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Gene therapy for cystic fibrosis
By Peter Hiatt, M.D.
Cystic fibrosis (CF) is the most common fatal autosomal
recessive disease among Caucasian populations, with a frequency
of one in 2000 to 3000 live births. CF is caused by mutations in
the cystic fibrosis transmembrane conductance regulator (CFTR)
gene. The CFTR protein is a complex, regulated chloride channel
found in all exocrine tissues. Deranged chloride transport leads
to thick, viscous secretions in the lungs, pancreas, liver,
intestine, and reproductive tract, and to increased salt content
in sweat gland secretions. Respiratory complications account for
the major morbidity and mortality of CF, and are characterized
by persistent pulmonary infection leading to inflammation and
obstructive mucus. CF is currently inadequately treatable.
Existing treatments for CF lung disease are designed to control
and delay the progressive destruction of lung tissue, through
mechanical airway clearance, treatment of chronic and acute
infections, and reduction of inflammation and lung
transplantation. Although improved therapies have led to longer,
healthier lives for CF patients in the last 20 years, they
remain vulnerable to a wide variety of pulmonary infections and
lung damage ultimately advances to the stage of irreversible
bronchiectasis and progressive respiratory failure with a median
survival of 32.3 years of age. Therefore, development of novel
modalities is clearly needed and gene delivery to the lungs, for
correction of the CFTR defect holds tremendous potential.
When the CF gene was cloned in 1989, there was relatively
unbridled optimism that this would lead to a cure for the lung
disease in CF. A number of viral vectors such as E1-deleted
first generation adenoviral vectors (FG-Ad), adeno-associated
viral vectors (AAV) and lentiviral vectors have been
investigated for pulmonary gene transfer for CF gene therapy.
Although it was quickly demonstrated that CFTR could be
expressed in various cultured cell systems and animal models,
successful clinical intervention proved far more difficult. In
general, limitations of viral vectors include inefficient
transduction of the airway epithelium due to the basolateral
localization of viral receptors, vector-mediated toxicity and a
potent humoral immune response preventing vector
readministration. Nonviral vectors based on plasmid DNA are also
being investigated for pulmonary gene transfer for CF. Although
these nonviral vectors are generally less toxic than viral
vectors, the efficiency of gene transfer is even lower and
problems of successful readministration remain.
HD-AD vectors show promise
Significant improvement in the safety and efficacy of Ad-based
vectors came with the development of helper-dependent adenoviral
vectors (HD-Ad), which are deleted of all viral coding
sequences. In contrast to FG-Ad, HD-Ad are able to mediate
long-term, high-level transgene expression in the absence of
chronic toxicity owing to the absence of viral protein
expression in the transduced cells.
Doctors Ng and Beaudet, Baylor College of Medicine Department of
Genetics, have developed HD-Ads containing an epithelial
cell-specific expression cassette designed for airway gene
therapy. In studies conducted at Baylor, the HD-Ad was used to
deliver a transgene to non-human primate lungs expressing the
lacZ reporter gene. Non-human primates offer anatomy that
closely mimics humans, are amenable to instrumentation for
aerosol delivery similar to humans, and can be monitored closely
for inflammation by bronchoscopy and radiography.
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Fig 1.
The AeroProbe™ is a ~1 mm diameter multi-lumen catheter
where liquid is injected under pressure, using the LABneb™
control system (not shown), down a central lumen and sheared
into droplets at the distal tip from high pressure air
traveling down 5 peripheral lumens.
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Fig. 2
X-gal histochemistry of (A and B) the epithelium lining of a
large airway, (C) mid sized bronchiole and (D) respiratory
bronchiole. Submucosal gland indicated by the
yellow arrow.
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In order to deliver the HD-Ad vector a novel catheter (AeroprobeTM)
was used to aerosolize material directly into the lungs,
bypassing the pharynx and vocal cords, minimizing waste and
destruction of expensive material and minimizing exposure of
investigators/health care workers to aerosolized virus (Fig. 1).
Using the above methodology, HD-Ad containing the lacZ
reporter gene was delivered to the airways of non-human primates
with the aeroprobe catheter. Since lacZ can be easily
detected in pieces of gross lung tissue, as well as on
microscopic tissue sections, delivery to the airway allows the
observation of both transduction of airway cells as well as the
path of aerosol distribution within the lungs. Following aerosol
delivery extensive transduction of the airway epithelium of
large airways, medium size bronchioles and respiratory
bronchioles (Fig. 2) was observed. These results suggest HD-Ad
can be delivered to the airway with minimal toxicity with good
transduction of the targeted cells for CF gene therapy.
To determine the duration of transgene expression and the
absence of chronic toxicity a construct using α-fetoprotein was
delivered to non-human primate airways. These studies have
revealed high levels of α-fetoprotein expression measured in
serum and bronchoalveolar lavage fluid for 90 days following
airway delivery. Minimal to no toxicity has been observed to
date with administration of the HD-Ad vector or with the
long-term expression of α-fetoprotein. These studies suggest
that CFTR delivered by HD-Ad via aerosol may be expressed for 90
days following administration in humans. Studies conducted to
date have been designed to achieve effective HD-Ad mediated
aerosol gene transfer as a precursor to trials in humans.
Further study is under way
Current studies are in progress to address the impact of the
immune response with recurrent administration of HD-Ad vectors
to the lung. Readministration will be required using this
methodology for CF gene therapy. Depending on the clinical
response following aerosol delivery, repeated therapy could be
required every 6 to 12 months. Successful readministration may
not be possible in the presence of high titers of neutralizing
antibodies and may require a period of time for titers to drop
below a selected protective threshold. Alternatively, adenovirus
serotype switching or shielding of the virion could be used to
evade neutralizing antibodies. These issues are currently being
examined in studies of repeated HD-Ad aerosol delivery to
non-human primates expressing a non-immunogenic transgene
(α-fetoprotein).
In summary, gene therapy for cystic fibrosis has been
disappointing to date; however, encouraging work continues here
at Baylor as well as other gene therapy centers in North
America, Europe and Australia. Several alternative therapies for
CF are in Phase I, II, and III human trials and include new
antimicrobial agents, medications to decrease chronic airway
inflammation, mucolytics, chloride channel agonists and agents
that increase airway surface liquid. It is expected that several
new therapies will be available to aid in the treatment of CF
until a cure can be found.
Peter Hiatt, M.D., is associate professor of Pediatrics,
director to the Cystic Fibrosis Center.
References
Boucher RC. An overview of the pathogenesis of cystic fibrosis
lung disease. Adv Drug Deliv Rev 2002; 54: 1359-1371.
Kerem B, Kerem E. The molecular basis for disease variability in
cystic fibrosis. Eur J Hum Genet 1996; 4:65-73.
Koehler DR, Hitt MM, Hu J. Challenges and strategies for cystic
fibrosis lung gene therapy. Mol Ther 2001; 4:84-91.
Koehler DR, Frndova H, Leung K, Louca E, Palmer D, Ng P,
McKerlie C, Cox P, Coates AL, Hu J. Aerosol delivery of an
enhanced helper-dependent adenovirus formulation to rabbit lung
using an intratracheal catheter. J Gene Med 2005;
7:1409-1420.
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