Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Abstract

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.

FIG. 1

AAV vectors. AAV TRs (hatched boxes), coding regions (open boxes), promoters and polyadenylation sequences (solid boxes), and transcriptional start sites (arrows) are indicated. Abbreviations: TR, AAV TR; MLV, Moloney murine leukemia promoter and enhancer; RSV, RSV promoter and enhancer; CMV, cytomegalovirus promoter and enhancer; SV40, simian virus 40 early promoter; SV40-pA, AAV-pA, and hGH-pA, simian virus 40, AAV, and human growth hormone polyadenylation sequences, respectively; AP, human placental AP; β-gal, bacterial β-Gal; neo, neomycin phosphotransferase. There are three related AxLAPSN vectors, where x represents the AAV type from which the vector was derived, that contain either AAV2, AAV3, or AAV6 TRs. All other vectors contain AAV2 TRs.

FIG. 2

Transduction by AAV vectors in cultured cells. Transduction rates in COS-1 and BHK-21 cells are expressed as the number of AP⁺ FFU per vector genome. The CWRAP vector contains AAV2 TRs and was pseudotyped with AAV2, -3, or -6 Rep and Cap proteins. The AxLAPSN vectors contain TRs from the same AAV serotype as that of the capsid proteins.

FIG. 3

Histochemical detection of AP expression in mouse lungs 1 month after vector exposure. Naive mouse lungs (control) did not exhibit AP⁺ cells, while AAV vector-treated lungs exhibited AP⁺ alveolar cells (arrowheads), airway epithelial cells (small arrows), and smooth muscle cells (large arrows). Lungs given AAV2 or -6 pseudotype vectors (10¹¹ genome-containing particles) are indicated. Original magnifications: top row, ×80; middle row and bottom row (left panel), ×100; bottom row (middle and right panels), ×200.

FIG. 4

AAV-neutralizing antibody titers in serum from mice after administration of AAV vectors. AAV2-, -3, or -6 pseudotype vectors were incubated with dilutions of pooled sera from mice given the same pseudotype vectors 3 weeks previously. The relative titer was determined by comparison to the titer of the vector incubated without mouse serum. The asterisks indicate that no stained cells were observed, and the underlying bars represent the limit of sensitivity of the assay in these cases. Duplicate wells in three repeat assays were scored. Mean values ± standard deviation are given.

FIG. 5

AAV-neutralizing antibody titers in mouse serum after administration of AAV2- or AAV6-pseudotyped vector. AAV2- or AAV6-pseudotyped ARAPGH vectors were incubated with dilutions of sera from mice given the AAV2- or AAV6-pseudotyped CWCZn vectors 3 weeks previously (n = 4 per group; see Table 2). The relative titer was determined by comparison to the number of AP⁺ FFU/ml of sample, in which vector was incubated with serum from a saline-treated mouse (n = 4). Duplicate wells for each animal serum were scored. Mean values ± standard deviation are given.