Durable transgene expression and efficient re-administration after rAAV2.5T-mediated fCFTRΔR gene delivery to adult ferret lungs

Abstract

The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRΔR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wild-type ferrets. Within the first 3 months following rAAV2.5T-fCFTRΔR delivery to the lung, CFTRΔR transgene expression declined ∼5.6-fold and then remained stable to 5 months at ∼26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRΔR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporter-only controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and ∼2.3-fold decline in fCFTRΔR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRΔR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.

Keywords: CFTR; T cell responses; adeno-associated virus; cystic fibrosis; durability; gene therapy; lung; neutralizing antibodies; repeat administration; transgene.

Graphical abstract

Figure 1

Durability of rAAV2.5T-fCFTRΔR transduction in adult ferret lung (A) Six groups of adult ferrets received rAAV2.5T-fCFTRΔR (1 × 10¹³ DRP/kg body weight) via intratracheal atomization. The lungs, plasma, bronchoalveolar lavage fluid (BALF), and splenocytes were harvested at the indicated time points. (B) Copies of transgene-derived fCFTRΔR and endogenous fCFTR mRNA normalized to GAPDH and quantified by qPCR with plasmid copy number standard curves. Six technical replicates were measured by independent sampling of the pulverized lung tissue from each animal, and values were averaged. (C) Ratio of the copy number of transgene-derived fCFTRΔR to endogenous fCFTR mRNA (i.e., fCFTRΔR/fCFTR). (D and E) (D) Number of rAAV2.5T-fCFTRΔR vector genome (vg) copies in 100 ng of total cellular DNA and (E) per cell following normalization to the genomic DNA content in a single ferret cell. Six technical replicates were measured from 100 ng of total cellular DNA generated from independent sampling of the pulverized lung tissue from each animal and values averaged. (F) Total endogenous fCFTR DNA copies found in 100 ng of lung tissue DNA. Three technical replicates were measured by independent sampling of the pulverized lung tissue from each animal and values averaged. (G) Endogenous fCFTR DNA copies per lung cell. Based on the endogenous genomic fCFTR copies from 100 ng of lung genomic DNA, a single diploid ferret cell is estimated to contain 5.78 pg of DNA, which is very close to the cellular content of a diploid human cell (∼6 pg/cell). Results show the mean ± SEM. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test using original data in (B) and (C) and using log₂(Y + 1) transformed data in (D) and (E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. n = 5–8 animals in each group as shown in (A).

Figure 2

Humoral and cellular immune responses following rAAV2.5T-fCFTRΔR administration to the ferret lung Neutralizing antibody (NAb) IC₅₀ titers measured by rAAV2.5T-fLuc preincubation with serially diluted (A) plasma or (B) BALF prior to incubation on A549 cells and assessment of firefly luciferase expression. (C) ELISpot measurement of IFN-γ levels from 2 × 10⁵ splenocytes following challenge with AAV2.5T capsid peptides. The dashed line in (A) indicates the average plasma NAb titer of all naive ferrets in the study prior to rAAV2.5T-fCFTRΔR transduction (10.21 ± 0.83). Results show the mean ± SEM. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test using log₂(Y + 1) transformed data. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. n = 5–8 animals in each group as shown in Figure 1A.

Figure 3

Capsid-binding immunoglobulin levels in plasma and BALF following rAAV2.5T-fCFTRΔR administration to the ferret lung AAV capsid-binding Ig titers were assessed by ELISA using purified vector as the capture antigen. (A) Plasma IgG levels (diluted 4,000-fold). (B) Plasma IgM levels (diluted to 1:2,000). (C) Plasma IgA levels (diluted to 1:20). (D) BALF IgG levels (diluted 50-fold). (E and F) BALF (E) IgM level and (F) IgA level were assessed using undiluted samples. Results represent the mean ± SEM. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. n = 5–8 animals in each group as shown in Figure 1A.

Figure 4

Transduction efficiency following repeat administration of rAAV2.5T (A) Schematic of the three experimental groups of ferrets used to evaluate repeat administration of rAAV2.5T. The repeat group received rAAV2.5T-fCFTRΔR (1 × 10¹³ DRP/kg body weight), were administered rAAV2.5T-gLuc (1 × 10¹³ DRP/kg body weight) 5 months later, and were euthanized 14 days later (5m.14d). The single group was only transduced with AAV2.5T-gLuc, while the control group was only transduced with rAAV2.5T-fCFTRΔR. (B) Secreted gLuc activity in BALF at 14 days after rAAV2.5-gLuc transduction. (C) gLuc activity in plasma within the first 14 days following rAAV2.5T-gLuc transduction. Results represent the mean ± SEM. Statistical significance was analyzed by Mann-Whitney test in (B) and using one-way ANOVA followed by Tukey’s multiple comparisons test in (C). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. n = 4 or 6 animals in each group.

Figure 5

Humoral and cellular immune response following repeat administration of rAAV2.5T Schematic of rAAV2.5T dosing in single, repeat, and control ferrets is as shown in Figure 4A. (A and B) Neutralizing antibody (NAb) titers in (A) plasma and (B) BALF from single and repeat groups. The dotted line in (B) indicates the average NAb titer of plasma from the control group of ferrets at 5 months post rAAV2.5T-fCFTRΔR administration. (C) ELISpot measurement of IFN-γ levels from 2 × 10⁵ splenocytes following challenge with AAV2.5T capsid peptides. The dotted line in (B) indicates the average NAb titer of plasma from the control group of ferrets at 5 months post rAAV2.5T-fCFTRΔR administration. (D) Copies of transgene-derived fCFTRΔR and endogenous fCFTR mRNA normalized to GAPDH from lung tissue harvested at 5 months after a single administration of AAV2.5T-fCFTRΔR (control) or at 14 days after repeat administration with AAV2.5T-gLuc in ferrets transduced with AAV2.5T-fCFTRΔR 5 months earlier (repeat). (E) Copy number ratio of transgene-derived fCFTRΔR mRNA to the endogenous fCFTR mRNA. (F) Number of AAV vg in 100 ng of lung cellular DNA from control and repeat groups tested. Results represent the mean ± SEM. Statistical significance was analyzed by Mann-Whitney test using log₂(Y + 1) transformed data in (A) and non-transformed data in (B–F). ∗p < 0.05, ∗∗p < 0.01. n = 4–6 animals in each group.

Figure 6

Capsid-binding immunoglobulin levels in plasma and BALF following repeat administration of rAAV2.5T to ferret lung Schematic of rAAV2.5T administration in single and repeat groups of ferrets is as shown in Figure 4A. AAV capsid-binding Ig titers were assessed by ELISA using purified vector as the capture antigen. (A) Plasma IgG levels (diluted 4,000-fold). (B) Plasma IgM levels (diluted to 1:2,000). (C) Plasma IgA levels (diluted to 1:20). (D) BALF IgG levels (diluted 50-fold). (E and F) (E) BALF IgM levels and (F) IgA levels assessed using undiluted samples. Results represent the mean ± SEM. Data were analyzed by Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01. n = 4 or 6 animals in each group.