Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways

Abstract

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression.

Keywords: cyclophosphamide; cystic fibrosis; gene therapy; transient immunosuppression.

Figure 1

Time frame of the helper-dependent adenoviral (HD-Ad) vector transduction and cyclophosphamide administration. All the mice were nasally delivered HD-Ad-CFTR at a dose of 1.5 × 10¹⁰ viral particles in 3 rounds, each indicated by a heavy vertical line. The date of the first round of vector delivery is called d0. The second and third round was given at day 60 and day 120, respectively, following the first round. A cyclophosphamide (cytoxan) injection was administered at 6 h before (−6 h) and 4 (d4) and 8 days (d8) following the vector delivery. However, there was a glitch in the scheme where the cyclophosphamide injection at day 4 for the second round of vector delivery was missed. The samples were collected at days 3 and 33 after the last round of vector delivery, as shown in the scheme. Deliv: delivery; sac: sacrifice.

Figure 2

CD3 (cluster of differentiation 3) (a), CD4 (b), and CD8 (c) gene expression in mouse lungs determined by real-time RT-qPCR. RNA was isolated from mouse lung tissues, which were collected at days 3 and 33 after the last round of vector transduction from both groups of mice with or without cyclophosphamide treatments. The expression of CD3 (left panel), CD4 (middle panel), and CD8 (right panel) genes was normalized with 18S. 3V-3d: samples collected at day 3 following the third round of vector delivery; 3V-3d+cyto: samples collected from mice with the same time frame as those of 3v-3d but with cyclophosphamide treatments; 3V-1m: samples collected from mice in one month following the last round of vector delivery; 3v-1m+cyto: samples collected from mice with the same time frame as those of 3V-1m, but with cyclophosphamide treatments. Data are presented as mean ± SD (standard deviation), n = 5 for all groups. #: p < 0.05; **: p < 0.01.

Figure 3

CD3 (a), CD4 (b), and CD8 (c) protein expression in mouse lungs. IHC (immunohistochemical) staining was performed in paraffin sections of mouse lungs and stained with antibodies against CD3, CD4, and CD8. The positive cells are shown in brown color. The sample labelling is the same as in Figure 2.

Figure 4

Host humoral immune responses to vector delivery. (a) The titer of anti-adenoviral antibodies in mouse bronchoalveolar lavage fluid (BALF). The total anti-Ad antibodies (IgA, IgE, IgGs, IgM) were detected with ELISA in all groups. Data were presented as mean ± SD (standard deviation). (b) Neutralizing antibody in mouse BALF. n = 5, #: p < 0.05; **: p < 0.01. (c) B-cell presence in mouse lungs. B-cells were detected in mouse lungs with antibodies against B220 by immunohistochemistry staining. The positive cells are shown in brown color. ns, no significant difference.

Figure 5

Expression of human cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in mouse lungs assessed by real-time RT-qPCR. (a) CFTR mRNA levels in lung tissues of mice at days 3 and 33 following the last round of HD-Ad-CFTR delivery with or without cyclophosphamide treatments. The transgene expression was normalized with 18S. Data were presented as mean ± SD. n = 5; #: p < 0.05; **: p < 0.01. (b) CFTR mRNA levels in mouselung tissues from 3 days of single dose delivery (1V-3d), and at days 3 (3V-3d) and 33 (3V-33d) of 3 dose delivery following the last round of transduction with cyclophosphamide treatment. Data were presented as mean ± SD. Ns indicates no significant difference.