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(Chest. 2002;121:98S-102S.)
© 2002 American College of Chest Physicians

Recombinant Adeno-Associated Virus Gene Therapy for Cystic Fibrosis and {alpha}1-Antitrypsin Deficiency*

Terence R. Flotte, MD

* From the Powell Gene Therapy Center, University of Florida, Gainesville, FL.

Correspondence to: Terence R. Flotte, MD, University of Florida Gene Therapy Center, Academic Research Building, Room R1-191, 1600 SW Archer Rd, Gainesville, FL 32610-0266

Recombinant adeno-associated vectors (rAAVs) have theoretical advantages as vehicles for human gene therapy because they are based on a virus that is nonpathogenic and has a natural mechanism for long-term persistence in human cells.1 2 3 The ability to manipulate single genes also has the potential to be a powerful research tool in animal models of human diseases. Our laboratory has developed rAAVs for the therapy of the two common single-gene disorders that affect the lung, cystic fibrosis (CF) and {alpha}1-antitrypsin (AAT) deficiency.4 5 6 In addition, we have developed vectors for either the constitutive or inducible expression of the important anti-inflammatory cytokine, interleukin (IL)-10, which could be therapeutically useful in patients with inflammatory diseases like CF, type-I diabetes mellitus, or inflammatory bowel disease. Preparations of rAAV-cystic fibrosis transmembrane conductance regulator (CFTR), rAAV-AAT, and rAAV-IL-10 have been extensively characterized in cell culture systems,7 8 9 animal models,4 10 11 and early phase I trials in CF patients.12 13 14 15 16 Studies with rAAV-CFTR and rAAV-IL-10 in CF bronchial cell cultures have been used to examine the functional consequences of CFTR complementation and IL-10 expression. In vivo studies in mice,6 17 18 rabbits,4 19 and monkeys10 11 with each of these vectors have demonstrated long-term gene transfer and expression (ie, > 6 months for CFTR and > 18 months for AAT) without any detectable pathologic findings. These studies also have demonstrated that therapeutic levels of AAT can be achieved in mice by delivery to muscle, liver, or lung. Interestingly, studies of both rAAV-CFTR in monkeys and rAAV-AAT in mice indicate that the vector DNA persists in long strings or concatemers that are episomal, that is, physically separate from the host cell chromosome (in contrast with the naturally occurring form of the virus) and that host cell factors, such as the DNA-dependent protein kinase, play arole in this process.10 18 20 21 22 This could allow for the DNA to persist without incurring the potential risk of disrupting host cell genes. Phase I trial results in CF patients are also encouraging in that DNA transfer and expression have been observed in the sinuses and the lung without vector-related toxicity. A phase II aerosol trial of rAAV-CFTR is planned in CF patients, as is a phase I trial of rAAV-AAT in AAT-deficient patients.


   Materials and Methods
TOP
 Materials and Methods
Results
 Discussion
 References
 
Generation of rAAV Vectors
rAAV vectors were prepared by generating proviral rAAV vector plasmids deleted for the viral proteins Rep and Cap, and by substituting the gene of interest (CFTR, AAT, or IL-10) between the rAAV2 inverted terminal repeats (Fig 1 ) with appropriate promoter and polyadenylation signal sequences. The packaging limit for such cassettes is approximately 5 kb. rAAV genomes are packaged into infectious virions using a cotransfection technique in which the vector plasmid is cotransfected into human embryonic kidney cells (HEK-293) along with the helper plasmid pDG encoding the rAAV2-rep and cap genes, as well as the necessary adenovirus helper functions.23 rAAV virions are released by lysing cells 48 to 72 h after the transfection and are purified by using a combination of density gradient ultracentrifugation and/or affinity chromatography.24 All vector preparations are characterized with respect to their physical titer (total deoxyribonuclease-resistant particles [drp]) and their biological titer (infectious units), and they are screened for the presence of any contaminating replication-competent AAV prior to use in transduction experiments.



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  		Figure 1.. Vectors were prepared by generating rAAV vector plasmids deleted for proteins Rep and Cap and substituting the gene of interest with appropriate promoter and polyadenylation signal sequences. ITR = inverted terminal repeat.

 
In Vitro Studies
For in vitro studies of CFTR complementation, the immortalized CF bronchial epithelial cell line IB3–1 (genotype deltaF-508/W1282X) was cultured in LHC-8E medium with 10% fetal bovine serum (37°C; 5% CO2).25 The constructs to be tested either were transfected using a reagent (Lipofectin; Invitrogen; Carlsbad, CA) or were infected at a multiplicity of infection ranging from 100 to 10,000 physical particles per cell. CFTR expression was assessed using the previously described 36Cl- isotope tracer efflux assay or was excised using the inside-out patch clamp analysis. The proinflammatory phenotype was studied by examining the IL-8 response to lipopolysaccharide (LPS) stimulation. In vitro AAT and IL-10 responses were characterized in the murine myoblast cell line C2C12.

In Vivo Studies
In vivo studies of rAAV-CFTR were performed in New Zealand white rabbits (Pasteurella-free, Hazelton; weight, 3 kg) with vector doses delivered to the posterior basilar segment of the right lower lobe under ketamine-xylazine sedation through the suction port of a 3.5-mm fiberoptic bronchoscope (Olympus; Tokyo, Japan). Similar rAAV-CFTR studies also were performed in rhesus macaques (Macacca mulatta; weight range, 3 to 4 kg). Studies of rAAV-AAT were performed in mice (C57Bl6 or C57Bl6-SCID) with vector doses administered either intramuscularly (in the quadriceps femoris muscle), intratracheally (under ketamine sedation), or by infusion into the portal vein (under aseptic conditions with tribromoethanol sedation). All studies were approved by the Johns Hopkins University or the University of Florida Animal Care and Use Committee.

Clinical Trials
Clinical trial data are summarized from institutional review board-approved studies that were performed at the General Clinical Research Centers at Johns Hopkins University, the University of Florida, and Stanford University. Adult male or female CF patients (age range, 18 to 47 years) were selected for studies if they had FVC values >= 60% and were free of colonization with pan-resistant pseudomonads, had experienced severe or recurrent hemoptysis, or had undergone recent (ie, < 1 month prior) hospitalization or IV antibiotic use. Doses of rAAV-CFTR for clinical use were prepared under cyclic guanosine 3',5'-monophosphate conditions at Targeted Genetics Corporation (Seattle, WA). Doses were administered by intranasal, bronchoscopic, intramaxillary sinus, or aerosol inhalation routes. DNA transfer efficiency was assessed by semiquantitative polymerase chain reaction. Safety assays included clinical examinations, complete blood cell counts, serum chemistries, chest radiographs, pulmonary function tests, and BAL fluid analysis for cell counts and proinflammatory cytokines.


   Results
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 Results
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In Vitro Studies
CFTR Complementation: A number of rAAV-CFTR constructs were tested for their ability to complement the CFTR defect in adenosine 3',5'-cyclic monophosphate-mediated chloride transport (Fig 2 ). As shown in Figure 2 , constructs with either the entire CFTR coding sequence or with amino-terminal truncations were able to correct both the original small (10pS) linear chloride channel defect that indicates a lack of CFTR channel activity and a secondary defect in the regulation of the higher conductance (40pS) outwardly rectifying the chloride channel. These data are helpful in that two separate functions of CFTR were defined and that CFTR minigenes could be generated that included a larger promoter, such as the cytomegalovirus (CMV)-enhanced chicken ß-actin promoter.



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  		Figure 2.. rAAV-CFTR constructs were tested for ability to complement the CFTR defect in adenosine 3',5'-cyclic monophosphate-mediated chloride transport. ATP = adenosine 5'-triphosphate; PKA = Protein kinase A. Reproduced with permission of Egan et al.7

 
Correction of the Proinflammatory CF Phenotype With rAAV-IL-10: There is a growing body of evidence to indicate that the CFTR defect is also primarily connected with a proinflammatory phenotype triggered by the mutant protein via an unfolded protein response. While there is evidence that rAAV-CFTR can correct this defect, it could be difficult to transduce a sufficient proportion of cells in the airway to reverse this effect. One option is to use a secreted anti-inflammatory molecule like IL-10. In support of this approach, IL-10-deficient mice share a very similar proinflammatory phenotype in response to Pseudomonas aeruginosa challenge as compared with CFTR knockout mice. In order to test this, we studied the secretion of IL-8 from IB3-1 (CF bronchial) cells in response to stimulation with bacterial LPS. IL-8 is the primary neutrophil chemoattractant in the CF lung and may be treated as an indication of the inflammation phenotype. As shown in Figure 3 , rAAV-IL-10 treatment of IB3-1 cells totally abrogated LPS induction of IL-8 secretion.



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  		Figure 3.. rAAV-IL-10 treatment of IB3–1 cells abrogated IL-8 secretion. GFP = green fluorescent protein.

 
rAAV-Mediated Secretion of AAT
Similar studies in C2C12 myoblasts indicated the rAAV-AAT constructs also resulted in efficient transgene expression. Secreted AAT responses were used to evaluate the various promoters, and the rAAV-CMV/ß-actin hybrid promoter (CB)-AAT construct was found to be the most efficient, as compared with the CMV, E1{alpha}, U1a, and U1b promoter constructs. The CB, CMV, and E1{alpha} promoter-containing vectors were used for subsequent in vivo studies.

In Vivo Studies
rAAV-CFTR vectors were tested in rabbits and rhesus macaques and were found to express human CFTR-messenger RNA in the lower airways at levels of approximately 1 copy per cell for > 6 months. There was no detectable pathologic finding in any of these studies. In rhesus macaques, chest radiographs, measurement of arterial blood gas levels, pulmonary function testing, and BAL fluid analyses were used to carefully examine the animals for any signs of vector-related inflammation, and none were seen. This is in marked contrast with previous and parallel studies with recombinant adenovirus vectors.

In the case of rAAV-AAT, studies were performed in C57Bl6 mice by intraportal, IM, IV, and intratracheal injection route. Representative data from the portal vein injection studies are shown in Figure 4 . These studies clearly show that a single injection of an rAAV-CB-AAT vector resulted in high-level, stable transgene expression over the life span of these animals. Once again, there was no detectable inflammation in the vector-treated animals.



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  		Figure 4.. Portal vein injection studies show that single injection of rAAV-CB-AAT (CB-AT) vector resulted in high-level, stable transgene expression. hAAT = human AAT. C-AT = CMV promoter-driven AAT vector; E-AT = elongation factor 1{alpha} promoter-driven vector; PV = portal vein; TV = tail vein. Reproduced with permission of Song et al.17

 
Clinical Trials
A total of four clinical trials of the rAAV-CFTR vector have now been completed. These encompass seven individuals with CF who were treated with doses from 6 x 104 to 1 x 1013 drp per administration to the surface of the nose, maxillary sinus, or bronchus. No vector-related adverse effects have been observed. Transgene expression has been detected at doses of 6 x 108 drp in the sinus or 1 x 1013 drp in the lung. Phase II trials are ongoing.


   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
Recombinant AAV vectors have proven to be useful for gene transfer and expression in tissue cultures, animal models, and early phase I trials in CF patients. In contrast with recombinant adenovirus vectors, the lack of inflammatory toxicity has allowed these vector studies to proceed into phase II trials in the case of CF and to approach phase I trials in AAT deficiency.

Despite these early successes, we and others have begun to define some potential limitations of this vector system. These limitations include the following: (1) the inhibitory effect of preexisting airway inflammation on rAAV transduction in the lungs26 ; (2) a relative paucity of receptors on the apical surface of airway epithelial cells27 28 ; (3) the relatively weak nature of the minimal promoters used in the first-generation rAAV-CFTR vectors8 ; and (4) the potential for adverse long-term effects from rAAV vector DNA persistence.

Each of these potential limitations is being addressed in different ways. Our group has determined that the existing barriers to rAAV in the CF airway most likely are related to neutrophil-derived {alpha}-defensins (HNP1 and HNP2) and are reversible by AAT protein delivery.26 This concept is being tested in a clinical experiment. The relative paucity of receptors is being addressed in a number of different ways. First, genetic manipulation of the rAAV2 capsid has resulted in enhanced targeting of the serpin enzyme complex receptor on IB3–1 cells (ie, CF bronchial cells).29 Alternatively, other rAAV serotypes may more efficiently target bronchial epithelial cells.30

Promoter effects are also being addressed using a number of different strategies. Our group has developed CFTR minigene constructs expressing the CFTR {Delta}-264M truncation from the very active CB or Rous sarcoma virus promoters and has seen effective increases in complementation. Meanwhile, other groups have exploited the ability of rAAV to concatemerize by packaging a vector with a superenhancer along with the rAAV-CFTR vectors with minimal promoter elements.31 32 Cotransduction of these vectors has greatly enhanced transgene expression by 100-fold to 600-fold.

Based on the very promising data from existing vectors and the prospects for improved transduction with the features described above, the future of gene therapy for monogenic lung diseases appears to be bright. In this context, the need for long-term safety studies to define any potential mutagenesis risk for rAAV is all the more pressing. The data from our laboratory strongly indicate that the bulk of rAAV DNA in the lung, muscle, and liver is episomal and that rAAV genomes interact with host cell proteins such as the DNA-dependent protein kinase in the formation of stable high-molecular weight concatemers. Additional long-term studies are clearly warranted to objectively define the potential risk posed by any vector DNA integration that does occur.


   Footnotes
 
Abbreviations: AAT = {alpha}1-antitrypsin; CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; CMV = cytomegalovirus; drp = deoxyribonuclease-resistant particles; IL = interleukin; LPS = lipopolysaccharide; rAAV = recombinant adeno-associated vector

This work was supported by the National Heart, Lung, and Blood Institute (grants HL51811 and HL59412), the National Institute of Diabetes and Digestive and Kidney Diseases (grants DK51809 and DK58327), the Cystic Fibrosis Foundation, and the Alpha One Foundation.


   References
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 Results
 Discussion
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