Expression of human alpha1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs

Abstract

We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.

Figure 1

Human AAT expression in normal and immunodeficient mice. (a) hAAT in blood plasma from C57BL/6 mice that received 5 × 10¹⁰ (closed symbols) or 1 × 10¹¹ vg (open symbols) of the ACAGhAAT(AAV6) vector. (b) Plasma hAAT in C57BL/6 Rag2 immunodeficient mice that received 10¹¹ (open symbols) or 2 × 10¹¹ vg (closed diamond) of the ACAGhAAT(AAV6) vector, or 10¹¹ vg of an AAV6 vector encoding alkaline phosphatase [ARAP4(AAV6)] (closed circles) as a negative control. AAT, α1-antitrypsin; AAV, adeno-associated virus; hAAT, human AAT.

Figure 2

Immunostain for hAAT expression in mouse lungs. Immunodeficient C57BL/6 Rag2 mice were given 10¹¹ vg of ACAGhAAT(AAV6) or ARAP4(AAV6) as a negative control and lungs were harvested at 3 months and stained with an antibody specific for hAAT in formalin-fixed mouse lung. Expression (purple stain) was seen in airway epithelium (top row), alveolar cells (bottom row), and in some vascular cells (data not shown). AAV6, adeno-associated virus type 6; hAAT, human α1-antitrypsin.

Figure 3

AP expression in lungs of dogs and mice following administration of ACAGAP(AAV6). ACAGAP(AAV6) or saline was administered to lungs of animals and lungs were stained for AP expression (purple/black stain) 2 months later. AAV6, adeno-associated virus type 6; AP, alkaline phosphatase.

Figure 4

Immune response to AAV vector and AP protein. (a) Neutralization of an AAV6 vector by 1:100 dilutions of blood plasma and (b) measurement of antibodies against AP by ELISA of plasma harvested at the indicated times from a dog given ACAGAP(AAV6). AAV6, adeno-associated virus type 6; AP, alkaline phosphatase.

Figure 5

Expression of hAAT in normal and immunosuppressed dogs. (a) Serum samples from normal and immunosuppressed dogs taken at various times after vector administration were analyzed by ELISA for hAAT levels. (b) Expression of hAAT in ELF of the immunosuppressed dog. Three BAL samples from each lung at each time point were analyzed by ELISA to determine hAAT concentration, and ELF levels were calculated from urea assays to determine fold dilution of ELF in BAL fluid. The means ± SD are shown. (c) BAL fluids from immunosuppressed dogs given ACAGhAAT(AAV6) or ACAGAP(AAV6) were obtained at day 44 and day 60, respectively, and western analysis was done to detect expression of hAAT. Ten µl portions of three BAL fluid samples obtained sequentially from the right (R) and left (L) lungs were analyzed. Ten nanogram of an hAAT standard is shown in the left lane. The arrow marks the position of hAAT. Canine AAT is shown in the lane containing the sample from the ACAGAP(AAV6)-treated dog. The rabbit antibody is specific for human AAT in ELISA. In denaturing conditions such as that used for the western analysis, the antibody also recognizes canine AAT. AAT, α1-antitrypsin; AAV6, adeno-associated virus type 6; BAL, bronchial alveolar lavage; ELF, epithelial lining fluid; ELISA, enzyme-linked immunosorbent assay; hAAT, human AAT.

Figure 6

Expression of hAAT in two immunosuppressed dogs. (a) Expression of hAAT in serum. (b,c) Expression of hAAT in epithelial lining fluid (ELF). hAAT, human α1-antitrypsin.