1. Stephanie M. Delgado BIOL 3095, Section 140, No. 804-09-2123
Dra. Eneida Díaz December 13, 2009
REVIEW
Gene Therapy for the Treatment of Cystic Fibrosis
Abstract
Cystic fibrosis is an autosomal recessive genetic disorder caused by various mutations in the
cystic fibrosis transmembrane conductance regulator (CFTR) gene. It affects over 70, 000
people worldwide, mostly of Caucasian descent. It should be possible to treat an autosomal
recessive disease such as cystic fibrosis with insertion of one copy of normally functioning DNA
into the affected cells, independent of the class of mutation the recipient had before gene
therapy. Although easy in concept (and in vitro), in practice gene therapy has proven to be quite
difficult in treating Cystic Fibrosis. There are biological barriers that must be surmounted as well
as different vectors that have been tried without encouraging results. This article focuses on
these aspects of gene therapy for Cystic fibrosis as well as the human clinical trials that have
been performed.
Table of Contents
1. Introduction 1
2. Degree of correction required 2
3. Barriers for gene delivery 2
4. Vectors 3
4.1 Viral Vectors 4
4.2 Nonviral vectors 4
5. Clinical studies successes and failures 4
6. Conclusions 9
7. References 10
1. Introduction
Cystic Fibrosis (CF) is an autosomal recessive genetic disorder. It’s caused by various
mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene on
chromosome 7q31.2. These mutations cause imbalanced ion and water movement across the

2. 2
airway epithelium, resulting in accumulation of sticky mucus, chronic bacterial infection and
inflammation. Although this disease also affects other organs, lung disease contributes mainly
to morbidity and mortality in CF patients. Gene therapy is a treatment that consists of using
genetic material with a curative purpose. Gene therapy aims to get rid of genetic diseases at
their source by targetting the faulty gene.The objective of this treatment for Cystic Fibrosis is to
insert a normal copy of the CFTR gene into the affected cells in order to restore normal
functions (Davies, 2006). With the discovery and cloning of the CFTR gene in 1989, CF was
one of the first diseases chosen to be treated with gene therapy. There are several reasons for
this, but mainly it’s because CF is a monogenic disease (caused by only one gene), the CFTR
gene is relatively large, and small quantities of the CFTR protein are needed to correct the
chloride transport. Although fairly simple in concept, after twenty years of intense research and
clinical trials, there are still many obstacles that must be overcomed before gene therapy for this
disease is successful. (Ostrowski, 2007).
2. Degree of correction required
One of the most important things to consider when administering gene therapy is the
minimal percentage of cells with a non-CF phenotype that will be required to normalize the
functions of the CF bronchial epithelial layer. One study concludes that only 10% of non-CF
cells are sufficient to normalize the abnormal Na+ absorption and Cl- secretion associated with
CF (Dannhoffer, 2009). This study is consistent with previous research that found that only
about 5-10% of corrected cells are needed to correct the abnormal Cl- transport, although by
measuring the IL-8 secretion and the basal Isc they also concluded that this percentage is
enough to correct the inflammation and Na+ transport (Ziady, 2006). Although we know the
percentage of cells needed to normalize the CFTR dependant functions in vitro, it’s still not clear
the percentage necessary in vivo or which specific cells should be transduced.

3. 3
3. Barriers for gene delivery
Humans are well protected against the invasion of introduced genetic material by
intracellular and extracellular barriers. This has been an evolutionary adaptation designed to
maintain our genetic stability. That’s why genetic transfer agents (GTA’s) or vectors have to be
used for gene therapy in cystic fibrosis. These delivery systems usually are made of a
polynucleotide, encoding the therapeutic gene, and a carrier which “should facilitate transport of
the therapeutic gene from the extracellular space into the nuclear compartment, where
transcription can take place” (Conese et al, 2007). But depending on the method of
administration these vectors have to penetrate many defense mechanisms like circulating
antibodies in the bloodstream, a mucus layer that is constantly cleared by cilliae in the airway,
macrophages, and immune responses which can clear the positively transduced cells (Price et
al, 2007).
4. Vectors
The target of CF gene therapy is the airway epithelium. Gene delivery systems have
been developed to reach this site. A carrier molecule called a vecto is used to deliver the
therapeutic gene to the patient's target cells in the airway epithelium. The vector then unloads
its genetic material containing the CFTR gene into the target cell. The generation of a functional
protein product from the therapeutic gene restores the target cell to a normal state.
Example of gene delivery system. From Eubanks, 2001.

4. 4
4.1 Viral vectors
Some gene transfer agents have been developed from viruses. These include
adenoviral vector, adeno-associated viral vectors, lentivirus vectors, and poxvirus and, Sendai
and herpes virus (Buckley et al, 2008; Ferrari et al, 2007; Rogers et al, 2008). Most of these
viral vectors have currently limited use for CF gene therapy because of inefficient gene
expression after repeat administration, but lentiviral vectors may be an exception (Buckley et al,
2008). Lentiviruses transfect non-dividing cells and may, therefore, be suitable for transfection
of terminally differentiated airway epithelial cells. The virus stably integrates into the genome of
transfected cells and expression is therefore likely to last for the life-time of the cell, but still
present some problems like risk of insertional mutagenesis and proto-oncogene activation (Yu,
2007).
4.2 Nonviral vectors
Non-viral gene transfer agents are generally less efficient than viral vectors in
transfecting lung epithelium. However, if free of “non-human” protein components, they are less
likely to induce an immune response, thereby allowing for repeat administration, which is a
crucial for the treatment of CF and other chronic diseases that will require life-long treatment
(Griesenbach and Eric, 2008). These synthetic vectors consist generally of DNA encoding the
therapeutic gene in the form of a plasmid, combined with a carrier generally of a synthetic
compound, which mimics the functions of a viral capsid, but should lack its immunogenicity.
While this development is of major interest, much work needs to be done to accomplish its full
therapeutic potential.
5. Clinical studies successes and failures
Since 1993, twenty-five phase I/II safety clinical trials have been carried out using a
variety of viral and nonviral gene transfer agents (GTA’s). These trials have involved around 450

5. 5
CF patients (Griesenbach and Alton, 2008). As we can see on Table # 1 on the appendix the
first trials mostly focused on the nasal epithelium, as it’s more secure and allows easy access
and sampling. When an acceptable safety profile had been achieved, the GTA’s were
administered directly into the lung. Adenovirus vectors were first used, until it was clear that
these were not efficient transducing into the human airway epithelial cells (target cells for CF
gene therapy) because there was no adenoviral receptor in their membrane. Also these
adenoviral vectors could not be administered repeatedly, something necessary for a life-long
chronic disease like CF (Griesenbach and Alton, 2007). The adenoviral vectors were replaced
with adeno-associated (AAV) vectors, but the most recent phase I/II trials were unsuccessful.
Other nine clinical trials have used nonviral GTA’s. These have shown more promise as they
are more likely to be repeatedly administrable and have shown approximately 25% correction of
the molecular defect in CF patients. Groups like the UK CF Gene Therapy Consortium and
Copernicus Inc. are currently preparing clinical trials focusing on nonviral GTA’s. It is hoped that
next-generation vectors will lead to effective gene transfer and sustained cure of the pulmonary
disease associated with cystic fibrosis (O’Sullivan and Freedman, 2009).
6. Conclusions
Cystic Fibrosis is a disease that affects over 70,000 individuals worldwide. It’s a single
gene disorder with insufficient treatment options and non-invasive access to the target organ,
which is the lung. For these reasons and because of its genetic features, it was one of the first
diseases considered for gene therapy. Although very promising at first, gene therapy for CF has
encountered many problems like the absence of an appropriate vector. But significant progress
is currently being made in the development of viral and nonviral vectors that are more effective.
It can be concluded that new and longer clinical trials are needed as well as the development of
new vectors to be tested in these clinical trials. Until then, gene therapy as a cure for the basic

6. 6
defect that could normalise the functions and life expectancy for those born with CFTR
mutations is still a great hope more than a reality.
7. References
Buckley SMK , Howe SJ, Sheard V, Ward NJ, Coutelle C, Thrasher AJ, Waddington SN,
McKay TR. 2008. Lentiviral transduction of the murine lung provides efficient pseudotype
and developmental stage-dependent cell-specific transgene expression. Gene Therapy 15:
1167–1175
Conese M, Boyd C, Di Gioia S, Auriche C, Ascenzioni F. 2007. Genomic Context Vectors and
Artificial Chromosomes for Cystic Fibrosis Gene Therapy. Current Gene Therapy 7: 175-187
Dannhoffer L, Blouquit-Laye S, Regnier A, Chinet T. 2009. Functional Properties of Mixed
Cystic Fibrosis and Normal Bronchial Epithelial Cell Cultures. American Journal of
Respiratory Cell and Molecular Biology 40: 717–723.
Davies JC. 2006. Gene and cell therapy for cystic fibrosis. Paediatric Respiratory Reviews
7(1):S163-S165.
Eubanks M. 2001. Gene Therapy for CF. Environmental Health Perspectives 109: A16
Ferrari S, Griesenbach U, Iida A, Farley R, Wright AM, Zhu J, Munkonge FM, Smith SN, You J,
Ban H, Inoue M, Chan M, Singh C, Verdon B, Argent BE, Wainwright B, Jeffery PK, Geddes
DM, Porteous DJ, Hyde SC, Gray MA, Hasegawa M, Alton EW. 2007. Sendai virus-
mediated CFTR gene transfer to the airway epithelium. Gene Therapy 14: 1371–1379.
Griesenbach U, Alton EW. 2007. Progress and Prospects: Gene Therapy Clinical Trials (Part 1).
Gene Therapy 14: 1439–1447.
Griesenbach U, Eric WF. 2008. Gene transfer to the lung: Lessons learned from more than 2
decades of CF gene therapy. Advanced Drug Delivery Reviews 61: 128–139.
Hyde SC, Pringle IA, Abdullah S, Lawton AE, Davies L, Varathalingam A, Nunez-Alonso G,
Green A, Bazzani RP, Sumner-Jones SG, Chan M, Li H, Yew NS, Cheng SH, Boyd C,
Davies JC, Griesenbach U, Porteous DJ, Sheppard DN, Munkonge FM, Walton EW, Gill
DR. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene
expression. 2008. Nature Biotechnology 26: 549-551
Liu X, Luo M, Guo C, Yan Z, Wang Y, Engelhardt JF. 2007. Comparative biology of rAAV
transduction in ferret, pig and human airway epithelia. Gene Therapy 14: 1543–1548.
O’Sullivan BP, Freedman SD. 2009. Cystic fibrosis. Lancet 373: 1891–904
Ostrowski LE, Yin W, Diggs PS, Rogers TD, O’Neal WK, Grubb BR. 2007. Expression of CFTR
from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in
CF mice. Gene Therapy 14: 1492–1501
Price AR, Limberis MP, Wilson JM, Diamond SL. 2007. Pulmonary delivery of adenovirus
vector formulated with dexamethasone–spermine facilitates homologous vector re-
administration. Gene Therapy 14: 1594–1604.
Rogers CS, Hao Y, Rokhlina T, Samuel M. 2008. Production of CFTR-null and CFTR-
[Delta]F508 heterozygous pigs by adeno-associated virus-mediated gene targeting and
somatic cell nuclear transfer. Journal of Clinical Investigation 118 (4): 1571-1577
Sumner-Jones SG, Davies LA, Varathalingam A, Gill DR, Hyde SC. 2006. Long-term
persistence of gene expression from adeno-associated virus serotype 5 in the mouse
airways. Gene Therapy 13: 1703–1713.
Yu Z, McKay K, Asperen P, Zheng M, Fleming J, Ginn SL, Kizana E, Latham M, Feneley M,
Kirkland PD, Rowe P, Lumbers ER, Alexander IE. 2007. Lentivirus vector-mediated gene
transfer to the developing bronchiolar airway epithelium in the fetal lamb. The Journal of
Gene Medicine 9: 429–439.

7. 7
Ziady AG, Davis PB. 2006. Current prospects for gene therapy of cystic fibrosis. Current
Opinion in Pharmacology 6(5):515-521.

8. 8
Appendix
Table 1. List of clinical trials
Reference Route Trial design Results
Adenovirus clinical trials
Zabner et al. (1993) Nose Dose-escalation 2 × 106– NPD: decreased baseline towards non-
5 × 107 pfu n = 1/dose CF values
mRNA: − ve, protein: − ve
Crystal et al. (1994) Nose Dose-escalation 2 × 105– NPD: inconclusive, too variable
5 × 107 pfu n = 2/dose
mRNA: 1/4 + ve, protein: 1/4 + ve
Lung Dose-escalation 2 × 106– mRNA: − ve, protein: 1/4 + ve
5 × 109 pfu n = 1–2/dose
Safety: no vector shedding. At highest
dose transient inflammation.
Knowles et al. (1995)Nose Dose-escalation 2 × 107– NPD: no change mRNA: 5/12 + ve
5 × 1010 pfu n = 3/dose
Vehicle control applied on Safety: no toxic effects at low doses. At
contralateral nostril highest dose mild mucosal inflammation
2/3 patients
Hay et al. (1995) Nose Dose-escalation 2 × 105– NPD: decreased baseline + amiloride
5 × 108.5 pfu n = 1–2/dose towards non-CF values. Partial
correction of Cl− transport
Zabner et al. (1996) Nose Dose-escalation repeat NPD: partial correction of Cl− in some
administration 2 doses patients, but reduced effect with
2 × 107–1 × 1010 pfu n = 4– subsequent administration. All patients
6/dose developed serum antibodies to vector,
but not CFTR
Bellon et al. (1997) Nose Dose-escalation 1 × 105– DNA: 6/6 + ve
4 × 108 pfu n = 2/dose
Dose-escalation 1 × 107–
5.4 × 108 pfu n = 2/dose
mRNA: 4/6 + ve, protein: 4/6 + ve
Lung Dose-escalation 1 × 107– DNA: 1/6 + ve
5.4 × 108 pfu n = 2/dose
mRNA: 1/6 + ve, protein: 2/6 + ve
Expression was transient in both nose
and lung
Harvey et al. (1999) [ Lung Dose-escalation Sampled 3 and 30 days after each
administration mRNA:
Repeat administration 3 1st administration + ve only with highest
doses 106–1 × 109 pfu dose, transient (− ve by day 30)
n = 2/dose
2nd administration + ve only with
intermediate dose, − ve by day 30
3rd administration no expression Anti-
Ad neutralising antibodies detected but
no close correlation with loss of
expression

9. 9
Zuckerman et al. Lung Dose-escalation 2 × 109– DNA: + ve on day 4, transient
(1999) 2 × 1011 pfu n = 2–3/dose
Immune response: Ad-specific T-cells,
mild humoral response
Safety: mild flu-like symptoms
observed, resolved about day 10
Joseph et al. (2001) Lung Dose-escalation 8 × 106– DNA: 4/5 + ve on day 2
2.5 × 1010 pfu n = 2–3/dose
mRNA: 3/5 + ve on day 2
Perricone et al. DNA: all + ve on day 2
(2001)
mRNA: 4/13 + ve on day 2
Expression transient, undetected by
day 7. < 3% of airway epithelial cells
transfected. Safety: Mild, non-specific
inflammatory response (fevers,
myalgia). Cleared within 24 h.
AAV2 clinical trials
Wagner et al. (1999) Nose Dose-escalation 1 × 102– DNA: 7/10 + ve day 14
1 × 105 RU
Single and two doses n = 5/ 1/10 + ve day 41
group
1/10 + ve day 70
CFTR mRNA: − ve
NPD: partial Cl− correction in some
cases, effect transient
Aitken et al. (2001) Lung Dose escalation 1010 DRP– DNA: 6/6 + ve up to day 30 with two
1013 DRP n = 3/group highest doses
mRNA: − ve
Safety: several adverse effects, three of
which possible related to study
(pneumonia, exacerbation)
Wagner et al. (2002) Nose 1 nostril 105 RU, No change in: rate of sinusitis relapse,
contralateral nostril placebo NPD, histopathology and IL-8.
(n = 23)
IL-10: increased vs. placebo at day 90
Flotte et al. (2003) Nose Dose escalation Nose:
Lung 3 × 101 RU–1 × 109 RU NPD: no change
placebo (n = 25)
contralateral nostril
Vector DNA: 2/25 + ve
Lung:
Vector DNA: some + ve, but cross-
contamination from nose
Safety: generally well tolerated, one
adverse effect possible linked to
treatment (pulmonary exacerbation)
Moss et al. (2004) Lung Repeat admin: 3 doses, DNA: 6/6 + ve (only assessed after 3rd
30 days apart 1 × 1013 DRP dose)
(n = 37)

10. 10
mRNA: − ve
FEV1: trend in improvement day 30
IL-8 and IL-10: sputum IL-8 reduced
after 1st dose, IL-10 no change
Safety and immune response: well
tolerated, active group developed
AAV2-neutralising antibodies
Moss et al. (2007) Lung Repeat admin: 3 doses No changes in spirometry, days of
30 days apart 1 × 1013 DRP antibiotic use or induced sputum
(n = 102) markers (IL-8 + neutrophil elastase)
Safety: well tolerated
Non-viral clinical trials
Caplen et al. (1995) DC- Dose escalation 10–300 mg Vector DNA: 7/8 + ve (some problems
Chol/ DNA n = 3/dose with false + ves)
DOPE
Nose
CFTR mRNA: − ve
NPD: partial correction (20%) of Cl−
defect towards normal at day 3,
undetected by day 7
Safety: well tolerated
Gill et al. (1997) DC- 40 + 400 μg DNA/nostril NPD: 2/8 transient correction of Cl− for
Chol/ n = 4/dose 7–15 days
DOPE
nose
SPQ: 5/8 showed CFTR function
Hyde et al. (2000) DC- Repeat administration 3 DNA: 6/9 + ve after ≥ one dose
Chol/ doses 400 μg DNA/nostril
DOPE
nose
1/9 + ve for all 3
mRNA: 7/9 + ve after ≥ one dose
1/9 + ve for all 3
protein: 6/9 + ve after ≥ one dose; 2–
15% epithelial cells transfected
NPD: partial Cl− correction in individual
patients
SPQ: 5/9 showed CFTR function after
≥ one dose
Bacterial adherence: no difference, but
technical problems
Immune responses: no response to
CFTR
Importantly, no loss of efficacy with
repeated dosing
Porteous et al. DOTA 400 μg DNA DNA: 7/8 + ve on days 3 and 7
(1997) P
nose
2/7 + ve on day 28
mRNA: 2/8 + ve on days 3 and 7

11. 11
NPD: 2/8 partial Cl− correction up to
4 weeks
SPQ: − ve
Noone et al. (2000) EDM Dose escalation 0.4–4 mg DNA: all + ve (some cross-
PC DNA placebo in contamination to placebo)
nose contralateral nostril
mRNA: − ve
NPD: no change
Zabner et al. (1997) GL67 1.25 mg DNA/nostril GL67/ DNA: 8/9 + ve
vs. DNA to one nostril, pDNA to
naked contralateral nostril
pDNA
nose
pDNA to one nostril, empty RNA: − ve, technical problems
plasmid to contralateral
NPD: statistically significant correction
of Cl− with both GL67 and naked pDNA.
No difference between vectors
Alton et al. (1999) GL67 Lung: 42.2 mg DNA Lung:
lung
and
nose
DNA: 8/8 + ve
Nose: 11.8 mg DNA RNA: − ve
PD: statistically significant Cl− correction
in active group, 25% of normal values
Lipid only SPQ: CFTR function in 5/6 patients
Placebo Bacterial adherence: 5/6 patients in
active group reduced bacterial binding
compared to pre-treatment values
(n = 16) Inflammation: significant reduction of
inflammatory cells in sputum in active
group similar results (DNA, mRNA, PD,
SPQ, bacterial adherence ) in the nose
Safety: 7/8 patients in active group
developed flu-like symptoms (fever,
headache), resolved within 36 h. 6/8
patients in both groups had mild airway
symptoms.
Ruiz et al. (2001) GL67 7.9–21.12 mg DNA mRNA: 4/8 + ve, 3/4 received highest
lung dose
Inflammatory response: 4/8 pronounced
fever, myalgia within 6 h post-
administration. Serum IL-6 increased,
but no changes in IL-8, IL-1, TNF-α or
IFN-γ. No antibodies to lipid or plasmid.
(n = 2–4/dose) Lipid and DNA have synergistic effect
on inflammation.
Konstan et al. (2004) DNA Dose escalation 0.8–8.0 mg DNA: 12/12 + ve in active but cross-
nanop DNA n = 2–6/dose contamination in placebo
article contralateral nostril placebo

12. 12
s
nose
NPD: partial to complete Cl− correction
8/12, up to day 6
Safety: well tolerated, no adverse
effects related to treatment
Table # 1. From Griesenbach and Eric, 2008.