CF Gene Therapy Clinical Trials

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CF gene therapy approaches have been based on the assumption that the target cell population is the respiratory epithelial cells lining the lung, although the majority of clinical trials have used delivery of the GTA to the nasal and maxillary sinus epithelia of CF patient volunteers as a surrogate tissue. In total, there have been 25 published clinical trials for CF and non-viral approaches have accounted for nine of these (Griesenbach & Alton 2009).

The first non-viral clinical trials assessed the safety and efficacy of a single nasal delivery of plasmid DNA containing the CFTR cDNA under the transcriptional control of the SV40 (Caplen et al. 1995) or RSV 3'LTR promoters (Gill et al. 1997) complexed with DC-Chol:DOPE liposomes. Evidence for plasmid DNA transfer, vector-derived CFTR mRNA expression and partial correction of the CFTR Cl- channel defect was obtained in a subset of subjects. Alternative formulations, in which plasmid DNA contained the human CMV immediate early enhancer/promoter, were complexed with DOTAP liposomes (Porteous et al. 1997), EDMPC:Chol liposomes (Noone et al. 2000), or a peptide consisting of the sequence CK30 (Konstan et al. 2004) and were broadly shown to be similarly effective.

Crucially, no important safety considerations were raised after nasal application of any of these formulations. The target cells of the respiratory epithelium are mostly slowly dividing or terminally differentiated, thus ultimately repeated administration of the GTA will be required to treat the chronic lung disease.

The repeated administration of viral vectors appears to be limited by the existence of pre existing neutralising antibodies (Rosenecker et al. 1996), or antibodies produced in response to vector delivery (Halbert et al. 1997). Successful repeat administration of non viral vectors without loss of efficacy has been demonstrated in the human airways after delivery of three doses of plasmid DNA complexed with DC-Chol:DOPE liposomes (Hyde et al. 2000).

Collectively, these clinical studies provided 'proof-of-principle' for CF non viral gene therapy, but highlighted the need for development of formulations with enhanced efficacy.


Figure from Hyde et al 2000. (a) CFTR staining (red) from a non-CF individual, (b) a CF individual before treatment and (c & d) post-treatment.


Adenovirus Clinical Trials: 1993-2001 (10)

Zabner, 1993 Nose Dose-escalation
2e6 – 5e7 pfu
n = 1/dose
No No 3 NPD: decreased baseline towards non-CF values
mRNA: −ve, protein: −ve
Crystal, 1994 Lung Dose-escalation
2e6 - 5e9 pfu
n = 1–2/dose
No No 4 mRNA: − ve
protein: 1/4 + ve
Safety: no vector shedding
At highest dose transient inflammation.
Crystal, 1994 Nose Dose-escalation
2e5 – 5e7 pfu
n = 2/dose
No No 4 NPD: inconclusive, too variable
mRNA: 1/4 + ve, protein: 1/4 +
Hay, 1995 Nose Dose-escalation
2e5 – 5e8.5 pfu
n = 1–2/dose
No No 9 NPD: decreased baseline + amiloride towards non-CF values
Partial correction of Cl− transport
Knowles, 1995 Nose Dose-escalation
2e7 – 5e10 pfu
n = 3/dose
Vehicle control applied on contralat nostril
No No 12 NPD: no change mRNA: 5/12 + ve
Safety: no toxic effects at low doses
At highest dose mild mucosal inflammation 2/3 patients
Zabner, 1996 Nose Dose-escalation repeat administration 2 doses 2e7 –1e10 pfu
n = 4–6/dose
No Yes 6 NPD: partial correction of Cl− in some patients, but reduced effect with subsequent administration. All patients developed serum antibodies to vector, but not CFTR
Bellon, 1997 Nose Dose-escalation
1e5 – 4e8 pfu n = 2/dose
Dose-escalation 1e7 –5.4e8 pfu n = 2/d
No No 6 DNA: 6/6 + ve
mRNA: 4/6 + ve, protein: 4/6 + ve
Expression was transient in nose
Bellon, 1997 Lung Dose-escalation
1e7 – 5.4e8 pfu
n = 2/dose
No No 6 DNA: 1/6 + ve
mRNA: 1/6 + ve, protein: 2/6 + ve
Expression was transient lung
Harvey, 1999 Lung Dose escalation
Repeat administration 3 doses 1e6 – 1e9 pfu
n = 2/dose
No Yes 14 Sampled 3 and 30 days after each administration mRNA:
1st administration + ve only with highest dose, transient (− ve by day 30)
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
Zuckerman, 1999 Lung Dose-escalation 2e9 - 2e11 pfu
n = 2–3/dose
No No 11 DNA: + ve on day 4, transient
Immune response: Ad-specific T-cells, mild humoral response
Safety: mild flu-like symptoms observed, resolved about day 10
Joseph, 2001 Lung Dose-escalation
8e6 – 2.5e10 pfu
n = 2–3/dose
No No 36 DNA: 4/5 + ve on day 2
mRNA: 3/5 + ve on day 2
Perricone, 2001 Lung Dose escalation
8e6 - 2.5e10 IU/patient
Lobar instillation or aerosol
No No 14 DNA: all + ve on day 2
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: 1999-2007 (6)

Wagner, 1999 Sinus Dose-escalation
1 × 102–1 × 105 RU
Single and two doses n = 5/group
No No 10 DNA: 7/10 + ve day 14
DNA: 1/10 + ve day 41
DNA: 1/10 + ve day 70
CFTR mRNA: − ve
NPD: partial Cl− correction in some cases, effect transient
Aitken, 2001 Lung Dose escalation
1e10 – 1e13 DRP n = 3/group
No No 12 DNA: 6/6 + ve up to day 30 with two highest doses
mRNA: − ve
Safety: several adverse effects, three of which possible related to study (pneumonia, exacerbation)
Wagner, 2002 Nose 1 nostril 1e5 RU
contralateral nostril placebo (n = 23)
No No 25 No change in: rate of sinusitis relapse, NPD, histopathology and IL-8
IL-10: increased vs. placebo at day 90
Flotte, 2003 Nose Dose-escalation
3e1 RU – 1e9 RU placebo (n = 25) contralateral nostril
No No 25 Nose:
NPD: no change
Vector DNA: 2/25 + ve
Moss, 2004 Lung Repeat admin:
3 doses, 30 days apart 1e13 DRP (n = 37)
Yes Yes 37 DNA: 6/6 + ve (only assessed after 3rd dose)
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, 2007 Lung Repeat admin:
3 doses 30 days apart
1e13 DRP
Yes Yes 102 No changes in spirometry, days of antibiotic use or induced sputum markers (IL-8 + neutrophil elastase)
Safety: well tolerated

Non-Viral Clinical Trials: 1997-2012 (9)

Caplen, 1995 Nose DC-Chol/DOPE
Dose escalation
10–300 µg DNA n = 3/dose
Yes No 15 Vector DNA:
7/8 + ve (some problems with false + ves) CFTR mRNA: − ve NPD: partial correction (20%) of Cl− defect towards normal at day 3, undetected by day 7
Safety: well tolerated
Gill, 1997 Nose DC-Chol/DOPE/pDNA
40 + 400 μg DNA/nostril
n = 4/dose
Yes No 15 NPD: 2/8 transient correction of Cl− for 7–15 days
SPQ: 5/8 showed CFTR function
Porteous, 1997 Nose DOTAP
400 µg DNA
Yes No 16 DNA: 7/8 + ve on days 3 and 7
DNA:2/7 + ve on day 28
mRNA: 2/8 + ve on days 3 and 7
NPD: 2/8 partial Cl− correction up to 4 weeks
SPQ: − ve
Zabner, 1997 Nose GL67A v naked pDNA No No 12 DNA: 8/9 + ve
RNA: − ve, technical problems
NPD: statistically significant correction of Cl− with both GL67 and naked pDNA
No difference between vectors
Alton, 1999 Lung & Nose GL67ApDNA
Lung: 42.2 mg DNA
Nose: 11.8 mg DNA
Lipid only placebo
Yes No 16 Lung:
DNA: 8/8 + ve
RNA: − ve
PD: statistically significant Cl− correction in active group, 25% of normal values
SPQ: CFTR function in 5/6 patients
Bacterial adherence: 5/6 patients in active group reduced bacterial binding compared to pre-treatment values
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
Hyde, 2000 Nose DC-Chol/DOPE/pDNA
Repeat administration 3 doses 400 μg DNA/nostril
Yes Yes 12 DNA: 6/9 + ve after ≥ one dose
DNA: 1/9 + ve for all 3
mRNA: 7/9 + ve after ≥ one dose
mRNA: 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
Noone, 2000 Nose EDMPC/pDNA
Dose escalation
0.4–4 mg DNA placebo in contralateral nostril
Yes No 12 DNA: all + ve (some cross-contamination to placebo)
mRNA: − ve
NPD: no change
Ruiz, 2001 Lung GL67A/pDNA
7.9–21.12 mg DNA
No No 8 mRNA: 4/8 + ve, 3/4 received highest 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
Lipid and DNA have synergistic effect on inflammation
Konstan, 2004 Nose DNA Nanoparticles
Dose-escalation 0.8–8.0 mg DNA
n = 2–6/dose contralateral nostril placebo
Yes No 12 DNA: 12/12 + ve in active but cross-contamination in placebo
NPD: partial to complete Cl− correction 8/12, up to day 6
Safety: well tolerated, no adverse effects related to treatment


A pellet of E.coli containing a plasmid expressing a pink fluorescent protein.


Sheep lung parenchyma (cell nuclei blue) transduced with an adenoviral vector (green).


E.coli from a large scale industrial production of our clinical trial plasmid pGM169.


Schematic diagram of the large human airways.


Large scale lentivirus production in suspension culture.


Mouse lung large airway (cell nuclei blue) transduced with an adenoviral vector (green).


Purifying mRNA from tissue samples.


DNA fragments being cut from an agarose gel exposed to UV.


Proposed 3D model of the CFTR protein.


Pellets of DNA following precipitation.


A CFTR Western blot, to confirm protein production in cell culture.


Light microscope image of a human airway liquid interface cultures. Dark patches are mucous.


Human airway liquid interface cultures transduced with a lentivirus expressing Luciferase.


A cake that only some of us got to enjoy!


A frozen vial of GL67A (left) and a frozen vial of pGM169 plasmid DNA (right)