Short-Term Steroid Treatment of Rhesus Macaque Increases Transduction

Abstract

Repeat dosing poses a major hurdle for the development of an adeno-associated virus (AAV)-based gene therapy for cystic fibrosis, in part because of the potential for development of an immune reaction to the AAV1 capsid proteins. Here, to dampen the immune response to AAV1, we treated Rhesus monkeys with methylprednisolone before and after the instillation of two doses of AAV1Δ27-264-CFTR into their airways at 0 and 30 days, followed by a single dose of AAV1-GFP on day 60. Animals were euthanized on day 90, except for one monkey that was sacrificed at 1 year. No adverse events occurred, indicating that the two AAV1 vectors are safe. rAAV1-CFTR and AAV1-GFP vector genomes and mRNA transcripts were detectable in all lung sections and in the liver and pancreas at day 90 and after 1 year at levels comparable with animals necropsied at 90 days. The numbers of vector genomes for cystic fibrosis transmembrane regulator (CFTR) and green fluorescent protein (GFP) detected here were higher than those found in the monkeys infected without methylprednisolone treatment that we tested previously.¹ Also, lung surface and keratin 5-positive basal cells showed higher CFTR and GFP staining than did the cells from the uninfected monkey control. Positive immunostaining, also detected in the liver and pancreas, remained stable for at least a year. All animals seroconverted for anticapsid antibodies by 90 days post-treatment. The neutralizing antibody titer declined in the animal necropsied at 1 year. Conclusion: AAV1 safely and effectively transduces monkey airway and basal cells. Both the presence of vector genomes and transduction from AAV1-CFTR and AAV1-GFP virus seen in the monkeys 4 months to 1 year after the first instillation suggest that repeat dosing with AAV1-based vectors is achievable, particularly after methylprednisolone treatment.

Keywords: AAV1; CFTR; Rhesus; basal cells; cystic fibrosis; lung liver; pancreas; repeat dosing; surface cells.

Figure 1.

Distribution of AAV1-CBA-Δ27-264 CFTR and AAV1-CBA-GFP vector genomes in lung tissue. Vector-specific genomes per microgram of lung DNA were measured by real-time DNA PCR in the trachea, bronchus, carina, cranial, middle, and caudal regions of five Rhesus monkeys and one untreated control. The data from the untreated control were used as a tissue blank and subtracted from each of the experimental samples. (A, B) Measurements taken in each lung region of the five animals. (C, D) Measurements taken in all of the five lung segments of each animal. (E, F) Summary data for all the lung segments of four animals (#34, 45, 52, 62) sacrificed at 90 days and one animal (#22) sacrificed at 1 year. Dots represent each independent measurement. Summary data are averages ± SE. Distribution of AAV1-CBA-Δ27-264 CFTR and AAV1-CBA-GFP vector genomes in liver and pancreatic tissue. Vector-specific genomes per microgram of genomic DNA were measured in the liver and pancreas of five monkeys and one untreated control, which was used as a tissue blank. (G, H) Measurements taken in one liver sample from each of the five animals. (I, J) Summary data for all the livers of the four animals sacrificed at 90 days and animal #22, sacrificed at 1 year. (K, L) Measurements taken in the pancreas of each of the five animals. (M, N) Summary data for all the pancreata of the four animals sacrificed at 90 days and animal #22, sacrificed at 1 year. Dots represent each independent measurement. Summary data are averages ± SE. AAV, adeno-associated virus; CFTR, cystic fibrosis transmembrane regulator; GFP, green fluorescent protein; PCR, polymerase chain reaction; SE, standard error.

Figure 2.

CFTR or GFP mRNA expression in lung tissue. Copies per microgram of lung DNA were measured by real-time DNA PCR in the trachea, bronchus, carina, cranial, middle, and caudal regions of five animals and one untreated control. (A, B) Are measurements taken in each lung region of the five animals. (C, D) Represents the measurements taken in all of the five lung segments of each animal number. (E, F) Are the summary data of all lung segments of four animals (#34, 45, 52, 62) sacrificed at 90 days and one animal (#22) sacrificed at 1 year. CFTR or GFP mRNA expression in liver and pancreas tissue. Copies per microgram of lung DNA were measured in the liver and pancreas of five animals and one untreated control. (G, H) Are measurements taken in one liver sample of each of the five animals. (I, J) Are the summary data of all the livers of four animals sacrificed at 90 days and animal #22 sacrificed at 1 year. (K, L) Are measurements taken in the pancreas of each of the five animals. (M, N) Are the summary data of all the pancreas of four animals sacrificed at 90 days and animal #22 sacrificed at 1 year. Dots represent each independent measurement. Summary data are averages ± SE.

Figure 3.

CFTR expression in whole lung: (A) Western blots showing CFTR expression in total lysate samples of lung tissue (pool of trachea, bronchus, carina, cranial, middle, caudal) in a vector-treated monkey (52) sacrificed at 90 days (+AAV) and the one animal (#22) vector treated and sacrificed at 1-year (+AAV-1Y) versus a nontreated (#30) control monkey (−AAV). (B) Columns represent the means ± SE of CFTR expression in lung tissue from the one control monkey (−AAV); four vector-treated animals (#34, 45, 52, 62) sacrificed at 90 days (+AAV) and from the one animal vector treated and sacrificed at 1 year (+AAV-1Y). (C) GFP expression in whole lung: Western blots showing GFP expression in total lysate samples of lung tissue (pool of trachea, bronchus, carina, cranial, middle, caudal) in a vector monkey (#34) sacrificed at 90 days (+AAV) and the one animal (#22) vector treated and sacrificed at 1-year (+AAV-1Y) versus a nontreated (#30) control monkey (−AAV). (D) Columns represent the means ± SE of GFP expression in lung tissue from the one control monkey (−AAV); four vector-treated animals sacrificed at 90 days (+AAV) and from the one animal vector treated and sacrificed at 1 year (+AAV-1Y). (E) CFTR expression in lung (trachea, bronchus, and carina) surface and basal cells: confocal images of lung tissue stained for CFTR (red) or cytokeratin 5 (KRT5-green). The merged image is only shown. (F) Summary of CFTR expression as red-fluorescence mean intensity. The representative control image (−AAV) and the treated image (+AAV) are from monkey #45. The 1-year animal (+AAV-1Y). (G) GFP expression in the lung (trachea, bronchus, and carina) surface and basal cells: confocal images of lung tissue sections, stained for GFP (green) and cytokeratin 5 (KRT5-red). The merged image is only shown. (H) Summary of GFP expression as green-fluorescence mean intensity. The representative control image (−AAV) is from monkey #30, and the treated image is from monkey #62 (+AAV) and 1-year animal (#22) (+AAV-1Y). Three individual images were captured for each animal sample. Two random areas were analyzed from each image, and at least six values were averaged for each monkey. The scale bar is 10 μm. Arrows are included to highlight the luminal surface. One-sample t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Color images are available online.

Figure 4.

CFTR or GFP expression in hepatic duct epithelial cells: (A, B) Images of tissue stained for CFTR (red) and GFP (green). (C, D) Summary of CFTR or GFP expression as fluorescence mean intensity. CFTR and CK7 colocalization in hepatic duct epithelial cells: (E) Images of liver tissue stained for CFTR (red) and CK7 (green). (F) Summary of CFTR colocalization with CK7 as Pearson's correlation coefficient. GFP and CK7 colocalization in hepatic duct epithelial cells: (G) Images of liver tissue stained for GFP (green) and CK7 (red). (H) Summary of GFP colocalization with CK7 as Pearson's correlation coefficient. Statistical analysis was performed using a one-sample t-test. **p < 0.01, ****p < 0.0001. −AAV; +AAV; and AAV1Y as in Fig. 3. Arrows are included to highlight the luminal surface. Color images are available online.

Figure 5.

CFTR or GFP expression in pancreatic duct epithelial cells: (A, B) Images of tissue stained for CFTR (red) or GFP (green). (C, D) CFTR or GFP expression as fluorescence mean intensity. CFTR and chymotrypsin colocalization in pancreatic duct epithelial cells: (E) Images of tissue stained for CFTR (red) and chymotrypsin (green). (F) Summary of CFTR colocalization with chymotrypsin as Pearson's correlation coefficient. GFP and chymotrypsin colocalization in pancreatic duct epithelial cells. (G) Images of tissue stained for GFP (green) and chymotrypsin (red). (H) Summary of GFP colocalization with chymotrypsin as Pearson's correlation coefficient. Statistical analysis was performed using a one-sample t-test. ***p < 0.001, ****p < 0.0001. Scale bar is 10 μm. AAV; +AAV; and AAV1Y as in Fig. 3. Arrows are included to highlight the luminal surface. Color images are available online.

Figure 6.

Neutralizing antibody: the neutralizing antibody titer increased significantly after the second vector instillation and continued to increase thereafter. (A) N = samples from five monkeys. The data show large variation among the monkeys and are not statistically significant when compared with day 0 using ANOVA followed by a multiple comparisons test. (B–E). Ex vivo IFN-γ ELISPOT assays. For AAV1: representative raw data are shown for each of the five monkeys. VP1 capsids were divided into three groups (A–C). Data are reported as the number of SFCs per million PBMCs at each of the time points indicated. Each point represents PBMCs from a single monkey stimulated with an AAV1 peptide pool. Each graph depicts the results from each peptide pool. PBMCs were assessed using an IFN-γ ELISPOT assay (see the Methods section) by stimulation with the respective AAV peptide pools or medium alone (no AAV = no stimulation, NS). A result >55 (marked by an arrow) was considered to be positive. Some of the data points had high backgrounds and were not included as positive (see Supplementary Table 4 for details). PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell. Color images are available online.