Cystic fibrosis, existing and emerging therapies
Thomas Ferkol MD
Markey Pathway Conference
Cystic fibrosis: a historical timeline
1938
Cystic fibrosis (CF) of the pancreas was described by Andersen.
1953
The sweat defect was discovered by diSant'Agnese and colleagues when they noticed
that many of the infants presenting with heat prostration during the “great summer
heat wave” in New York City had CF.
Cystic fibrosis was identified as an autosomal recessive disease.
1965
The fundamental physiologic defects were clearly established by Knowles and
colleagues and Quinton as the failure of cAMP regulation of chloride transport.
The genetic defect for CF was located on chromosome 7.
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR)
was identified by positional cloning.
1983
1985
1989
1990
Cystic fibrosis transmembrane conductance regulator was established to be a cAMPregulated chloride channel by complementation studies.
Cystic fibrosis: clinical presentations
Gastrointestinal
• meconium ileus
• meconium plug syndrome
• distal intestinal obstruction syndrome
• rectal prolapse
• neonatal hyperbilirubinemia
• failure to thrive
• hypoproteinemic edema
• hypovitaminosis
• recurrent pancreatitis
• biliary cirrhosis and portal hypertension
Endocrine
diabetes
Genitourinary
• male infertility
Sweat Gland Dysfunction
• hypochloremic, hyponatremic alkalosis
Respiratory
• chronic cough
• recurrent sinopulmonary infections
• bronchiolitis/asthma
• nasal polyposis
• Staphylococcus aureus pneumonia
• Pseudomonas aeruginosa endobronchitis
Cystic fibrosis: epidemiology
Population
Epidemiologic
Newborn screening
Caucasian (US)
1 in 1,900-3,700
1 in 3,400-3,800
Caucasian (Great Britain)
1 in 2,400-3,000
1 in 2,200-3,200
Hispanic
1 in 8,000-9,000
--
African American
1 in 15,300
--
Native American
1 in 40,000
--
Asian (US, England)
1 in 10,000
--
Israel
1 in 5,000
--
1 in 2,000-4,000
--
Southern Europe
Median survival age (years)
Cystic fibrosis: median survival age, 1940-2007
37.8
35
30
25
20
15
10
5
0
1940
1950
1960
1970
1980
Year
1990
2000
Cystic Fibrosis Foundation Registry, 2007.
2010
Cystic fibrosis: a historical timeline
1938
Cystic fibrosis (CF) of the pancreas was described by Andersen.
1953
The sweat defect was discovered by diSant'Agnese and colleagues when they noticed
that many of the infants presenting with heat prostration during the “great summer
heat wave” in New York City had CF.
Cystic fibrosis was identified as an autosomal recessive disease.
1965
The fundamental physiologic defects were clearly established by Knowles and
colleagues and Quinton as the failure of cAMP regulation of chloride transport.
The genetic defect for CF was located on chromosome 7.
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR)
was identified by positional cloning.
1983
1985
1989
1990
Cystic fibrosis transmembrane conductance regulator was established to be a cAMPregulated chloride channel by complementation studies.
Cystic fibrosis: airway inflammation
Normal
Na+
Cl-
Cystic fibrosis
Na+
Cl-
Cl-
Cl-
ENaC
CFTR
Cl-a
K+
Na+
K+
Na+
2Cl-
K+
H2O
Na+
Na+
2Cl-
K+
H2O
Cystic fibrosis: nasal transepithelial potential difference
amiloride
Cl- free
forskolin
ATP
-60
PD (mv)
-50
normal
-40
PD
-30
-20
CF
-10
0
0
4
6
Time (m)
8
10
Cystic fibrosis: a historical timeline
1938
Cystic fibrosis (CF) of the pancreas was described by Andersen.
1953
The sweat defect was discovered by diSant'Agnese and colleagues when they noticed
that many of the infants presenting with heat prostration during the “great summer
heat wave” in New York City had CF.
Cystic fibrosis was identified as an autosomal recessive disease.
1965
The fundamental physiologic defects were clearly established by Knowles and
colleagues and Quinton as the failure of cAMP regulation of chloride transport.
The genetic defect for CF was located on chromosome 7.
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR)
was identified by positional cloning.
1983
1985
1989
1990
Cystic fibrosis transmembrane conductance regulator was established to be a cAMPregulated chloride channel by complementation studies.
Classes of cystic fibrosis-causing mutations
ADP
ADP
ATP
Class 3: regulatory
mutants that fail to
respond normally to
activation signals,
e.g., G551D
PKA
ATP
Class 4: CFTR
mutants that have
altered channel
properties,
e.g., R117H
ATP
ADP
Endosome
Class 2: CFTR
degradation in the
endoplasmic
reticulum, e.g., F508
Golgi
Class 1: premature
termination of CFTR
mRNA translation,
e.g., S489X
ER
Nucleus
Class 5: decreased
functional CFTR
synthesis or transport,
e.g., A455E
Cystic fibrosis: clinical phenotype associated with CFTR mutations
Severe lung disease
Milder lung disease
Mild lung disease
Pancreatic insufficiency
Pancreatic sufficiency
Pancreatic sufficiency
Abnormal sweat chloride
Abnormal sweat chloride
Equivocal sweat chloride
F508
R117H (5T)
R117H (7T)
G542X
3849 + 10kB C-to-T
3849 + 10kB C-to-T
G551D
2789 + 5 G-to-A
G551S
W1282X
R334W
D1152H
N1303K
G85E
A455E
R553X
G91R
3120 + 1G-to-T
R347P
1078 del T
R347H
R75X
R347L
Prospects for correcting cystic fibrosis: level of correction
Chillon M, et al. N Engl J Med. 1996; 332:1475.
Tissue affected
CFTR activity
100% (wt, 9T/9T)
unaffected
vas deferens
50% (wt, 9T, and mutant CFTR)
10% (wt protein, 5T/5T)
5% (wt protein, 5T, and severe mutant)
sweat duct
airway
4% (R117H, 7T, and severe mutant)
1% (R117H, 7T, and severe mutant)
pancreas
<1% (G551D, F508)
Pathogenesis of lung disease in cystic fibrosis
Davis PB, et al. J Respir Crit Care Med. 1996;154:1229.
Defective CF gene
Defective/deficient CFTR
Abnormal airway surface milieu
Bronchial obstruction
Infection
Inflammation
Bronchiectasis
Treatment of cystic fibrosis lung disease
Defective CF gene
Defective/deficient CFTR
Abnormal airway surface milieu
Chest physiotherapy
Mucolytics (rhDNase)
Hypertonic saline
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation
Treatment of cystic fibrosis lung disease
Defective CF gene
Defective/deficient CFTR
Abnormal airway surface milieu
Chest physiotherapy
Mucolytics (rhDNase)
Hypertonic saline
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation
Cystic fibrosis: organisms isolated from the lower respiratory tract
100
P. aeruginosa
Percentage positive
80
60
S. aureus
40
H. influenzae
20
B. cepacia
0
0-1
2-5
6-10
11-17
18-24
25-34
35-44
>45
Age (y)
Data compiled from Cystic Fibrosis Foundation Patient Registry, 2007.
Effect of Pseudomonas aeruginosa acquisition in cystic fibrosis
Demko CA, et al. J Clin Epidemiol. 1995;48:1041
Late acquisition (>6y)
Cumulative survival
1.0
0.9
0.8
Early acquisition (<6y)
0.7
0.6
males
females
6
8
10
12
Age (y)
14
16
Cystic fibrosis: bacterial colonization
Impaired mucociliary clearance
asialoGM1
Increased adherence
Impaired antimicrobial activity
CFTR
Antibacterial proteins
Impaired phagocytosis
Treatment of cystic fibrosis lung disease
Defective CF gene
Defective/deficient CFTR
Abnormal airway surface milieu
Chest physiotherapy
Mucolytics (rhDNase)
Hypertonic saline
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation
Cystic fibrosis: pathology
Cystic fibrosis: radiological findings
Cystic fibrosis: airway inflammation
mac
pmn
NE
O2-
TNF-a
IL-8
IL-1b
respiratory epithelium
Normal
Cystic fibrosis
Anti-inflammatory agents in cystic fibrosis: corticosteroids
Eigen H, et al. J Pediatr. 1995;126:515.
Lai HC, et al. N Engl J Med. 2000;342:851.
FVC (% predicted for age
A four year, randomized double-blind, placebo-controlled trial that compared the
efficacy of two doses (1 mg/kg/d and 2 mg/kg/d) of alternate-day prednisone therapy
with placebo in children with CF.
4
3
2
1
0
-1
-2
-3
-4
-5
-6
p = 0.0001
2 mg/kg
6
12
18
24
30
high-dose arm stopped
1 mg/kg
36
42
48 mos
placebo
Anti-inflammatory agents in cystic fibrosis: azithromycin
Saiman L, et al. JAMA. 2003;290:1749.
FEV1 (% predicted for age)
An five-month, randomized, double-blind, placebo-controlled trial that
examined the efficacy of azithromycin in patients with CF (age > 6 years, N =
251), chronically colonized with P. aeruginosa, and lung disease (FEV1 > 30%
predicted for age).
5
4
3
2
1
0
-1
-2
-3
-4
-5
-6
p = 0.009
azithromycin
28
84
168
196 days
placebo
Drug stopped
Cystic fibrosis: median survival age, 1940-2007
Median survival age (years)
inhaled mucolytics
35
30
25
37.8
anti-Staphylococcus
antibiotics
airway clearance
inhaled antibiotics
20
15
10
anti-Pseudomonas
antibiotics
5
0
1940
1950
1960
1970
1980
Year
1990
2000
Cystic Fibrosis Foundation Registry, 2007.
2010
Treatment of cystic fibrosis lung disease
Defective CF gene
Defective/deficient CFTR
Block
uptake
Increase Cl- efflux
Abnormal airway surface milieu
Amiloride
UTP/ATP
Hypertonic saline
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Mucolytics (rhDNase)
Chest physiotherapy
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation
Na+
Cystic fibrosis: alternative therapies to effect bioelectric properties of the
respiratory epithelium
CF
Altering other channels
Na+
Cl-
ENaC
Na+
Cl-
ClCa
CFTR
Cl-
Amiloride
UTP/ATP
Hypertonic saline
Aerosolized hypertonic saline for the treatment of cystic fibrosis
Elkins MR, et al. N Engl J Med. 2006;354:229.
An 48-week, randomized, double-blind, parallel-group trial that examined the efficacy
of inhaled hypertonic saline in patients with CF over 6 years of age.
Survival free of symptomdefined exacerbations (%)
100
75
p = 0.001
9.2 w
50
36 w
hypertonic saline
25
control
0
0
12
24
36
Period of observation (w)
48
Treatment of cystic fibrosis lung disease
Defective CF gene
Defective/deficient CFTR
VX809
VX770
PTC124
Block Na+ uptake
Increase Cl- efflux
Abnormal airway surface milieu
Amiloride
UTP/ATP
Hypertonic saline
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Mucolytics (rhDNase)
Chest physiotherapy
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation
Increase CFTR protein
Activate mutant form
Cystic fibrosis: correcting CFTR dysfunction
Zeitlin P. N Engl J Med. 2004;351:606
F508 CFTR
G551D CFTR
Cell membrane
Cell membrane
VX770
Endosome
Endosome
Proteasome
apical trafficking
degradation
Golgi
post-translational folding
translation
ER
Golgi
Low temperature
Glycerol
ER
transcription
Nucleus
VX809
Nucleus
Cystic fibrosis: correcting G551D CFTR dysfunction
Accurso FJ, et al. N Engl J Med. 2010;363:1991.
A four-week, randomized placebo-controlled trial that compared the effect of
regular treatment with VX770 with placebo in CF patients with G551D mutation.
[Sweat chloride] (mmol/L)
120
placebo
100
80
VX770, 150 mg
60
VX770, 250 mg
40
20
0
3
14
21
28 days
Cystic fibrosis: potentiating delF508 CFTR dysfunction
A two-week, randomized double-blind, crossover trial that compared the effect of
regular treatment with VX809 with placebo in CF patients with delF508 mutation.
[Sweat chloride] (mmol/L)
0
-2
-4
-6
-8
-10
25
50
100
200
Gentamicin-induced correction of CFTR function in patients with
cystic fibrosis and CFTR stop mutations
Response of nasal PD to chloridefree isoproterinol (mV)
Wilschanski M. N Engl J Med. 2003; 349:1433.
-8
-6
p = 0.03
pre-treatment
-4
-2
0
0
0.3
0.6
0.9
Gentamicin concentration (%)
1.2
post-treatment
Treatment of cystic fibrosis lung disease
Defective CF gene
Gene therapy
Increase CFTR protein
Activate mutant form
Defective/deficient CFTR
VX809
VX770
PTC124
Block Na+ uptake
Increase Cl- efflux
Abnormal airway surface milieu
Amiloride
UTP/ATP
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Mucolytics (rhDNase)
Chest physiotherapy
Hypertonic saline
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation
Provide normal gene
Active or completed human gene therapy protocols
Infectious diseases (40)
Human immunodeficiency virus (37)
Other viral diseases (3)
Monogenic diseases (58)
Alpha1-antitrysin deficiency (2)
Chronic granulomatous disease (3)
Cystic fibrosis (23)
Familial hypercholesterolemia (1)
Fanconi anemia (4)
Gaucher disease (3)
Hunter syndrome (1)
Ornithine transcarbamylase deficiency (1)
Purine nucleoside phosphorylase deficiency (1)
Severe combined immunodeficiency disease (6)
Leukocyte adhesion deficiency (1)
Canavan disease (3)
Hemophilia (5)
Muscular dystrophy (1)
Amyotrophic lateral sclerosis (1)
Junctional epidermolysis bullosa (1)
Neuronal ceroid lipofuscinosis (1)
Cancer (405)
Other diseases(66)
Peripheral artery disease (24)
Arthritis (4)
Arterial restenosis (3)
Congestive heart failure (1)
Coronary artery disease (21)
Alzheimer disease (2)
Ulcer (3)
Bone fracture (1)
Peripheral neuropathy (1)
Parkinson disease (2)
Eye disorders (4)
Erectile dysfunction (1)
Intractable pain (1)
Administration of an adenovirus containing the human CFTR cDNA to the
respiratory tract of individuals with cystic fibrosis
Crystal RG, et al. Nat Genet. 1994;8:42.
Pretreatment
Pretreatment
In vivo
transfection
In vivo
transfection
In vitro
transfection
In vitro
transfection
A controlled study of adenovirus-vector-mediated gene transfer in the nasal
epithelium of patients with cystic fibrosis
Knowles MR, et al. N Engl J Med. 1995;333:823.
CFTR mRNA
Patient No.
Cohort 1
1
2
3
Cohort 2
1
2
3
Cohort 3
1
2
3
Cohort 4
1
2
3
MOI
vehicle-treated
vector-treated
1
1
1
no
no
no
no
no
no
10
10
10
no
no
no
no
no
yes
100
100
100
no
no
no
yes
no
yes
1000
1000
1000
no
no
no
yes
no
yes
A controlled study of adenovirus-vector-mediated gene transfer in the nasal
epithelium of patients with cystic fibrosis
-40
-40
-30
-30
PD (mV)
PD (mV)
Knowles MR, et al. N Engl J Med. 1995;333:823.
-20
-10
-20
-10
0
0
10
10
-5
0
5
Days
10
-5
0
5
Days
10
Effectiveness of current gene transfer vehicles used in cystic fibrosis
Gene transfer vehicles
Adenovirus
Adeno-associated virus
Retrovirus
Murine leukemia virus
Lentivirus
Cationic liposomes
Molecular conjugates
DNA delivery
yes
yes
RNA expression
yes
no
CFTR function
no
no
ND
ND
yes
yes
ND
ND
maybe
no
ND
ND
no
maybe
Prospects for gene therapy of cystic fibrosis: submucosal gland
Engelhardt JF, et al. J Clin Invest. 1994;93:737.
epithelium (+)
duct (++++)
submucosal glands (++)
Sites of CFTR expression in the human airway
http://www.medicine.mcgill.ca/dynhist/histoimages
Prospects for gene therapy of cystic fibrosis: obstacles






Respiratory epithelial cells vs submucosal glands.
Unavailable target receptors.
Inability to bypass physical and functional barriers in the airway.
Possible biologic unsuitability of the airway epithelium as a target tissue.
Immunologic consequences.
Relevant outcome measure.
Treatment of cystic fibrosis lung disease
Provide normal gene
Defective CF gene
Gene therapy
VX809
VX770
PTC124
Amiloride
UTP/ATP
Hypertonic saline
Increase CFTR protein
Activate mutant form
Defective/deficient CFTR
Block Na+ uptake
Increase Cl- efflux
Abnormal airway surface milieu
Decrease mucus viscosity
Augment clearance
Bronchial obstruction
Mucolytics (rhDNase)
Chest physiotherapy
Decrease bacterial load
Infection
Antibiotics
Macrolides
Reduce host response
Inflammation
Corticosteroids
Ibuprofen
Replace damaged lungs
Bronchiectasis
Transplantation