HMG Advance Access published March 21, 2011

Genetic Therapies for Cystic Fibrosis Lung Disease

Patrick L. Sinn1, Reshma Anthony1, Paul B. McCray, Jr. 1*

Program in Gene Therapy, Department of Pediatrics, Carver College of Medicine, The

University of Iowa, Iowa City, Iowa 52242

Department of Pediatrics
200 Hawkins Drive
The University of Iowa College of Medicine
The University of Iowa
Iowa City, IA 52242
Tel: (319) 356-4866
Fax: (319) 356-7171
Email:paulmccray@uiowa.edu

TheAuthor2011.PublishedbyOxfordUniversityPress.Allrightsreserved.
ForPermissions,pleaseemail:journals.permissions@oup.com

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*Corresponding Author

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Abstract

The aim of gene therapy for cystic fibrosis (CF) lung disease is to efficiently and safely express
the cystic fibrosis transmembrane conductance regulator (CFTR) in the appropriate pulmonary
cell types. While CF patients experience multi-organ disease, the chronic bacterial lung
infections and associated inflammation are the primary cause of shortened life expectancy. Gene
transfer-based therapeutic approaches are feasible, in part, because the airway epithelium is

and the development of novel vectors, as well as emerging technologies and new animal models,
are propelling exciting new research forward. Here we review recent developments that are
advancing this field of investigation.

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directly accessible by aerosol delivery or instillation. Improvements in standard delivery vectors

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INTRODUCTION
Since the discovery of the CFTR gene in 1989, cystic fibrosis (CF) has been in the sights of
scientists hoping to prevent or delay the onset and progression of lung disease through the use of
gene transfer. While loss of CFTR function adversely affects multiple cells and tissues,
progressive lung disease accounts for the majority of the morbidity and mortality. For this
reason, most effort in the field has focused on gene transfer to the airways. CFTR is expressed in
multiple epithelial cell types in the surface and submucosal glands of the conducting airways

channel that is regulated by nucleotides and phosphorylation (1-3). Loss of CFTR function likely
alters the volume and composition of airway secretions, but key details of the molecular
pathogenesis of CF lung disease remain the subject of intense study.

Complementation of this autosomal recessive disease by the delivery of a CFTR cDNA to the
airway epithelium with a viral or non-viral vector holds appeal, as the envisioned target cells are
accessible via direct instillation or aerosol delivery approaches. Furthermore, early studies
indicated that complementation of as few as 6-10% of CF epithelia generated wild type levels of
chloride transport in vitro (4). However, since the completion of the first human gene therapy
trial in 1993, the achievement of this goal has proved challenging.

There is controversy regarding which cells to target for CF gene therapy. Arguments can be
made in support of correcting cells of the surface epithelium, the submucosal glands, or both (58). A heterogeneous population of cell types express CFTR in the airways including ciliated
cells within the surface epithelium and a subpopulation of cells in submucosal gland ducts and

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where its mRNA is expressed in low abundance. The gene product is an apical membrane anion

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acini. There appear to be several epithelial cell types in the lung that provide progenitor
functions, providing the possibility of long term correction if such cells can be targeted with
integrating vectors (9). These cells may represent a pluripotent population or serve as progenitors
for a specific lineage. Experiments from several species and model systems identify potential
progenitor populations, including: basal cells (10, 11) and non-ciliated columnar cells of the
airways (10, 12, 13), submucosal gland epithelia (14-16), Clara cells (17, 18) and alveolar type II
cells in the distal lung (19, 20). Studies using integrating vectors (Moloney murine leukemia

vitro and in vivo, long-term expression can be attained (21-25).

RECENT VECTOR DEVELOPMENTS

Enveloped Viral Vectors
Current lentiviral vector technology has made considerable progress toward the aims of
efficiently, safely, and persistently expressing CFTR in the appropriate pulmonary cell types.
Studies are beginning to examine the consequences of repeated administration of lentiviral-based
vectors in the airways. Sinn and colleagues repeatedly administered (7 doses, 1 dose/week) a
baculovirus envelope (GP64) pseudotyped feline immunodeficiency virus (FIV)-based lentiviral
vector to the nasal epithelia of mice (26) and observed dose-dependant increases in reporter gene
expression that persisted for the 80 week duration of the experiment (Figure 1). The observed
innate or adaptive immune responses to the vector or vector encoded transgenes were minimal
and failed to curtail reporter or therapeutic gene expression. In contrast, Limberis and coworkers
reported that gene transfer with a VSV-G pseudotyped HIV vector resulted in activation of
transgenespecific T cells in mice (27). Transduction by VSV-G pseudotyped HIV vectors can

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virus (MLV)- and lentivirus-based) suggest that if cells with progenitor capacity are targeted in

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be further improved by formulations including magnetofectins (28), polyethylenimine (29), or
lysophosphatidylcholine (30, 31). Such methods may prove vital for achieving transduction
efficiencies sufficient to correct the chloride transport defect in vivo. Careful preclinical studies
in large animal models will be needed to further assess the safety and efficacy of the different
lentiviral vector platforms under investigation for pulmonary gene transfer.

Fetal and neonatal re-administration of a GP64-pseudotyed HIV vector was also investigated in

administrations (1 fetal/2 neonatal), and 2 neonatal administrations. The levels of macrophage

transduction increased with neonatal re-administration. The authors speculated that following the
initial dose of lentiviral vector, macrophages are recruited to the pulmonary lumen and are
subsequently transduced by the second and third doses. The authors further concluded that intraamniotic administration of GP64-pseudotyped HIV was the most efficient mode of delivery for
achieving airway epithelial cell transduction in the mouse model.

Mitoma and colleagues described a simian immunodeficiency virus (SIV)-based lentiviral vector
pseudotyped with the Sendai virus envelope proteins, hemaglutinin-neuraminidase (HN) and
fusion (F) protein (33). F/HN-pseudotyped SIV vector transduced nasal epithelial cells, resulting
in sustained transgene expression for the duration of the experiment (8-12 months) in vivo.
Similar to studies with GP64-pseudotyped FIV (26), re-administration was feasible with F/HNpseudotyped SIV, where transgene expression remained stable after 3 vector doses. In addition,
F/HN-pseudotyped SIV conferred functional CFTR expression in vitro as determined by iodide
efflux assay.

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mouse lung (32). Buckley et al. compared a single fetal intra-amniotic administration, 3

Paramyxovirus family members with known airway tropism are currently being explored as
potential CFTR delivery vehicles for the treatment of CF lung disease using reverse genetics
systems. Kwilas and colleagues recently demonstrated that a respiratory syncytial virus (RSV)based vector could deliver CFTR and correct the chloride transport defect in primary cultures of
human CF airway epithelia (34). In addition, a human parainfluenza virus (PIV)-based vector
mediated detectable but transient expression of GFP and a-fetoprotein in rhesus macaques (35).

to replication attenuated vectors that are further engineered to reduce the expression of cytotoxic
and/or immunogenic proteins. If such engineering is feasible, it could improve the duration of
gene expression, address the obstacle of pre-existing immunity, allow for repeat administration,
and make these vectors suitable for clinical studies.

Encapsidated Viral Vectors

Helper dependent-adenoviral (HD-Ad) vectors do not express viral-coding sequences and elicit
reduced cell-mediated immune responses, as compared to earlier generations of Ad vectors.
However, HD-Ad capsid proteins remain targets for neutralizing antibodies and may trigger
cytokine responses from innate immune effector cells. Recently, Cao and colleagues
demonstrated that transient immunosuppression significantly enhanced the efficiency of
transgene expression and facilitated readministration of HD-Ad vectors to mouse lungs (36). In
addition to immunosuppression, serotype switching is a proposed technique to allow for redosing
of CFTR expressing HD-Ad vectors in vivo (37). Granio and colleagues delivered an Ad vector
expressing GFP tagged CFTR to primary cultures of human CF airway epithelia. They observed

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Both the RSV and PIV-based vectors are replication competent; however, these studies may lead

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that swapping Ad5 fiber with serotype 35 fiber conferred more effective apical transduction and
correction of the Cl- transport defect (38). These data suggest that Ad vectors such as Ad5 that
use the coxackie and adenovirus receptor (CAR) are less effective at transducing the apical
surface of airway epithelial cells than CAR-independent vector serotypes such as Ad35. Taken
together, these studies outline strategies for using HD-Ad, immunosuppression, serotype
switching, and optimal fiber selection to improve the safety and long-term efficacy of adenovirus
for gene transfer to the airways.

several preclinical and clinical trials. Flotte and colleagues demonstrated that AAV1 offered
advantages over AAV5 in the chimpanzee airways, both in terms of gene transfer efficiency and
reduced immunogenicity (39). Importantly, this observation was validated by studies in welldifferentiated human airway epithelia, suggesting that the dual reporter virus co-infection
approach can help predict efficacy of AAV vectors in vivo. Progress has also been made in
engineering minimal CFTR expression cassettes that can be accommodated by the AAV vector
(40).
Additional strategies to improve the efficiency of AAV transduction to airway epithelia include
using different capsid serotypes or capsid mutants with a greater affinity for airway epithelial
cells (41). As discussed below, other novel AAV capsid variants have resulted from directed
evolution and sequence shuffling (42-44). Although standard triple transfection methodology
remains an option, new developments in baculovirus-based methods are better suited to meet
AAV production requirements (45-47). In conclusion, improvements in AAV engineering,
capsid serotype design, and production methods have made AAV an attractive vector choice for

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Recombinant adeno-associated virus (AAV) has been used for pulmonary gene transfer in

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delivering CFTR to the airways. Studies of efficacy and vector readministration in large animal
models will help guide vector development.

Non-Viral Vectors
Considerable progress has been made toward developing non-viral vectors for gene transfer to
the lung. Typically, non-viral vectors fall into two categories: 1) non-integrating, such as
plasmids (48), nanoparticles (49), and mini-circles (50), or 2) integrating, such as transposons

many of the same delivery and transduction obstacles in vivo. Optimizing the delivery efficiency
of DNA-based vectors to the in vivo airways remains a focus of the field (54, 55). Doxorubicin
(56), carboxymethylcellulose (57), and chitosan (58) improve plasmid-based gene transfer and
expression in the airways. As an alternative, some groups are investigating hybrid vector systems
combining features of adenovirus (59) or lentivirus with transposon-based vectors to improve
delivery (60).

RECENT DEVELOPMENT OF CLINICAL STUDIES FOR CF GENE THERAPY

Since 1993, approximately 25 phase I/II trials using either viral or non-viral vectors for CF have
been conducted (61). A currently ongoing clinical trial in the field was initiated by the UK CF
Gene Therapy Consortium, funded by the Cystic Fibrosis Trust, using an aerosolized non-viral
gene transfer agent (62). CF patients are receiving a single dose of a plasmid carrying the CFTR
cDNA that is complexed to the cationic lipid GL67. The plasmid, termed pGM169, is devoid of
putative pro-inflammatory sequences (CpG islands) and gene expression is regulated by a hybrid
elongation factor-1a promoter. When complexed to the cationic lipid GL67A, pGM169 led to >4

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(51) and phage phiC31 (52, 53). Both integrating and non-integrating non-viral vectors face

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week expression in mouse models upon single dosing (48). The initial single dose clinical trial
will assess safety and duration of expression in patients and will guide a planned (approximately
100 patients) multi-dose placebo controlled trial. The planned trial, due to start in autumn 2011
(personal communication, Uta Griesenbach), will determine if repeated non-viral CFTR gene
transfer (12 doses over 12 months) improves CF lung disease (61).

NEW TECHNOLOGIES

As vehicles for gene therapy applications, all viral vectors have potential weaknesses, such as
immunogenicity, tropism, transient transgene expression, and production to high titers. The
success in exploiting viral vectors will depend on the ability to overcome these limitations.
Directed evolution of viruses is a method for generating new or improved viral protein properties
using selection-based approaches. AAV and retroviruses have been the subject of combinatorial
engineering approaches in the past decade. AAV has been attractive due to its safety profile, low
immunogenicity, and ability to transduce both dividing and non-dividing cells. The AAV capsid
determines infectivity and cell tropism (63) and is therefore the target of modification by directed
evolution (42) or phage panning (64). The breadth of naturally occurring AAV serotypes
suggests that the capsid is tolerant to changes (65). Directed evolution of the AAV capsid by
PCR-based mutagenesis combined with high throughput in vitro recombination generated a
library of chimeric cap genes with components from two diverse serotypes. These serotypes,
AAV2 and AAV5, use distinct receptors, heparan sulphate and sialic acid, respectively (42).
Further selection of this library for improved transduction of human airway cells in culture
identified a novel AAV variant, AAV2.5T, a chimera between AAV2 (aa1128) and AAV5

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Directed evolution of viral vectors

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(aa129725) with a single point mutation (A581T) that exhibited enhanced binding to the apical
surface of airway epithelia and improved gene transfer (42). Furthermore, AAV2.5T could
efficiently express the CFTR cDNA in human airway cells in culture and correct the Cl- transport
defect in human CF epithelia (42).

Gamma retrovirus vectors are also efficient gene delivery tools but insertional mutagenesis and
potential oncogenesis due to preferential integration at transcriptional start sites (TSS) are

engineered zinc finger domains throughout the MLV Gag-Pol region and selection of viable
variants resulted in a shifting of the integration preferences of these vectors (70). Furthermore,
these modified integration patterns did not favor TSS. This approach could be extended to
lentiviruses and may serve as a powerful method to improve the safety profile of retroviruses as
gene transfer vectors for clinical use.

Gene repair and gene addition

A technique known as genome editing enables efficient and precise modification of a target
sequence in a genome by introduction of a double stranded break (DSB) followed by
modification of the locus during subsequent DSB repair by homologous recombination. The
DSB is induced by a zinc finger nuclease (ZFN) (71-74), a specifically engineered endonuclease
designed to cleave a chosen target in the genome. The ZFN consists of two components, the zinc
finger protein (ZFP) and the non-specific cleavage domain of Fok1 endonuclease. The ZFP
binds to the target sequence and contains a tandem array of Cys2-His2 fingers (75) each
recognizing 3 bp of DNA. The arrays generally contain three or four fingers that bind a 9 or 12

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limitations for their clinical use (66-69). Lim et al. recently showed that the random insertion of

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bp target, respectively. The Fok1 domains must dimerize to cleave the DNA (76), consequently
the specificity of the recognition site is doubled from 9 to 18 bp for a 3 finger ZFN.

Homology-based genome editing can be exploited for correction of mutated genes responsible
for monogenic disorders (Figure 2). ZFN-based genome editing requires delivery of a donor
DNA repair template along with the target specific ZFN pair. Methods for generating ZFN pairs
targeting specific genomic loci are becoming widely available and include modular design

ZFNs designed using OPEN technology have been shown to bind the genomic DNA encoding
CFTR (78) and create double strand breaks near the F508 mutation in exon 10. Provision of a
wild-type donor DNA template with a non-integrating vector, such as integrase-deficient
lentiviral vector (81), can facilitate repair of this mutation by homologous recombination.

Other repair strategies using homing endonucleases (82-84) or transcription activator-like

effector (TALE) nucleases (85) provide alternative mechanisms for creating DSBs in genomic
DNA and allowing for gene repair by co-delivery of a homology repair template. A potential
advantage of each of these gene repair approaches is that correction of CFTR in progenitor cell
types could preserve the native regulatory elements and allow for correction in subsequent
daughter cells. Reagent development and delivery to airway epithelia will be important for this
field to advance.

An alternative to the gene repair approach is termed targeted gene addition (Figure 2). Here,
ZFNs may be used to create DSBs at potential safe harbor loci such as AAVS1 (86), CCR5

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approaches (77-79) and the selection-based oligomerized pool engineering (OPEN) strategy (80).

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(87), or the mouse Rosa26 locus (88). In this approach, the entire therapeutic transgene with
flanking homology arms would be inserted into the safe harbor loci by homologous
recombination.

Modification of such a locus is reasoned much less likely to perturb the

expression of neighboring genes or disrupt the function of other genetic elements (89).

Applications of RNA interference to treat CF

The recent explosion of knowledge in the field of small interfering RNAs has led to applications

contribute to steps in wild type and mutant CFTR biogenesis including ER and Golgi trafficking,
residence time in the cell membrane, and its removal by proteosomal degradation (90-92). This
has raised the possibility that RNAi-based strategies might be developed to increase the
expression of F508 CFTR, to rescue F508 CFTR from proteosomal degradation, or enhance
its residence time in the cell membrane. Any of these approaches might provide sufficient
residual CFTR function to be therapeutically relevant.

Similarly, targeting other cellular

pathways, such as those involved in inflammation, might offer a means to ameliorate disease
symptoms and progression.

A significant hurdle for translational studies in this area is

identifying the methods to efficiently deliver RNAi to well-differentiated airway epithelia.

Another area of investigation relevant to the field is the identification of the microRNA
repertoire in airway epithelia and other CFTR expressing cells, as well as their respective target
gene products. Knowledge in this area may identify new targets for therapeutic manipulation.

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of direct relevance to CF. First, RNAi has been used as a tool to identify gene products that

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Lung tissue engineering
Lung transplantation is currently the only definitive treatment for end stage CF lung disease. The
supply of donor lungs is limited and transplantation achieves only a 10 to 20% survival at 10
years (93). Recently, two groups independently used similar tissue engineering strategies to
develop an autologous bioartificial lung that may begin to help to overcome the limited
availability of donor tissues (94, 95). The bioartificial lungs were created by first generating a
whole lung scaffold by perfusion and decellularization of the adult rat lung, followed by

gas exchange within the resulting grafts was demonstrated. With the further development of this
technology, one could envision the ex vivo correction of patient derived cells, followed by lung
tissue engineering, and transplantation. Although these initial results are very exciting, several
steps need to be further optimized before long-term tissue-engineered lung function can be
translated to the clinic (96).

NEW ANIMAL MODELS OF CF DISEASE

A significant bottleneck for the development of new CF therapeutics has been the lack of animal
models that recapitulate key features of lung and other organ disease pathogenesis. The mouse
models with CFTR null alleles and specific disease mutations available since the early 1990s
have contributed greatly to disease understanding but fail to develop spontaneous lung disease
similar to humans with CF. Recently, two groups used somatic cell targeting of the CFTR gene
with AAV vectors, followed by nuclear transfer and cloning to develop novel models in pigs (97,
98) and ferrets (99, 100). These new animal models recapitulate key features of CF disease (98,
100). At birth, the lungs of CFTR null pigs are free of inflammation but manifest a bacterial host

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reseeding of the endothelial and epithelial surfaces of the scaffold with new cells. Evidence for

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defense defect without the secondary consequences of infection (98, 101). CF pigs spontaneously
develop a lung disease phenotype mirroring key features of human CF lung disease in the first
months of life including infection with bacteria, airway remodeling, and mucus hypersecretion
(Figure 3). CFTR null ferrets also develop multiorgan system disease and neonatal animals
manifest a pulmonary host defense defect in the airways associated with colonization by bacteria
(100). There is also early evidence that adult CF ferrets develop a lung disease phenotype with
similarities to human CF, including bacterial colonization (personal communication, John

studies. They offer the unique opportunity to test gene therapy interventions prior to the onset of
lung disease and monitor the outcomes for prevention-based treatment strategies.

CONCLUSIONS
This is an unprecedented time in the development of new therapies for CF. The near universal
availability of newborn screening for CF in developed nations has made early diagnosis
commonplace, allowing the potential for treatment of healthy lungs before the onset of chronic
lung disease. However, this opportunity comes at a time where there is a dearth of sensitive and
specific markers of early disease that can be used to assess lung disease onset and monitor
responses to therapy. Additional work is needed to develop new specific and sensitive measures
of the early stages of lung disease suitable for monitoring the response to therapies for use in
infants and young children. Parallel developments in improved gene transfer tools should further
aid the field and lead to new clinical trials.

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Engelhardt). These phenotypic features make these new models very attractive for gene therapy

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ACKNOWLEDGEMENTS
We thank John Engelhardt, and Uta Griesenbach for their helpful discussions, as well as David
Meyerholz for providing the images presented in Figure 3. This work was supported by NIH
grants: R01 HL-075363 (P.B.M.), PO1 HL-51670 (P.B.M.), R21 HL-91808 (P.B.M.), the Cystic
Fibrosis Foundation (P.L.S.), and the Roy J. Carver Charitable Trust (P.B.M.). We also
acknowledge the support of the In Vitro Models and Cell Culture Core and Cell Morphology
Cores, partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-

CONFLICT OF INTEREST STATEMENT

None declared.

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54759) and the Cystic Fibrosis Foundation.

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FIGURE LEGENDS
Figure 1. Repeat administration of lentiviral vector results in persistent transgene expression in
the nasal airways. Seven doses of GP64 pseudotyped FIV expressing luciferase were delivered at
1 week intervals for 7 consecutive weeks to mice via nasal instillation (arrows). At the indicated
time points, light release was captured using bioluminescent imaging and data quantified with
living image software. Stable luciferase expression was documented for 80 weeks. Data are
expressed as mean standard error. n = 5.

pairs bind to and cleave the target genomic locus near the disease causing mutation. ZFNs are
composed of chimeric zinc fingers (blue) and Fok1 endonuclease domains (red). Introduction of
a double stranded break (DSB) promotes homologous recombination, using the repair template
donor. B) For targeted gene addition, the ZFN pair cleaves a predetermined safe harbor locus.
The expression cassette with a therapeutic gene (green) is flanked by homology arms to the safe
harbor locus.

Figure 3. Pigs with CFTR mutations develop pulmonary disease with similarity to humans with
CF. In this example from a pig of 5.5 months of age, key pathogenic features observed include
mucus accumulation in the airways, airway obstruction with purulent material containing
neutrophils and bacteria (right panel, black arrows), and airway remodeling. The bacterial
cultures from this animal were positive for Bordetella bronchiseptica. The wild-type pig is an
age matched control. Hemotoxylin & eosin stain. Both images are 100X magnification.

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Figure 2. Schematic of targeted gene correction and gene addition strategies. A) Expressed ZFN

Figure 1

7 doses

photons/s/cm2

60000
50000
40000
30000
20000
10000
0
0

10

15

20

25

30

35

40

45

Weeks

50

55

60

65

70

75

80

Figure 2
A) Targeted correction

ZFN

Repair template
ZFN cleavage

Homologous recombination

disease mutation

Corrected gene

B) Gene addition
cDNA of wild-type gene
Homology arm

Homology arm
ZFN cleavage

Wild-type gene added to safe harbor locus by homologous recombination

Figure 3

Wild-type pig

CF pig