Seventeenth Annual North American Cystic Fibrosis Conference, Anaheim, California, October 2003

Scientific Report - Seventeenth Annual North American Cystic Fibrosis Conference, Anaheim, California, October 2003

Dr Tim Lee (MRC Fellow) . Nov, 2003. [online]. Seventeenth Annual North American Cystic Fibrosis Conference, Anaheim, California, October 2003. St James's University Teaching Hospital, Leeds, UK. Available from http://www.cysticfibrosismedicine.com

Summary

•Criteria improved to judge the success of new therapies enhancing Cystic Fibrosis Transmembrane Regulator protein (CFTR) function.

•North American CF Foundation Drug Discovery Programme identifies 4-6 promising potential new treatments for CF.

•European IMMUNO trial of vaccine against P. aeruginosa gives mixed results.

• ENaC Sodium channel in CF lungs may be more important than previously thought.

•Encouraging pre-clinical progress towards gene therapy for CF.

Detailed Report

Criteria improved to judge the success of new therapies enhancing CFTR function

Up to now, most laboratory and clinical studies of CF gene therapy and agents increasing CFTR function have used increased cyclic-AMP mediated chloride conductance as the endpoint to judge success. This has been because it is known that CFTR functions as a cyclic-AMP dependent chloride channel. In the laboratory this increase in chloride conductance can be measured in individual cells and across epithelial sheets grown in tissue culture. In clinical trials involving volunteers with CF the same measurement can be made by measuring nasal or rectal potential difference (PD).

However, there are a number of potential problems in using such measurements as the primary endpoint in such studies [1].

1) Whilst it is known that people with CF have abnormalities in chloride conductance (indeed this forms the basis of two diagnostic tests for CF), it is not yet known how much correction would be required to successfully treat the condition.

2) Nasal PD measurements require experienced operators, and to compare results between centres standardised protocols must be used. Even using the same operator and protocol reproducibility is limited, with variation of about 15%. Up to now, many centres have used different protocols for measuring nasal PD, which has made it virtually impossible to accurately compare results between different clinical trials.

3) CF is a complex disease, and severity does not depend purely on the level of impaired chloride conductance. For example, people with the A455E CF mutation have nasal PD measurements indistinguishable from those with DF508, but much better long-term lung function. If the aim of new therapies (such as gene therapy) is to improve lung function to the level seen with A455E, then clearly nasal PD could not be used as the only measure of success.

Whilst nasal PD may be a useful initial test of success, it doesn’t really address the important issues for people with CF, such as reducing chronic lung infection and inflammation.

Thus it was recommended that future trials should look carefully for changes in bacterial adherence, bacterial load in sputum over at least 6 months, changes in mucus quality, inflammatory markers [2], and possibly changes in imaging such as high resolution CT scans. It would also be useful to measure improvements in lung function, however, such changes are usually subtle, and would potentially need to be measured for at least 4 years to be able to detect a significant difference [3]. These improved measurements of success should enable researchers to make faster progress towards developing further treatments that will be of real benefit to people with CF.

New CF Drug Discovery Programme

The opening Plenary session of the conference focussed on the huge effort that is being made by the North American Cystic Fibrosis Foundation to identify and develop new therapeutic agents to improve CF treatment [4]. They have invested 100 million dollars in a High Throughput Screening Programme for new drugs that has been in progress for the last 3 years.

Essentially this involves testing over 4 million known drugs and related chemical compounds on DF508 CF cells grown in tissue culture using a substantial automated facility. The CF cells are incubated with each compound and then measurements are made of DF508 CFTR function, inflammatory markers, and bacterial adherence. This identifies promising compounds, which can then be tested in more detail.

So far this approach has identified 4-6 promising drugs that have entered a further stage of laboratory testing.

Drugs that are already available in the UK that have been developed and tested by the North American Cystic Fibrosis Foundation include Pulmozyme (Dnase); TOBI; and Azithromycin. These therapies are all useful agents for those with chronic pulmonary Pseudomonas aeruginosa infection.

In addition to this screening programme, there are many other therapeutic agents currently under development:

INS37217: This drug under test at the University of North Carolina, USA improves chloride secretion via a non-CFTR mechanism and inhibits sodium transport. A Phase II clinical trial in 21 volunteers (10 with CF) demonstrated that when it is perfused into the nose it is safe and effective [5]. A further Phase II clinical trial testing inhalation of this agent into the lungs is currently underway.

SPI-8811: Also improves chloride secretion, and currently undergoing clinical trial at Johns Hopkins University, Baltimore, USA [3].

Moli-1901: This agent also increases chloride conductance, and has been shown to be safe when nebulised into the lungs of 16 volunteers with CF. A trial to test effectiveness is awaited. [6].

Cox-2 inhibitors: It is known that increased inflammation in the lungs of those with CF is a major determinant of pulmonary damage. These selective anti-inflammatory agents inhibit the enzyme COX-2 and are already widely used to treat arthritis, as they are effective as anti-inflammatories but have a lower incidence of gastro-intestinal side effects than non-selective agents. The North American CF Foundation plan to assess these agents in CF [4].

Tobramycin inhaler: Nebulised antibiotics are known to be effective in eradication of new growth of P. aeruginosa [7,8] and against chronic P. aeruginosa infection [9], but are time-consuming to take and add to the burden of treatment. A new inhaler that delivers Tobramycin has recently been tested in 14 volunteers and demonstrated comparable efficacy to the nebulised drug, with the major advantage that just 2 minutes are required per dose, as compared to 15-20 minutes using a nebuliser [10].

Cardiac Glycosides: These drugs, particularly Oleandrin, are effective at reducing secretion of the inflammatory mediator IL-8 by CF cells in tissue culture. Certain of the active cardiac glycoside drugs have also been shown to activate corrected trafficking of DF508 to the surface of the cell [11]. Overall these agents seem demonstrate great early promise, and will be moved on to further laboratory based studies. As they are already licensed for use to treat cardiac conditions progress to a large clinical trial could potentially be rapid.

Vaccination to protect against Pseudomonas aeruginosa

Results from the European IMMUNO Trial on vaccination against Pseudomonas aeruginosa were presented at the Anaheim meeting [12]. Chronic infection with the bacteria P. aeruginosa is a major cause of lung damage in CF, so it would be extremely useful to develop an effective vaccine that would offer protection against this organism. The IMMUNO Trial ran across Europe between 1997-2002 and enrolled 483 people with CF. This vaccine was raised against P. aeruginosa flagella.

The results demonstrated that the intramuscular injection was safe, produced a good rise in serum vaccine-specific protective antibodies, and significantly reduced the rate of first ever growth of P. aeruginosa in the sputum.

However, disappointingly there was no difference in the amount of chronic P. aeruginosa infection after 2 years. This may be for 3 reasons:

1) The vaccine doesn’t protect against chronic P. aeruginosa.
2) Other effective treatments against new growth of P. aeruginosa such as eradication therapy using ciprofloxacin and colomycin are now widely used in Europe, consequently the vaccination was unable to show additional benefit.
3) More follow-up of these patients (e.g. at 5 years) will be needed to show a significant difference.

It remains to be seen which of these 3 reasons are responsible for the disappointing result of the trial, but the early data suggest that further analysis at 5 years will show a benefit.

In the meantime other clinical trials of other types of vaccine against P. aeruginosa are ongoing.

Importance of ENaC Sodium Channel in CF

There was a lot of interesting research presented at this year’s conference regarding the importance of another cell-surface ion channel, not CFTR, called ENaC. This channel transports sodium from the surface of the lungs into the lung cells. Because CFTR regulates ENaC function the amount of sodium transported is thought to be increased in people with CF, resulting in the build up of sticky mucus in the lungs and increased susceptibility to bacterial infections.

Of most interest at the conference was a new laboratory model of cystic fibrosis lung disease, caused not by “knocking-out” CFTR function, but instead by over-expressing ENaC [13]. This model demonstrated many of the key features of cystic fibrosis lung disease (reduction in airway surface liquid, reduced muco-ciliary clearance, increased mucus plugging of the lungs), without any direct impairment of CFTR function.

Therefore, if drugs could be developed that would block the transport of sodium through this channel, they could reduce the amount of sputum and lung infection in CF. Amiloride has such activity, and showed some promise in early clinical trials [14], but was not sufficiently potent or selective. Early laboratory data concerning a number of new ENaC blocking agents was presented at the conference [15]. Critical requirements are a long duration of blockade, and selectivity to minimise renal toxicity leading to systemic electrolyte imbalance.

Recent developments in CF gene therapy research

This year was more encouraging than last year in terms of pre-clinical progress made towards effective gene therapy for cystic fibrosis. The key problem remains developing an effective delivery system to deliver the replacement gene into the cells. Because lung epithelial cells only last about 120 days before being replaced by new cells, it is likely that any future gene therapy treatment for cystic fibrosis would have to be given at least 3 times a year. Therefore a delivery system has to avoid causing any immune response and inflammation, as any such response would probably get worse with each dose.

Adeno-associated virus (AAV)

Currently the most promising delivery system appears to be adeno-associated virus (AAV) [16]. This virus is not known to cause disease in humans, and inserts the replacement gene into chromosome 19 of the target cell genome allowing expression of the replacement gene for the lifetime of the cell. Early trials used serotype AAV-2, which only just had sufficient capacity to carry the gene encoding CFTR. Consequently there was no room for an efficient promoter to drive gene expression, but despite this limitation CFTR mRNA expression could be detected in pre-clinical trials.

A modified version of this AAV-2 CFTR vector has been used safely in 95 human volunteers with CF in a series of Phase I and Phase II clinical trials. Results have demonstrated that CFTR function is improved, and inflammatory markers reduced. The most recent trial, in 37 volunteers with CF, showed a significant improvement in FEV1 and significant fall in IL-8 levels compared to placebo [17]. However, the effectiveness reduces with each subsequent dose, due to the immune response developing.

Problems encountered with AAV-2 include the following:

1) A paucity of receptors for AAV-2 on the apical surface of the lung epithelial cells.
2) The mucus barrier.
3) Once the AAV-2 enters the cell, many vector particles are re-routed to the proteasome for degradation, rather than delivering the CFTR gene to the cell nucleus for successful transcription.
4) Weak promoter activity.
5) Immune response to AAV-2 with repeat dosing.

Many strategies to overcome these problems were presented at the conference.

One such strategy is to explore the use of other serotypes of AAV [18]. Initial work demonstrated that serotypes AAV-5, AAV-1, and AAV-6 were more efficient than AAV-2, and now more than 40 AAV serotypes have been discovered and are being tested. As a result, clinical trials are now planned using AAV-6 [19].

Promoter activity of the AAV-2 vector system can be enhanced up to five-fold in tissue culture by adding a further 83 base-pair transcriptional element [17].

Agents that modulate proteasome function can re-route the AAV vectors from the proteasome to the nucleus [20]. This can have an enormous effect on the amount of therapeutic gene expressed, by up to 1000-fold. One such agent is the anti-cancer drug doxorubicin. Side-effect profiles will have to be carefully evaluated but this could potentially be a major breakthrough. Such agents could be given at the same time as the gene therapy, perhaps three times a year.

Promising results were presented that changing the proteins on the surface of the AAV-2 (called “pseudotyping”) could increase effectiveness by a further 30-fold in pre-clinical studies, with the potential of a reduction in immune response, particularly if the doses were spaced out [21].

Pseudotyped lentiviral vectors

Lentiviral vectors also offer the possibility of providing sustained expression of the therapeutic gene, as they integrate into the DNA of the target cell. Again, they are hampered by a lack of suitable receptors on the apical surface of the airway cells, but data was presented that the addition of modified proteins from Ebola virus could increase uptake from the apical surface in pre-clinical studies [22,23].

This vector system has proved partially successful in treating another single gene defect, Severe Combined Immunodeficiency, although because it integrates the therapeutic gene randomly into the genome of the host cell there is a danger of side effects such as leukaemia [24]. This problem of random integration will have to be solved prior to a clinical trial in CF volunteers.

Parainfluenza Virus type 3

This vector system has the considerable advantage that it specifically targets the airway epithelial cells from the apical surface and specifically targets ciliated cells. This vector, containing the CFTR gene, has been shown to efficiently correct the abnormalities seen in CF cells in tissue culture [25]. Development will now be directed at generating forms that can be safely delivered to the human airway in vivo.

Non-viral gene delivery

Whilst viral vectors show promising efficiency, the immune response remains problematic. Delivery systems based on plasmid DNA (a circle of double stranded DNA encoding the therapeutic gene with appropriate promoter) have the potential advantage of being less immunogenic. Data from a clinical trial involving 12 volunteers with CF was presented at the conference. CFTR plasmid DNA compacted with the basic protein polylysine linked to polyethylene glycol was shown to be safe when administered to the nose, with partial correction of nasal potential difference, and no evidence of immune response [26].

Agents to rescue mutant CFTR

Various drugs are under test that can increase the cell surface expression and function of the mutant CFTR found in people with CF [27,28]. Clinical trials are ongoing and results awaited.

Conclusion

This year the basic science and gene therapy side of the conference was more encouraging than the previous 2 years. The increased consensus on important endpoints for the evaluation of new therapies, coupled with important pre-clinical progress in the vector field, were particular highlights. An increased understanding of the underlying defects in CF, particularly with regard to the role of ENaC, will provide stronger foundations for the ongoing development of new therapies.

References

All references unless otherwise stated from Pediatric Pulmonology, Supplement 25, 2003.

1) Alton EW. Evaluation of CFTR functions in humans. Symposium session S3.2, page 101.
2) Accurso FJ et al. Anti-inflammatory endpoints in CF: Lessons from clinical trials. Symposium session S3.4, page 104.
3) Moss RB. Disease progression in CF: Can we gain the upper hand? Plenary session III, page 55.
4) Collins FS et al. From genes to drugs: The CF master plan. Plenary session I, page 24.
5) Noone P et al. Dose-effect for a novel P2Y2 agonist (INS37217) to induce chloride secretion across normal and CF airway epithelia. Abstract 185, page 248.
6) Zeitlin P et al. A safety and pharmacokinetic assessment of inhaled administration of Moli-1901 in CF patients. Pediatric Pulmonology 2002, Supplement 24, page 259.
7) Fredericksen B et al. Antibiotic treatment of initial colonisation with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in CF. Pediatric Pulmonology 1997; 23: 330-335.
8) Gibson RL et al. A randomised controlled trial of inhaled tobramycin in young children with CF: Eradication of Pseudomonas aeruginosa from the lower airway. Pediatric Pulmonology 2002; Supplement 24, page 300.
9) Conway SP. Evidence for using nebulised antibiotics in CF. Archives of Disease in Childhood 80: 307-309.
10) Newhouse MT et al. Inhalation of a dry powder Tobramycin Pulmosphere formulation in healthy volunteers. Chest 2003; 124: 360-366.
11) Pollard HB et al. Pharmacogenomics of cardiac glycosides: Candidate CF drugs discovered by a high throughput screen for suppression of IL-8 secretion. Abstract 203, page 254.
12) Döring G. Relevant issues in bacterial vaccine development for patients with CF. Symposium session S9.1, page 128.
13) Mall M. Overexpression of ENaC in mouse airways: A novel animal model for chronic bronchitis and CF lung disease. Symposium session S7.3, page 121.
14) Knowles MR et al. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl J Med 1990; 322: 1189-1194.
15) Hopkins S. Development of long acting sodium channel blockers for the treatment of CF lung disease. Symposium session S7.4, page 122.
16) Flotte TR. Introduction: CF gene therapy with AAV. Symposium session S15, page 156.
17) Carter BJ et al. AAV-CFTR gene therapy for cystic fibrosis: Retrospect and prospect. Symposium session S15.3, page 159.
18) Wilson JM. Novel vectors for gene therapy of cystic fibrosis. Symposium session S15.1, page 157.
19) Miller AD. AAV reporter vector trial. Symposium session S15.2, page 158.
20) Zhang N. Intracellular barriers to rAAV-mediated CFTR correction in the CF airway can be overcome using proteasome modulating agents. Symposium session S15.4, page 160.
21) Weiner DJ et al. Novel pseudo-typed adeno-associated virus vectors for lung-directed gene transfer. Abstract 208, page 255.
22) Kobinger GP et al. Stable and efficient gene transfer in airway of non-human primates with HIV vector pseudotyped with deletion mutant of the Ebola envelope glycoprotein. Abstract 209, page 256.
23) Sinn P et al. Targeting apical entry in airway epithelia using pseudotyped FIV-based lentiviral vectors. Abstract 212, page 257.
24) Hacein-Bey-Abina S et al. Sustained correction of X-linked severe combined immunodeficiency by ex-vivo gene therapy. N Engl J Med 2002; 346: 1185-93.
25) Zhang L et al. Restoration of perciliary liquid volume to normal levels in human CF airway epithelium after targeted expression of CFTR in ciliated cells with a parainfluenza virus type-3 CFTR gene transfer vector. Abstract 211, page 256.
26) Konstan M et al. Single dose escalation study to evaluate safety of nasal administration of CFTR001 gene transfer vector to subjects with CF. Abstract 215, page 258.
27) Verkman AS et al. Small-molecule activators (potentiators and correctors) of ?F508 CFTR identified by high-throughput screening. Abstract 14, page 190.
28) Wright JM et al. Microarray assessment of phenylbutyrate correction of CFTR trafficking in IB-3 cell line suggests pulsed drug application may be therapeutically effective. Abstract 15, page 190.