It’s been a decade and more since research geneticist Francis Collins discovered the mutant gene for cystic fibrosis (CF) in 1989.

That discovery led many gene therapists to have a field day at the prospect of correcting CF by inserting intact gene sequences into the genomes of patients suffering from that genetic disease. But so far they’ve been rained out, and the field remains wide open to effective gene therapy strategies against the malady.

CF is the commonest inherited disease in Caucasian populations. It afflicts some 30,000 new people a year in the U.S. alone with chronic, eventually fatal, lung infections. The gene defect is a mutated protein called CFTR cystic fibrosis transmembrane regulator. In healthy people, this protein transports chloride ions across cell membranes, thus guarding their electrolyte and fluid balance. That allows pathogenic bacteria to be cleared from lungs and airway epithelial cells, thus preventing infection.

“Traditional gene therapy,” observed cell biologist and gene therapist John Engelhardt at the University of Iowa, Iowa City, “approaches correcting genetic disease by overexpressing an unmutated version of the defective gene. That overexpression may or may not correct the genetic defect, depending on where, what cell types, and at what level that gene product has to be expressed.

“For instance,” he continued, “if a patient has a genetic disease, the gene, if overexpressed, may prove toxic. Or, if expressed in the wrong place, it might not reverse the pathophysiology of that disease. That’s the sort of traditional machine-gun approach to blasting as much gene product into the target organism as possible, which may not be the best solution. And in CF, given the complexities associated with the way CFTR functions, effective treatment of this disease may require proper regulation of the gene product.”

Engelhardt is senior author of an article in the January 2002 issue of Nature Biotechnology titled: “Partial correction of endogenous DF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.”

Traditional Gene Therapy For CF Rained Out

“The significance and the difference between our gene therapy approach and what’s traditionally been applied,” Engelhardt told BioWorld Today, “is that we corrected a CF genetic defect at the level of messenger RNA, not DNA. That, in and of itself, is not novel. But in this particular study we were able to report, for the first time, correcting the endogenous-derived CFTR mRNA in polarized airway epithelial cells.”

Engelhardt explained: “The airway is a barrier for protecting lungs from the external environment. At the same time it has to let air pass through. Polarity means that if a number of cells line up against each other, their top side of the membrane is different from the bottom. The reason is that the top is a seal, like a Ziploc bag around the edges of the cells, where they touch at the top of the membrane. That’s necessary,” he pointed out, “to protect the lung from invading bacteria.”

The acronym for “Spliceosome-mediated RNA trans-splicing,” as cited in the journal paper’s title, spells “SMaRT” the novel gene therapy approach developed by Engelhardt’s team and his collaborators at Intronn Inc. in Raleigh, N.C. Spliceosomes are protein complexes that clip out the otiose introns in a gene and splice the remaining functional exons into an integrated RNA sequence.

“The DNA encodes a carbon copy called pre-mRNA,” Engelhardt said. “This contains both exons and introns. The process of removing the nonfunctional introns involves splicing, and generates a functional mRNA transcript. This is transported out of the cell nucleus, and translated by the ribosomes into proteins.

“Trans-splicing,” Engelhardt recalled, “is where we’ve tricked this spliceosome complex into moving across two different mRNA templates, and splicing them together into one intact RNA strand. That allowed us to express part of a mRNA that was within the region of the mutant CF gene. Then we replaced part of that mRNA by the endogenous transcript, thereby giving rise to a fully corrected RNA molecule.”

To test their SMaRT concept in vivo, the co-authors constructed a mouse model that mimicked cystic fibrosis. “This is not to be confused with the current CF mouse knockout model,” Engelhardt pointed out, “which puts the same mutation a human has into the mouse. That mouse doesn’t get lung disease. It’s a huge problem for the field, because there’s no intact animal model of CF.

“Ours is a xenograft model,” he recounted, “in which we’ve made a small human airway in a mouse. This is not grafted into the animal’s lungs, but subcutaneously implanted under its skin. It gets vascularized and begins to behave like an airway. That bronchial segment, about 1 centimeter long, allowed us to access the lumen of that airway, do gene therapy to it, and evaluate correction at an electrophysiological level.”

Approach Might Work In Other Disorders, Too

“The gene-delivery vehicle was a recombinant adenovirus [AV] rendered avirulent,” Engelhardt went on. “The gene was the back end of the sequence that makes CFTR, plus the sequences required to fool the spliceosomes. This experiment demonstrated that we could indeed correct the resident mRNA in a human CF model.

“It’s important to understand the relevance of this,” he observed. “We’re not suggesting we could take adenovirus, do a clinical trial and cure a CF patient with this approach. Adenovirus is inherently an inefficient vector in the lung. That vector and that approach could never be used in a clinical trial. We’re using it to test the concept, not the delivery method. The gene therapy field doesn’t have the right delivery method yet,” he concluded. “That’s part of the problem.”

Engelhardt deems the potential of using this approach in other diseases besides CF “very good.”

“There are some disorders that are not amenable to traditional DNA-based gene therapy approaches. Those include dominant-negative disorders, such as blistering keratin skin diseases, for which the SMaRT-mediated approach will have applications. The other application will be in diseases where the gene is very large. Take Duchenne muscular dystrophy: The dystrophin gene is a bit too big to fit into a recombinant AV or AAV virus. In this case, using the SmaRT approach, one can splice in part of the gene.”