Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes

Abstract

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.

Figure 1

Locations of deletions made in the CFTR gene; 16 of the 20 deletions are shown. The CFTR gene is represented by patterned boxes. The gray regions represent loop regions, solid black boxes indicate transmembrane regions, and the dotted oval represents the globular portion of the R-domain. The two nucleotide binding domains are represented by dashed boxes. Deletions are represented by horizontal bars. Black bars represent internal deletions. Gray bars and hatched bars represents N- and C-terminal truncations, respectively. The numbers indicate the sizes of the deletion in nucleotides. Deletions that retain CFTR function are enclosed.

Figure 2

Iodine efflux assays of CFTR constructs stably transfected into human 293 cells. Samples were taken at 1-min intervals. Forskolin was added at the dashed vertical line. The slope of the initial increase in rate of loss was taken as an index of CFTR activity. The ΔF508 is a negative control. WT is the 4.7-kb wild-type CFTR cDNA, and S2 is the cDNA containing the entire coding sequence but with only a 30-bp untranslated region at the 5′-end.

Figure 3

Comparison of promoter strengths. The promoter activities are represented by CAT activities indicated on the vertical axis. Each promoter is indicated on the horizontal axis. CATBasic is a CAT gene containing plasmid without a promoter. In AVCAT, the CAT gene is expressed from the AAV ITR. P5CAT contains the AAV ITR and P5 promoter. SVCAT and CMVCAT express the CAT gene from SV40e and CMVie promoter, respectively.

Figure 4

Patch–clamp studies of HeLa cells infected with rAAV vectors containing functional CFTR cDNAs. (Left, A) Excised membrane patch of cells infected with AAVP5ΔC2. PKA treatment readily activated a current carried by multiple channels in this patch. I/V protocol applied during PKA treatment (middle graph) and I/V relations (right graph) show that the current is time- and voltage-independent. The total activated conductance in this patch was 64pS, equivalent to 16–20 CFTR channels. (B and C) Cell-attached, single-channel patch–clamp recording on HeLa cells infected with AAVp5ΔC2 or AAVp5D4.1C2, respectively. Cells were stimulated with forskolin and clamped to potentials as indicated. In both B and C, top and bottom curves show recordings containing at least three channels. Right graphs show linear current–voltage relations yielding a single channel conductance of 6.1pS + 0.27 (n = 6) for AAVp5ΔC2 or 8.7 pS + 0.16 (n = 6) for AAVP5D4.1C2. Whole-cell patch–clamp analysis of AAV–CFTR transduced cells.

Figure 5

Patch–clamp recording of cells transduced with (A) AAV-CFTR in which a full length CFTR is driven by ITR, (B) AAVp5ΔC2, and (C) AAVp5D4.1C2. The durations of forskolin treatment are indicated. Downward curves indicate forskolin-activated Cl⁻ current. (D) Whole-cell current–voltage relations of each CFTR Cl⁻ channel, triangle: ITR–CFTR; open circle: AAVp5ΔC2; filled circle: AAVp5D4.1C2. Minimal conductance was detected in cells transduced with AAV–CFTR, and highest conduction was detected in cells transduced with AAVp5D4.1C2 as indicated by the slope of the I/V curve.