradford method with Protein Assay CBB Solution (Nacalai Tesque, Kyoto, Japan). The β-galactosidase activity was assayed three times to verify its reliability.

In the in vitro chemiluminescence assay, HEK293 cells and BEAS-2B cells were seeded on 96-well plates at a density of 2 × 104 cells per well 24h before transduction. These cells were transduced with AAV1, 2, 6 or 9-CAG-Luc at 3 × 105 vg per cell. After 48h, luciferase assays were performed with Fluoroskan Ascent FL (Thermo Fisher Scientific) using the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. To compare the promoters in vitro, five respiratory cell lines were seeded 24h before transduction, as described above, and transduced with AAV6-CAG- (or CMV)-Luc at 3 × 105 vg per cell for all cell types, except MH-S (1 × 106 vg per cell). After 48h, luciferase assays were performed as described above. The total protein concentration of the supernatants was measured as described above, and luciferase expression was represented as relative light units per mg protein. The luciferase assay was performed three times to verify its reliability.

In vivo gene transfer and in vivo imaging

All animal experiments were approved by the Jichi Medical University (Tochigi, Japan) Ethics Committee and performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Male C57BL/6J mice aged 8–12 weeks (from SLC Japan, Shizuoka, Japan) were used, and the sample size was limited to four to seven for each condition to allow for the detection of only highly significant differences. To evaluate the efficiency of in vivo gene expression according to the route of administration, the AAV6-CAG-Luc vector was administered to mice intravenously (150μl, 1 × 109 vg per mouse), intranasally (50μl, 1 × 109 vg or 1 × 1011 vg per mouse) or intratracheally (50μl, 1 × 109 vg per mouse). To investigate the efficiency of serotypes and promoters, the AAV1, 2, 9-CAG-Luc vector and AAV6-CMV-Luc vector were administered to mice intratracheally (50μl, 1 × 109 vg per mouse). In histological studies, the mice were intratracheally injected with the AAV6-CAG-hrGFP vector (50μl, 1 × 1011 vg per mouse). The control animals were intratracheally injected with the same volume of HN buffer. No randomization was used in the animal studies.

The mice were anesthetized with 2% isoflurane and oxygen. The d-luciferin substrate (Ieda Chemical, Tokyo, Japan) was injected intraperitoneally at a dose of 75mgkg−1 body weight. After 5min, luciferase activity was detected from the ventral surface using a noninvasive bioimaging system (IVIS100; Xenogen, Hopkinton, MA, USA). Luminescence levels in the region of interest (average radiance: photonss−1cm−2 per steradian) were analyzed with Living Image software (Xenogen, Alameda, CA, USA).

Vector biodistribution and expression

Two months after the administration of the AAV1, 2, 6, 9-CAG-Luc and AAV6-CMV-Luc vectors, the mice were killed and the lungs and livers were subjected to vector biodistribution and expression analyses. DNA was isolated from tissues using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The copy numbers in 300ng of DNA were measured by qPCR using Thermal Cycler Dice Real Time System II (Takara Bio, Shiga, Japan) following the manufacturer’s instructions. PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA) with sets of primers and probes specific for the luciferase gene. The gene-specific primers used were: forward, 5′-GAAAAAGTTGCGCGGAGGAG-3′; and reverse, 5′-CCGCCCTTCTTGGCCTTTAT-3′. Plasmid DNA pCMV-Luc (described above) was used as a reference standard at 10-fold serial dilutions ranging from 101 to 105 copies. Quantitative values were obtained from the threshold cycle (Ct) number that indicated exponential amplification of the PCR product. To normalize each sample, the copy number of the mouse ribosomal protein large P1 gene (Rplp1) was also quantified as a reference,63 by using the following primers: forward, 5′-TACGATTTCCACCACCAGCCTTG-3′; and reverse, 5′-CTCATTCTCAAGCCATGGACCGT-3′.

In the mRNA measurement, total RNA was isolated from tissues using an RNeasy Plus Mini Kit (Qiagen) and reverse-transcribed into single-stranded complementary DNA (cDNA). Five hundred nanograms of total RNA was reverse-transcribed into first-strand cDNA using the PrimeScript RT reagent Kit with the gDNA Eraser (Takara Bio) according to the manufacturer’s protocol. The cDNA was amplified with SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio) and Thermal Cycler Dice Real Time System II (Takara Bio). qPCR was performed using primer pairs specific for Luc (forward: 5′-CTATGAAGAGATACGCCCTG-3′ and reverse: 5′-TTCGAAGTACTCAGCGTAAG-3′) and the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) (forward: 5′-GTTCCAGTATGACTCCACTC-3′ and reverse: 5′-CCTCACCCCATTTGATGTTA-3′). The expression levels of target genes were analyzed using the comparative Ct method, and GAPDH was used as an internal control.

Luciferase enzyme was measured with the PicaGene Luminescence Kit (Toyo Ink, Tokyo, Japan). Specimens (100mg each) from the dissected organs were homogenized in 300–500μl of distilled water and centrifuged at 10000g at 4°C for 10min. The supernatant was mixed with an equal volume of lysis buffer (Toyo Ink) and incubated at room temperature for 15min with occasional vortexing. An aliquot of 100μl of the mixture was placed onto a 96-well plate in duplicate, 100μl of the substrate (luciferase assay reagent; Toyo Ink) was added to each well, and luminescence was captured with Fluoroskan Ascent FL (Thermo Fisher Scientific). A calibration curve was made with luciferase standard in the kit, and luciferase enzyme in tissues was expressed as ng luciferase per mg of total protein. The total protein concentration of the supernatants was measured using the Bradford method as described above.

Histology and immunohistochemistry

Pulmonary transgene expression was visualized with hrGFP. The mice were killed 1 month after the intratracheal injection of the AAV6-CAG-hrGFP vector. The lungs were fixed with 4% paraformaldehyde at 4°C overnight, transferred to 30% sucrose in 0.1moll−1 phosphate buffer (pH 7.4), and incubated at 4°C overnight for cryoprotection. The lung tissues were frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan) and 10μm-thick sections were cut using a cryostat. Fluorescence microscopy (BX63; Olympus, Tokyo, Japan) was used to directly monitor hrGFP fluorescence in bronchial sections. Alveolar epithelial cells were immunostained with a mouse monoclonal anti-pan Cytokeratin antibody (AE1/AE3, ab27988; Abcam, Cambridge, MA, USA), biotin-conjugated secondary antibody (M.O.M. Immunodetection Kit; Vector Laboratories, Burlingame, CA, USA), and Rhodamine Avidin D (Vector Laboratories). To detect macrophages, the sections were incubated with a rat monoclonal anti-F4/80 antibody (ab6640; Abcam) and Alexa Fluor 568-conjugated secondary antibody (ab175710; Abcam). To identify mesenchymal cells, αSMA was immunostained with a rabbit polyclonal anti-αSMA antibody (ab5694; Abcam) and Alexa Fluor 568-conjugated secondary antibody (ab175471; Abcam). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). No signal was detected from negative controls using only secondary antibodies. Images were obtained with a fluorescence microscope (BX63; Olympus). The percentage of positive cells was calculated by counting positive and negative cells in three lung sections per mouse.

Statistical analysis

Data are summarized as the mean±s.e.m. Regarding vector distribution and transgene expression in the dissected tissues, undetectable levels were defined as zero. Comparisons were made using Welch’s t-test (two-sided) or a one-way analysis of variance where appropriate. Depending on sample variance, Tukey’s correction or Games-Howell’s correction was applied to probability values for multiple comparisons. Statistical analyses were performed with StatMate V software (ATMS, Tokyo, Japan) and SPSS software (IBM Japan, Tokyo, Japan). In all the tests, P<0.05 was considered to denote significance.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Miyoko Mitsu, Satomi Fujiwara and Tomonori Tsukahara for their technical assistance; and Dr Ryoko Saito (Tohoku University) for thoughtful advice and suggestions. This work was supported by a Jichi Medical University Graduate Student Start-up Grant for Young Investigators to FK in 2015. This study was also supported by the Research Program on HIV/AIDS and the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development.

Supplementary Information accompanies this paper on Gene Therapy website