By David N. Leff

Two keys on your word processor — "cut" and "paste" — remove and rearrange words, sentences or paragraphs of text.

Computer programmers borrowed those words from old-time newspaper editors, who used scissors and paste to accomplish the same process. Both plagiarized those terms from a far older practice, one that goes back to the dawn of life on earth.

When a gene unwinds to express its pre-programmed protein, it slices its transcribed RNA into segments consisting of structural coding sequences called exons, separated by apparently non-functional stretches called introns. A complex machinery then splices the sliced-up exon bits and pieces into a continuous stretch of messenger RNA, on its way to synthesize the end product.

RNA splicing is far from a mutation-proof procedure, even if it is nature's way of making proteins.

"Overall," observed molecular biologist Ryszard Kole, at the University of North Carolina, in Chapel Hill, "an estimated 15 percent of all mutations that cause genetic diseases are splicing mutations."

Kole is senior author of a research report in the current Proceedings of the National Academy of Sciences (PNAS), dated April 28, 1998. Its title: "Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs."

Antisense Technology Applied To Critical Mutation

He and his co-authors have applied antisense technology to correct a critical mutation in hemoglobin genes gone wrong in beta-thalassemia. In its full-blown form, this inherited disease affects populations of victims in southern Italy, Cyprus, the Middle East, North Africa and Southeast Asia.

"As it happens," Kole told BioWorld Today, " there are about 100 mutations in beta-thalassemia that disturb the expression of beta-globin genes. And of this number, the major mutations, in terms of numbers of people affected, are splicing mutations. They interfere with the proper splicing of the gene's exons after removal of the noncoding introns."

Specifically, the PNAS co-authors focussed on a mutation in the intron that creates a defective sequence recognized by the cellular machinery as a splice site. This, Kole explained, "is a place where the cutting and pasting should take place. Because of the mutation, splicing is incorrect. A piece of the intron RNA remains in the final product, and therefore you don't get a correct messenger RNA. It's like a surgeon leaving a sponge behind in the incision when sewing up his patient."

As described in his PNAS paper, Kole continued, "what we find we can do, at the cell-culture level so far, is target a sequence complementary to this mutated splice site that binds to and blocks the RNA, thus preventing incorrect splicing from taking place. So it forces this special machinery to go back to good old intact splice sites that are still there and have not been used because of the new ones. They now restore production of correct beta-globin RNA."

It takes four genes to make a molecule of hemoglobin. Its protein consists of two alpha and two beta subunits.

"If you have this aberrant splicing," Kole pointed out, "beta globin is not made in proper quantities, or not at all, but the alpha protein is still being produced in the bloodstream. So, two things happen.

"First of all, you as a thalassemia patient do not make hemoglobin, so you have anemia," Kole said. "But an even worse result is the fact that alpha subunit proteins, because they don't have beta globin proteins to complex with, to play with, precipitate, thus destroying the red blood cells in the hemoglobin.

"Because of that," he continued, "the bone marrow expands and starts to make more red blood cells — which are no good to begin with. So not only are you missing hemoglobin but you also now have too much alpha globin, with nowhere to go.

"By our approach," Kole said, "if we are correcting or improving splicing, and restoring production of beta globin, we are allowing the existing alpha globin subunits to complex back to beta globin, correcting the imbalance."

To accomplish this repair job, Kole's team constructed a gene in a vector, which codes for a small nuclear RNA, called U7. They used it to replace the end fragment of that RNA, with antisense sequences complementary to that aberrant splice site in the beta globin gene.

Then, they put this construct into their model cell line, which produced the thalassemic beta globin RNA. "When the small nuclear RNA was made in the cell's nucleus," Kole said, "it hybridized with the beta globin RNA, and because it had a sequence complementary to the mutated splice site, it blocked that aberrant splicing and forced correct splicing to take place.

"Adding these modified molecules," he went on, "led to increased levels, about 65 percent, of correctly spliced mRNA carrying the code for beta globin."

Kole made the point that "because it's in a vector and this RNA is now made constantly in the cell, we had permanently modified splicing from incorrect to correct — sort of like a gene-therapy approach."

In previous work, the team used antisense oligonucleotides to block the same splice site.

"That approach raised some interest," Kole recalled. "For quite some time we had a collaboration going on with Hybridon Inc. [of Cambridge, Mass.]."

'Knock-In' Mice Wait Their Turn

"That, in a sense, was a pharmacological therapy," he observed. "In clinical practice, one would have to give the oligo[nucleotide] from time to time or constantly, to keep the splicing corrected. The step we made this time made the same sequence into a gene, which is expressed, and so is a one-time permanent treatment."

Meanwhile, the North Carolinians have constructed what they call a "knock-in" mouse model of a splicing-mutation thalassemia.

"What we and colleagues did," Kole said, "was to replace the normal mouse beta globin genes with a human beta globin gene. It contains this thalassemic mutation in the intron with the splicing defect in it. And those mice have thalassemia symptoms, by the way." The scientists expect to replay their cell-culture gene-repair experiment in these murine knock-ins "within a few months, if things work out," he said.

Kole and his co-authors "are now looking at a mutation in the cystic fibrosis gene, CFTR, where similar splice-site mechanisms operate." He also points to such mutations in phenyl ketonuria and adenosine deaminase deficiency, the inherited disorder that launched gene therapy in human patients a decade ago.

"Besides," Kole suggested, "what one can do is to affect splicing pathways in general, not necessarily in mutations. Rather, let's say, a method of affecting cancer-causing genes. (See BioWorld Today, May 15, 1997, p. 1.)

There are about 2 million asymptomatic carriers of beta-thalassemia in the U.S., mostly among immigrants from the disease's geographical focus.

"But there are not so many actual patients," Kole said, "because of genetic counseling," which helps carrier parents avoid the birth of children with the disease.

"The point is," he concluded, "if you had an effective treatment, you wouldn't need to do that. And in fact I know that among people with thalassemia this is a serious issue. 'Why do we have to subject ourselves to that avoidance,' they ask, 'when, people with diabetes, say, do not?'" *

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