Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans

Abstract

The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans.

Figure 1.

The first demonstration of in vivo gene expression from an rAAV vector in a living animal was this RT-PCR from rabbit bronchial epithelial cells harvested at intervals from 3 days to 6 months after instillation of rAAV2-CFTR through a fiberoptic bronchoscope in a 1993 publication. A Southern blot (above) and an ethidium bromide-stained gel (below) depict the products of RT-PCR on total cellular RNA extracted from lung tissue homogenates collected 3 days, 10 days, 3 months, and 6 months after vector administration. The lung homogenate harvested from the vehicle-treated animal (vehc), along with the reactions without reverse transcriptase (− RT), act as control. rAAV, recombinant adeno-associated virus; CFTR, cystic fibrosis transmembrane regulator; RT, reverse transcriptase. [Reproduced with permission from Flotte et al.]

Figure 2.

Discovery of episomal persistence in vivo in the rhesus lung. [Reproduced with permission from Afione et al.] Primary rhesus bronchial epithelial cells harvested from animals after in vivo AAV-CFTR delivery were cultured and evaluated by Southern blot with (+) and without (−) Ad5 and AAV2 supernatants. Lane G shows the genomic DNA after BamH1 digestion. The left Southern blot was analyzed by an internal specific CFTR cDNA probe and the right by an AAV2 internal specific probe. AAV-CFTR was determined to persist episomally, as Hirt supernatants without AAV2 resulted in episomal bands with sizes of AAV-CFTR and wild-type AAV2 replicating-form dimers.

Figure 3.

Correlation between rAAV2-CFTR gene transfer and restoration of cAMP-activated chloride flux (A). Cells were harvested from rAAV2-CFTR trial patients after in vivo gene delivery and then grown in primary culture for analysis. Primary cultures taken by bronchial or nasal brushings (B1, L9, R9) or from nasal polypectomy (PL9, PR9) were analyzed by DNA PCR for rAAV2-CFTR sequences (B). The primary culture from L9 (left nasal sample of patient 9), which had shown strong positive DNA signal, was then analyzed by whole cell current analysis after stimulation with a cAMP activation cocktail. [Reproduced with permission from Flotte et al.]