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A Shortened Adeno-Associated Virus Expression Cassette for CFTR Gene Transfer to Cystic Fibrosis Airway Epithelia

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A Shortened Adeno-Associated Virus Expression Cassette for CFTR Gene Transfer to Cystic Fibrosis Airway Epithelia A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia Lynda S. Ostedgaard*†, Tatiana Rokhlina*†, Philip H. Karp*†, Philip Lashmit‡, Sandra Afione§, Michael Schmidt§, Joseph Zabner†, Mark F. Stinski‡, Jay A. Chiorini§, and Michael J. Welsh*†¶ʈ *Howard Hughes Medical Institute and Departments of †Internal Medicine, ‡Microbiology, and ¶Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242; and §National Institute of Dental and Craniofacial Research, National Institutes of Health, 10 Center Drive, Building 10͞Room 1N113, Bethesda, MD 20892 Contributed by Michael J. Welsh, December 30, 2004 Adeno-associated viruses (AAVs) such as AAV5 that transduce (17). With the addition of the two AAV inverted terminal airway epithelia from the apical surface are attractive vectors for repeats (ITRs), 3Ј and 5Ј untranslated regions, and enhancer͞ gene transfer in cystic fibrosis (CF). However, their utility in CF has promoter elements, the expression cassette can easily surpass the been limited because packaging of the insert becomes inefficient packaging limits. A number of strategies have been explored to when its length exceeds Ϸ4,900–5,000 bp. To partially circumvent circumvent this limitation. Some studies have used short endog- this size constraint, we previously developed a CF transmembrane enous AAV sequences as a promoter (with or without proteo- conductance regulator (CFTR) transgene that deleted a portion of somal inhibitors to increase expression) with variable results in the R domain (CFTR⌬R). In this study, we focused on shortening the differentiated airway epithelia (15, 18, 19). Other studies have other elements in the AAV expression cassette. We found that produced AAV vectors with a CFTR cDNA that deletes the N portions of the CMV immediate͞early (CMVie) enhancer͞promoter terminus (15), and other deletions have been proposed for AAV could be deleted without abolishing activity. We also tested vectors (16, 18). However, their function in airway epithelia various intervening sequences, poly(A) signals, and an intron to requires additional study. A different approach has involved develop an expression cassette that meets the size restrictions coadministration of two independent vectors followed by inter- imposed by AAV. We then packaged these shortened elements molecular joining of the DNA or RNA to generate expression with the CFTR⌬R transgene into AAV5 and applied them to the (20–23). Although promising, such strategies require that each apical surface of differentiated CF airway epithelia. Two to 4 weeks cell be infected with two different vectors. later, the AAV5 vectors partially corrected the CF Cl؊ transport In an earlier study, we developed a transgene encoding a defect. These results demonstrate that a single AAV vector can CFTR ClϪ channel with a shortened R domain (deleting residues complement the CF defect in differentiated airway epithelia and 708–759) (24). For simplicity, we call this CFTR⌬R. The thereby further the development of effective CF gene transfer. CFTR⌬R variant showed normal biosynthesis, targeting to the apical membrane, and ClϪ channel activity. Like the wild type, CMV ͉ promoter ͉ gene therapy CFTR⌬R complemented the ClϪ transport defect in differen- tiated CF airway epithelia in vitro and in nasal mucosa of CF mice ystic fibrosis (CF) is an autosomal recessive disease resulting in vivo. Although shortening the CFTR cDNA takes us closer to Cfrom mutations in the gene encoding the CF transmembrane a workable expression cassette, by itself it is not sufficient to conductance regulator (CFTR) ClϪ channel (1). Airway disease meet the size constraints imposed by AAV. In this study we causes most of the morbidity and mortality in CF. Transfer of the endeavored to minimize the length of the other components of CFTR cDNA to airway epithelia could potentially offer a new an AAV expression cassette. After the CFTR cDNA, the next prevention and treatment for the people who suffer from this largest element is the enhancer͞promoter. Therefore, we at- disease (for review see refs. 2–5). Previous studies established tempted to design a small but effective promoter. In addition, we the feasibility of gene transfer to CF airway epithelial cells, both minimized the size of other required elements in the expression in vitro and in people with CF. However, such studies also vector. Then we tested the hypothesis that the expression revealed numerous barriers, which have so far prevented gene cassette would function in well differentiated human airway transfer from becoming an effective treatment. epithelia and correct the CF ClϪ transport defect. Adeno-associated viruses (AAVs) overcome some of these barriers and have advantages that make them attractive vectors Materials and Methods for CF gene transfer (5–10). AAVs cause no known human Plasmids. We began with pCMV5, which contains a full-length disease, and AAV vectors have a good safety record. AAV CMV immediate͞early (CMVie) enhancer͞promoter with a vectors retain no viral genes, thereby minimizing cell-mediated multiple cloning site and a human growth hormone (hGH) immune responses. They transduce nondividing cells, such as poly(A) signal (25). We inserted the coding sequence for ␤-gal airway epithelia. Transgene expression from AAV vectors can be from pCMV␤ (Clontech) to make pCMV5–␤-gal and used this prolonged. In addition, recent studies have identified AAV types for making all of the other plasmids. that can efficiently transduce well differentiated human airway The truncated promoter constructs 348CMV and 222CMV epithelia, including AAV5, which has a receptor on the apical surface of airway epithelia (11–13). A limitation of AAV vectors is the relatively small AAV Abbreviations: CF, cystic fibrosis; CFTR, CF transmembrane regulator; AAV, adeno- genome. Studies testing the insert size suggest that 4,100–4,900 associated virus; ITR, inverted terminal repeat; CMVie, CMV immediate͞early; hGH, human growth hormone; IVS, intervening sequence; SPA, synthetic poly(A) signal; moi, multiplicity bp is the optimal genome size for packaging (14). Other studies of infection. and our own unpublished data also suggest that packaging ʈTo whom correspondence should be addressed at: Howard Hughes Medical Institute, 500 becomes very inefficient whenever insert sizes exceed 4,900– EMRB, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 5,000 bp (15, 16). This poses a problem for genes with large 52242. E-mail: [email protected]. coding sequences, such as CFTR, which has a cDNA of 4,450 bp © 2005 by The National Academy of Sciences of the USA 2952–2957 ͉ PNAS ͉ February 22, 2005 ͉ vol. 102 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409845102 Downloaded by guest on October 2, 2021 were made by excising the enhancer region from Ϫ587 to Ϫ348 or Ϫ222, respectively. We produced construct ⌬470–173 by deleting nucleotides 470–173 from pCMV5–␤-gal. We generated construct 173CMV by deleting nucleotides from Ϫ587 to Ϫ173 and construct 113CMV by deleting nucleotides from Ϫ587 to Ϫ113. All truncated promoter constructs in plasmids retained nucleotides Ϫ600 to Ϫ587, which contain no known transcription factor binding sites. These nucleotides were removed when we generated the AAV5 vector. Fig. 1. AAV5 genome and expression cassette. The schematic shows the We varied the length (44, 51, and 69 bp) of the intervening relative length of the left and right ITRs and the coding sequence for the Rep sequence (IVS) between the transcription start site and the and Cap genes. Also shown is the relative size of the elements of a CMVie CFTR translation start site of 173CMV and 113CMV. The 19S͞16S expression cassette. intron (96-bp) from pCMV␤ (Clontech) was excised with intact splice sites and ligated into 173CMV and 113CMV (with a 44-bp Results and Discussion IVS). The synthetic poly(A) signal (SPA) (26) was introduced An AAV5 Expression Cassette. Fig. 1 graphically depicts the into pCMV5–␤-gal, 173CMV, and 113CMV by using PCR 4,642-bp AAV5 genome (27), including the left and right mutagenesis (QuikChange, Stratagene). All constructs were flanking ITRs and the sequences encoding Rep and Cap genes. sequenced from promoter to poly(A) signal for verification. Below that we show a potential starting point for a CFTR expression cassette, which includes the viral ITRs, a CMVie AAV Vectors. To make AAV5-173CMV-CFTR⌬R and AAV5- enhancer͞promoter, the IVS between the promoter and the 113CMV-CFTR⌬R, we excised CFTR⌬R from pAd-CFTR⌬R transgene, the CFTR cDNA, and a poly(A) signal. The 6,065-bp (24) and ligated it into 173CMV and 113CMV in place of the ␤ total length exceeds the packaging capacity of AAV (refs. 14–16 -gal coding sequence. The NotI restriction sites were engi- and unpublished observations). Substituting the recently devel- neered upstream of the 173CMV and 113CMV promoters and oped shortened CFTR transgene, CFTR⌬R (4,287 bp) (24), downstream of the SPA in 173CMV-CFTR⌬R and 113CMV- ⌬ reduced the cassette length to 5,902 bp. However, this still CFTR R. We also engineered a NotI site into the multiple exceeds the packing limits. To achieve additional reduction, we cloning site of the AAV5 plasmid (27, 28) to insert 173CMV- first focused on the next largest element in the expression CFTR⌬R and 113CMV-CFTR⌬R. AAV5-173CMV-CFTR⌬R ͞ ⌬ cassette, the enhancer promoter. and AAV5-113CMV-CFTR R were produced as previously We began these studies by using the CMVie enhancer͞ described (12) with the following modification: vectors were promoter because it has been successfully used in both viral and produced by transfection of 293T cells and harvested 48–60 h nonviral vectors, and it drives transgene expression in many posttransfection. DNase-resistant particle titers were deter- different types of cells and tissues, including airway epithelia (24, mined by quantitative PCR (29) with the following primers: 32–35).
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