By David N. Leff

Breathing can be hazardous to your health.

Every breath of air you inhale, whether by nose or mouth, is freighted with a potentially pathogenic payload of bacteria. What stops them, in most cases, from infecting the lungs and bronchial tubes are the epithelial cells lining these airways.

The exterior surfaces of those cells are studded with a dense mass of hair-like cilia, all beating in one direction, like the phalanx of oars on a racing shell. That direction is upward and outward, sweeping those invading microbes back upstream for expectoration out of the body.

Some people lack this ciliary protection, notably those suffering from cystic fibrosis (CF), which is the principal genetic disease afflicting Caucasian (i.e., white, Western) populations. The gene mutated in CF is known as CFTR, which stands for the protein it encodes: cystic fibrosis transmembrane conductance regulator. That defect is a prime target for efforts to correct the inherited gene mutation by gene replacement.

One such gene therapist is Eric Alton, at the British National Heart and Lung Institute of Imperial College in London. Here is how he laid out the cascade of events that explain why CF patients die young:

¿If you take an epithelial cell in the lining of the lungs,¿ Alton began, ¿one of its functions appears to be to move chloride ions from inside that cell onto the lining of the lung, outside the cell. The apparent reason it does that is because it is moving water with it by osmosis onto the airway lining. And the reason you need water there is because we have a defense mechanism called mucociliary clearance. So, when you inhale bacteria, the cilia, the hairs, flick it up toward the throat. And you appear to need an optimal volume of water to make that happen. The reason that you add water back to your lungs with chloride is because you sometimes dehydrate your airways when you¿re exerting yourself. It¿s a sort of moisture-replenishment mechanism.

¿CF patients don¿t appear to be able to move chloride,¿ Alton said, ¿because that chloride moves through the protein that the CFTR gene makes as its transmembrane chloride channel. If you have a defective gene, then you don¿t have the protein. Then, you don¿t have the chloride movement, so you don¿t have the water movement, so you can¿t optimally clear bacteria, which leads to the recurrent infections, mainly by Pseudomonas aeruginosa, that eventually end up in the death of the patient. That¿s one possible link between chloride movement and a CF life expectancy of around 30 years of age.¿ (See BioWorld Today, March 3, 1999, p. 1.)

But that¿s only half the story. Those chloride ions are hitched to sodium ions; together they make up the sodium chloride that accounts for the salt imbalance in the bodies of CF victims.

¿Sodium does the opposite from chloride,¿ Alton explained. ¿It goes the other way. Sodium comes from the airways¿ surface liquid into the cell. It also drives water with it, so it¿s reducing the water level. And those two ions appear to be the key factors that alter the volume of the airway surface cells¿ cilia-supporting liquid.¿

Alton is lead author of a paper in the current Lancet, dated March 20, 1999. It bears the title: ¿Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial.¿ The article¿s penultimate author is pulmonologist David Meeker, senior vice president of medical affairs at Genzyme Corp. in Cambridge, Mass.

That Phase I clinical trial, at the Imperial College-affiliated Royal Brompton Hospital in London, treated eight male CF patients in their early 20s with a gene-therapy plasmid consisting of a recombinant complementary DNA sequence of the CFTR gene complexed to a liposome vector. Eight control patients received only the lipid carrier as a placebo.

Both the gene and its proprietary vector were provided by Genzyme, which supported the British trial.

¿One of the major things Alton and his colleagues had developed,¿ Meeker told BioWorld Today, ¿was the ability to measure electrical potential differences in the lower airway. That was one of the most significant things about that study that¿s different from some of the earlier CF trials. It¿s a good measurement that tells us, hopefully, whether we¿ve been successful in getting CFTR in there.¿

That gene therapy experiment inserted its CFTR construct via two ports of entry, nasal and oral. ¿First,¿ Alton said, ¿we nebulized the plasmid as an aerosol into the mouth, inhaled like a conventional asthma nebulizer and aimed at the lungs. A week later we also did a nose study in the same patients. That was through a nasal delivery device.

¿We used the nose as a surrogate marker for the lungs,¿ he added. ¿They both attained about 25 percent correction up to normal levels of chloride conduction. There were no side-effect safety problems in the nasal route, but some transient and mild flu-like symptoms, and inflammation in the lungs.

Chloride Correction Up; Inflammation, Infection Down

¿We measured, in their sputum, markers with the inherent inflammation that these patients suffer,¿ Alton continued. ¿It was significantly reduced in the treated cohorts, but not in the placebo group. So, the 25 percent correction appears to correlate somehow with extent of lung inflammation. But whether the reduction of inflammation then makes the CF patients better, I don¿t know.¿

He and his co-authors didn¿t look at that in the lung, but ¿did do a time course in the nose, and the correction lasted up to three weeks,¿ he said. ¿It was completely gone after that.¿

Another result of the Phase I trial was marked diminution of the Pseudomonas bacterial flora found binding, in vitro, to cells from the CF patients¿ airway linings. ¿That¿s one of the reasons that we went for a clinical trial,¿ Alton said. ¿It¿s not, of course, the electricity; it¿s the bacteria sticking on the cells that kill these patients. We were encouraged to see that the bacterial adherence to the respiratory epithelium also decreased.¿

For now, neither Alton nor Meeker has any plans for more collaborative clinical trials. ¿Not until we know,¿ Alton said, ¿that we can get better gene transfer efficiency. So, we¿re working on that in our lab.¿

Meeker defined the hurdles ahead before patient studies can resume: ¿There were positive results from our Lancet paper,¿ he said, ¿but its conclusion was that significant improvements need to be made in that vector system before it¿s going to reach the level of viability as a product. What we decided at the end of that trial was that we were going back to the lab and really work on things like efficiency of delivery, which is still what plagues the cationic lipid system.¿

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