116. Functional Comparison of Four CFTR rAAV Vectors with Alternative Transcriptional Elements in Cell Lines and Polarized Human CF Airway Epithelia

Liang Zhang; Benjamin Dutzar; Keith Munson; Barrie Carter; John F Engelhardt

doi:10.1016/j.ymthe.2005.06.121

The limiting packaging capacity (<5 kb) of recombinant adeno-associated virus (rAAV) has restricted the use of large promoters for the delivery of the cystic fibrosis transmembrane conductance (CFTR) cDNA in gene therapy of cystic fibrosis (CF). The relatively large size of the CFTR cDNA (4.5 kb) leaves little space for conventional, strong, promoters to enhance transcription. In clinical trials, the promoter activity of the ITR has been used to drive expression of CFTR. In the present study, we sought to compare different regulatory elements within rAAV2 vectors for their ability to express CFTR mRNA and functionally correct CFTR chloride channel defects. The vectors used in our study included: the current ITR promoter vector used in clinical trials (AVtgCF), a vector with a 83-bp synthetic promoter (AVCF83), a vector utilizing the ITR promoter and a 55-bp consensus sequence optimized intron derived from the rabbit Beta-globin gene (AV55globinCF), or a vector with a synthetic TATA box upstream of the 55-bp Beta-globin gene intron (AVmTATA55globinCF). These vectors were initially compared for their ability to express vector-derived CFTR mRNA following infection of HeLa and IB3 cells. Results from this analysis demonstrated significantly improved mRNA expression for most vectors as compared to the ITR-driven CFTR vector (AVtgCF). The approximate fold increase over that seen with AVtgCF was as follow: AVCF83: 1.1-fold in HeLa and 4.1-fold in IB3; AV55globinCF: 5.7-fold in HeLa and 42-fold in IB3; and AV-mTATA55globinCF: 4.7-fold in HeLa and 32-fold in IB3. These findings suggested that incorporation of the beta-globin intron might significantly improve CFTR correction in polarized human CF airway epithelia. Following apical infection of polarized CF airway epithelia, these regulatory elements showed improvements in the expression of CFTR mRNA as compared to AVtgCF (2.3-fold for AVCF83, 4.0-fold for AV55globinCF, and 3.3-fold for AVmTATA55globinCF). The functional correction of CFTR-mediated chloride transport was then assessed for each vector type as the cAMP-sensitive short-circuit current (Isc) in response to IBMX (100 μM) and forskolin (10 μM). The differences in the level of functional correction between the alternative promoter vectors and AVtgCF correlated with the increases in vector-derived CFTR mRNA. The delta-Isc in response to IBMX/forskolin for the AVCF83, AV55globinCF and AVmTATA55globinCF groups were 1.59-, 1.48-, and 1.83-fold higher than that observed in the AVtgCF group, respectively. Additionally we examined the kinetics of expression of AVtgCF and AV55globinCF following intranasal delivery to BALB/c mice. CFTR mRNA expression paralleled the in vitro data. AV55globinCF had higher levels of expression at all time points through 90 days ranging from 3-fold to greater than 10-fold. These studies provide further improvements in CFTR AAV vector design.

116. Functional Comparison of Four CFTR rAAV Vectors with Alternative Transcriptional Elements in Cell Lines and Polarized Human CF Airway Epithelia

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