A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

Abstract

Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds Ϸ4,900 -5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTR⌬R). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate͞early (CMVie) enhancer͞promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTR⌬R transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl ؊ transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer.

Directed evolution of adeno-associated virus to an infectious respiratory virus

“…4F). This is in contrast to AAV2, which does not correct this model, and AAV5, which requires a substantially higher multiplicity of infection (MOI) (26). Furthermore, a dose-response with AAV2.5T-CFTR⌬R in CF epithelia showed, surprisingly, that as little as 10 vg/cell was sufficient for chloride transport correction, and a dose of 100-1000 vg/cell was equivalent to adenovirus carrying the wild-type CFTR gene (Fig.…”

“…We next investigated potential therapeutic applications of AAV2.5T by analyzing whether it could efficiently express CFTR and correct the CF chloride transport defect. CF airway epithelia were transduced with AAV2.5T encoding a shortened CFTR expression cassette (CFTR⌬R, 50,000 vg/cell), then analyzed in Ussing chambers 30 days posttransduction, as previously described (26,27). Normal epithelia demonstrated chloride transport (Fig.…”

“…CFTR transgene in AAV2.5T contains a deleted portion of the R domain (CFTR⌬R, 708Ϫ759), a shortened CMV immediate/early (173CMVie) enhancer/ promoter, and minimal poly(A) signal as described by Ostedgaard et al (26,27). Ad-CFTR contained the full length CMV promoter and wild-type CFTR.…”

“…The short-circuit current (Isc) was measured, using a Cl Ϫ gradient in modified Ussing chambers (Jim's Instruments) as previously described (26). Epithelia were treated with forskolin (10 Ϫ5 M) and IBMX (10 Ϫ4 M) for 18 -24 h before study in Ussing chambers to minimize basal CFTR current.…”

Long-term persistence of gene expression from adeno-associated virus serotype 5 in the mouse airways

“…Persistent expression could be useful for long-term therapy of chronic lung diseases such as CF; and recent studies have shown that rAAV5/5 vectors can be used to correct CF chloride transport defects in airway epithelial cells grown in culture. 51 However, data presented here suggest that when rAAV5/5 is delivered to the mouse lung, it is unlikely to efficiently transduce airway cells which are the target for gene therapy of CF lung disease. In addition, animals demonstrating persistent luciferase expression for many months demonstrated a high level of anti-rAAV5 antibodies, including neutralizing antibodies, in serum (Figure 3), suggesting that it might be difficult to repeatedly administer rAAV5/5 for treatment of chronic conditions.…”