New Gene Therapy Method Corrects Genetic Defects In Human Cystic Fibrosis Lung Models

The particular genetic defect that the researchers sought to correct results in a mutated cystic fibrosis transmembrane regulator (CFTR) protein. This protein normally transports chloride ions across cell membranes, thus maintaining cells' electrolyte and fluid balance. In turn, this balance allows bacteria to be cleared from the surface of airway cells and prevents infection. A mutated CFTR protein disrupts normal ion transport, and people with CF are prone to chronic, and often fatal, bacterial lung infections. CF is the most common genetic inherited disease in the Caucasian population, affecting about 30,000 individuals in the United States each year.

In addition to providing a doorway for chloride ions to pass across cell membranes, the CFTR protein also appears to be involved in regulating the action of other ion channels. Proper regulation of the CFTR channel together with all its neighboring channels seems to be necessary for normal lung function and has been a challenge for gene therapists approaching genetic treatment of this disease.

"The biology of CFTR is so complex that even 10 years after the gene was cloned scientists are still puzzled about how patients with CF get airway disease," said John Engelhardt, Ph.D., UI professor of anatomy and cell biology and internal medicine, and principle investigator of the study.

Using two different model systems, Xiaoming Liu, D.V.M., Ph.D., a postdoctoral associate in Engelhardt's lab and lead author on the study, and his colleagues, applied a novel approach to partially restore normal chloride ion transport to the resident mutant CFTR protein in CF airway cells. The team's results are published in the January issue of the journal Nature Biotechnology.

"These are very significant results for us and for the field," said Gerard McGarrity, president and CEO of Intronn, Inc., the company that developed the proprietary technology used in the study. "This represents a new way to perform gene therapy."

The gene therapy technique used to correct the CF defect in these human airway models is known as SMaRT (spliceosome-mediated RNA trans-splicing) and was developed and patented by scientists at Intronn, Inc., who collaborated with the UI team on this latest study. SMaRT works by correcting genetic defects at the RNA level rather than at the DNA level and may be useful in circumventing some of the problems associated with traditional gene therapy.

DNA sequences contain the genetic information that encodes proteins. The decoding process of DNA to protein occurs through a nucleic acid intermediate called messenger RNA (mRNA).

The blocks of genetic sequence that encode amino acids (protein building blocks) are known as exons. Exons are separated from each other by blocks of sequence called introns. Cells use a large molecular complex called a spliceosome to clip out the introns and splice together the exons. The resulting messenger RNA molecule is the final template that cells use to make a protein.

The UI team used an engineered, defective common cold virus as a carrier to deliver the genetic information needed to produce a short piece of RNA that contained the CF genetic correction. This RNA molecule attaches to the defective RNA at the site of the mistake. The spliceosome complex of the target cells is then "tricked" into skipping across the two RNA molecules to produce a full-length, error-free RNA. This resulting molecule is the template for a normal CFTR protein.

"The major advantage of this approach stems from the fact that correction will occur only in cells that have the defective mRNA. In essence, the technology allows only for correction at the cellular sites it is needed," said Engelhardt who also is director of the UI Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases.

This targeted correction is an important advantage of this technology over traditional gene therapy for treating CF.

"Traditional gene therapies for CF have primarily used a machine gun approach, attempting to express as much of the corrected gene product in as many cells as possible," Engelhardt explained. "But more is not necessarily better, and in CF there are potential disadvantages to over-expressing the corrected protein in the wrong place. For example, if you had an infection on the skin of your arm that could only be treated with a drug that was toxic to your liver, would you take the drug orally? Of course not, your doctor would treat only the infected area with a topically applied drug to the skin."

In contrast to the "more is better" machine gun approach, the SMaRT technology is analogous to a sharp shooter targeting low levels of protein to the right places.

"We demonstrated that we could indeed correct the resident mRNA in two human CF airway model systems and, surprisingly, we saw much more functional correction than we would have anticipated based on the abundance of corrected mRNA and protein," Engelhardt said. "It suggests that properly regulated CFTR expression may be more effective at reversing CFTR abnormalities than over-expressing a huge amount of protein in the wrong cell types. This new technology allows the cells' biology to dictate where the corrected protein is expressed."

In addition to treating diseases like CF where precise targeting of protein expression is important, Engelhardt outlined several other instances where this technology might improve on traditional gene therapy. In certain "dominant" genetic diseases the mutated protein itself is harmful. The SMaRT gene therapy intervenes at the level of the resident RNA, which means that mutant defective RNAs are transformed into corrected versions, hence reducing the level of a harmful genetically defective protein. This technology also reduces the amount of genetic information that gene therapy viruses must deliver to cells, which means that the approach might be useful in treating diseases caused by mutations in very large genes, such as Duchennes muscular dystrophy.

In addition to Engelhardt and Liu, the research team included the following UI researchers: Qinshi Jiang, Ph.D., now at Cornell University; and Weihong Zhou, M.D., and Yulong Zhang, both research associates in Engelhardt's lab. The team also included S. Gary Mansfield, Ph.D., M. Puttaraju, Ph.D., and Lloyd G. Mitchell, M.D., all at Intronn, Inc., Raleigh, N.C.; and Mariano A. Garcia-Blanco, M.D., Ph.D., and Jonathan A. Cohn, M.D., both associate professors at Duke University Medical Center.

The study was funded by grants from the National Institutes of Health, the Cystic Fibrosis Foundation and the UI Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases.