CF Gene Therapy Clinical Trials

Introduction

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)

Author/Year	Organ	Design	Placebo	Repeat	N	Results
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)

Author/Year	Organ	Design	Placebo	Repeat	N	Results
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)

Author/Year	Organ	Design	Placebo	Repeat	N	Results
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 Dose-escalation 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