Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.
Over the last decade, adenoviruses have been widely used as vectors for gene therapy applications, including treatment of cystic fibrosis (CF). CF is an autosomal recessive monogenic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for an epithelial chloride channel.1 The major cause of morbidity and mortality in CF patients is lung disease, and hence the most important aim of CF gene therapy is to treat the respiratory system. By far, CF gene therapy has garnered most research due to the feasibility of theoretically correcting the disease by providing a single copy of the CFTR gene to airway epithelial cells. Among all the vectors available for gene delivery, vectors derived from adeno-associated virus (AAV) and adenovirus have been most widely used. Multiple clinical trials were initiated with AAV vectors; however, owing to unsatisfactory primary outcomes, most of them were discontinued. Similarly, adenoviral vector-mediated gene therapy suffered from the drawbacks of vector toxicity and low efficiency of gene transfer.
Adenoviral entry into the cells requires an initial binding of viral fiber to the CAR (Coxsackie and adenoviral receptor) receptor followed by internalization. Unfortunately, the localization of CAR receptors to the basolateral side of epithelial cells creates a new obstacle by reducing the efficiency of gene transfer.2 Additionally, the airways of CF patients are covered with a thick mucus layer due to chloride channel defects, which can further reduce the efficiency of gene delivery to the epithelial cells.3 Furthermore, the host innate and adaptive immune responses against adenoviral vectors also hinder their application in gene therapy protocols. Development of helper-dependent adenoviral (HDAd) vectors has reduced the adaptive immune response; however, owing to the presence of capsid protein, the host innate immune response remains a problem. Hence, there is a need to deliver adenoviral particles in a way that results in maximal gene transfer to the airways, while at the same time reduces the acute toxicity associated with the administration of these vectors.
Nacystelyn (NAL) is a derivative of N-acetyl-cysteine with an added L-lysine residue, and has been used for treatment of impaired mucociliary clearance and chronic mucus retention in CF patients.4, 5 NAL has been shown to possess antioxidant,6 mucolytic and anti-inflammatory properties, thus indicating that it may be beneficial for CF patients.7, 8 Clinical trials conducted with NAL in CF patients have demonstrated an improvement in symptoms, with a dose-dependent decrease in sputum viscoelasticity along with a decrease in sputum solids content, and an increase in chloride and sodium concentrations, thus indicating improved mucus clearance.9 At the same time, no adverse effects were observed in CF patients, even at a high dose of 24 mg given via inhaler.10 Presently, multiple phase I and phase II clinical trials are being undertaken in Europe and USA to assess the efficacy of NAL treatment in CF patients. Studies conducted in humans have also indicated that NAL prevents respiratory outburst of peripheral blood polymorphonuclear cells, indicating that it may have potential in reducing damage to lungs caused by an active inflammatory response,11 which could be of a potential benefit in the context of adenoviral vector-mediated CF gene therapy. Furthermore, NAL can inhibit the maturation of human dendritic cells,12 which in turn may limit the host adaptive immune response against adenoviral vectors. NAL has also been shown to enhance cationic liposome-mediated gene transfer to the airways.13, 14 In spite of this wealth of knowledge, no studies have assessed the efficacy of NAL in enhancing adenoviral vector-mediated gene delivery to the lung.
To further explore the potential properties of NAL in gene therapy, we investigated whether NAL can enhance adenoviral vector-mediated gene delivery to the airways of mice, and simultaneously reduce the acute toxicity induced by adenoviral vector particles. Our data suggest that pretreatment of the airways with NAL before adenoviral vector administration can enhance the efficiency of gene delivery. We propose a two-step delivery approach, where adenoviral vectors complexed with DEAE-Dextran are delivered after NAL pretreatment, resulting in maximum enhancement of mouse airway epithelial cell transduction by adenoviral vectors, along with a reduction in airway inflammation.
Initially, we assessed the ability of NAL to enhance adenoviral gene delivery in vitro by transducing A549, a lung carcinoma cell line. However, no enhancement effects were observed (data not shown). We also assessed the anti-inflammatory effects of NAL on A549 cells in response to lipopolysaccharide (LPS). Addition of LPS in the presence of NAL resulted in reduced expression of inflammatory cytokines (data not shown), confirming previous reports on the anti-inflammatory properties of NAL.15, 16 Therefore, we went on to assess the ability of NAL to enhance adenoviral vector-mediated gene delivery in vivo. We administered adenoviral vectors containing LacZ in saline to C57BL/6 mice, with or without NAL pretreatment. LacZ was chosen as a transgene due to a relative ease of quantification of expression at the protein level, along with the ease to assess localization of gene delivery/expression throughout the lung by performing X-gal staining. Moreover, first-generation adenoviral (FGAd) vectors were used for most of the experiments, since these vectors are much easier to produce than the HDAd vectors. We delivered 5 × 109 FGAd vector particles per animal, which we identified to be the optimum dose based on our preliminary studies. In accord, one of the aims of gene therapy is to use the lowest number of viral particles possible while obtaining maximal transduction, in order to reduce any toxicity associated with the viral vector itself. Furthermore, pretreatment with NAL did result in an enhancement of adenoviral gene delivery (Figure 1b) compared to control, which received adenoviral vectors in saline without NAL pretreatment (Figure 1a); however, the level of enhancement was not very high. Thus, we hypothesized that delivery of adenoviral vectors in other formulations, such as DEAE-Dextran, after NAL pretreatment may result in a significant enhancement of adenoviral vector-mediated airway epithelial cell transduction. DEAE-Dextran is a polycation, a derivative of dextran sugar, which has been shown to form complexes with adenoviral particles and enhance transduction of the airways in vivo.17, 18, 19 Additionally, DEAE-Dextran is clinically approved for human use and has an established safety profile,20 which makes it an attractive candidate to combine with NAL for airway gene delivery.
Adenoviral vectors containing LacZ were complexed with different concentrations of DEAE-Dextran and delivered to C57BL/6 mice with or without NAL pretreatment. NAL pretreatment led to a significant enhancement of LacZ transgene expression in the mouse airways upon adenoviral vector delivery (Figure 2a). NAL-mediated enhancement of transgene expression could be observed both in mice receiving viral formulation in 10 μg/ml as well as 20 μg/ml DEAE-Dextran, indicating that a combinatorial approach involving NAL pretreatment and DEAE-Dextran can be used for significant enhancement of adenoviral vector-mediated gene delivery to the airways. In contrast, viral delivery in saline after NAL pretreatment only led to a minor enhancement, indicating that NAL by itself may not open tight junctions to allow vector particles to reach the basolateral surface, and therefore, DEAE-Dextran is needed to partially fulfill this requirement. Pretreatment with NAL followed by adenoviral vector delivery in 20 μg/ml DEAE-Dextran resulted in approximately 64-fold enhancement in gene transfer compared to viral delivery in saline without NAL pretreatment (Figure 2a), whereas viral delivery in 20 μg/ml DEAE-Dextran without NAL pretreatment resulted in approximately 20-fold enhancement (Figure 2a), indicating a further 3- to 4-fold enhancement by NAL pretreatment over DEAE-Dextran without NAL pretreatment (Figure 2c). Similar 3- to 4-fold enhancement of LacZ transgene expression was observed upon comparing mice receiving NAL pretreatment followed by viral delivery in 10 μg/ml DEAE-Dextran with mice receiving viral delivery in 10 μg/ml DEAE-Dextran without NAL pretreatment (Figure 2a). Since DEAE-Dextran was needed for a significant enhancement of transgene expression upon NAL pretreatment and DEAE-Dextran by itself could also mediate significant enhancement of transgene expression, it can be speculated that DEAE-Dextran may transiently open tight junctions to allow vector particles to reach the basolateral surface of the airway epithelial cells.
The simplest approach for gene delivery would be a combination of NAL together with DEAE-Dextran and virus rather than any form of NAL pretreatment. To test this idea, we assessed the efficacy of NAL-mediated enhancement, by combining it with DEAE-Dextran and virus instead of pretreating with NAL. The results indicate that a combination of NAL administered together with virus in DEAE-Dextran ameliorated the enhancement effects seen with NAL pretreatment (Figure 2b). Hence, NAL can only enhance adenoviral vector-mediated gene delivery if it is administered before vector delivery. Since pretreatment with NAL followed by vector delivery in 20 μg/ml DEAE-Dextran resulted in higher LacZ expression, we chose the 20 μg/ml DEAE-Dextran concentration for further experimental analysis. We also assessed whether NAL-mediated enhancement of gene delivery was specific for FGAd vectors or whether it could be applied to third generation of viral vectors (HDAd vectors). We assessed the ability of NAL pretreatment to enhance gene delivery of HDAd vectors using LacZ construct driven by K18 promoter (HDAdK18LacZ). The K18 promoter is derived from the cytokeratin K18 gene, and it has been previously shown to mediate airway epithelium-specific transgene expression.21 Analysis of β-galactosidase activity confirmed that NAL pretreatment can also be used to enhance gene delivery mediated by HDAd vectors with airway epithelium specificity to similar levels as seen with FGAd vectors (Figure 2c).
Since the β-galactosidase assay relies on protein expression in the whole lung and cannot differentiate between localized and uniform expression, we performed staining of the whole lung to assess localization of LacZ expression after delivery of adenoviral vectors to the mouse airways in saline without NAL pretreatment, DEAE-Dextran without NAL pretreatment or DEAE-Dextran with NAL pretreatment (Figure 3). X-gal staining of the lungs isolated from mice that received saline without any virus did not show any positive X-gal staining throughout the airways as expected (Figure 3a). Delivery of FGAd vector particles in saline led to weak transduction of bronchioles in the lung (Figure 3b, left panel), whereas delivery of virus in DEAE-Dextran without NAL pretreatment led to transduction throughout the lung except for the lower extremities of the airways (Figure 3c, left panel). In contrast, pretreatment with NAL followed by viral delivery in DEAE-Dextran led to uniform transgene expression throughout the lung, including the lower extremities of the airways (Figure 3d, left panel). We also took a closer look at staining within the trachea, and in contrast to viral delivery in saline or DEAE-Dextran without NAL pretreatment, pretreatment with NAL followed by viral delivery in DEAE-Dextran resulted in maximum transduction of the trachea as indicated by an increase in X-gal staining intensity (Figure 3b–d, right panels). In contrast to the previous reports of DEAE-Dextran-mediated enhancement of gene transfer only in the lower lung,18 our observations indicated enhancement throughout the airways including the mouse trachea, which could be due to higher DEAE-Dextran concentration used in our experiments (Figure 3c, right panel). The whole-lung X-gal staining data in combination with results from β-galactosidase activity assay (Figure 2) confirmed that NAL pretreatment followed by viral delivery in DEAE-Dextran can significantly enhance gene delivery throughout the airway epithelium.
Having established the efficacy of NAL pretreatment in enhancing adenoviral vector-mediated gene transfer to the airways, we next assessed whether the use of NAL can potentially reduce the acute toxicity of adenoviral particles. Histological analysis of the mouse airways was conducted by a veterinary pathologist. Mild perivascular and peribronchiolar inflammation characterized by an infiltration of neutrophils, lymphocytes and macrophages was observed in the lungs of mice in all groups except for the control group, which appeared to have normal lung histology (Figure 4). For all groups, trachea appeared normal with no inflammation (data not shown). The data also showed that inflammation was more prominent in mice receiving adenoviral vector particles in saline or DEAE-Dextran without NAL pretreatment (Figure 4b and c) as compared to mice, which received pretreatment with NAL (Figure 4d). This clearly indicates that NAL pretreatment can reduce the acute toxicity of adenoviral vector particles. Moreover, we also confirmed anti-inflammatory effects of NAL by delivering NAL before LPS in mice intranasally and the results showed a reduction in the mRNA levels of inflammatory cytokines upon NAL pretreatment (data not shown). However, it needs to be noted that murine airways are different from human airways for they secrete low amounts of mucus due to limited number of submucosal glands.22 Therefore, enhancement of gene delivery seen in mice airways may be mostly due to a combination of antioxidant, mucolytic and anti-inflammatory effects of NAL. In contrast, human airways both in normal and particularly in CF patients secrete a lot of mucus; therefore, we can speculate that pretreatment with NAL may result in even higher levels of enhancement of adenoviral vector-mediated gene delivery than seen in mice, mostly due to its mucolytic activity in combination with anti-inflammatory effects.
In summary, our results demonstrate the utilization of NAL, a clinically used mucolytic agent in CF patients, in enhancing adenoviral vector-mediated gene delivery to the mouse airways. Additionally, NAL is able to reduce the acute toxicity associated with delivery of adenoviral particles in saline, indicating its importance as an anti-inflammatory agent with the potential of enhancing adenoviral gene delivery in a clinical setting for CF patients. Currently, studies are underway in large animal models, particularly rabbits, to assess the feasibility of using NAL pretreatment to enhance adenoviral vector-mediated gene delivery and reduce vector-induced acute toxicity.
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We gratefully acknowledge Cathleen Duan for assistance in performing β-galactosidase assays, and Dr Colin McKerlie (Hospital for Sick Children, Toronto, Canada) for assistance in histological assessment of inflammation in the mouse airways. This work was supported by Operating Grants from the Canadian Institutes of Health Research, the Canadian Cystic Fibrosis Foundation and the Foundation Fighting Blindness-Canada. J Hu is a CCFF Scholar and recipient of the CCFF Zellers Senior Scientist Award, and holds a Premier's Research Excellence Award of Ontario, Canada.
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