Antisense therapy has made its name as a sort of molecular hired gun,aimed at disrupting rogue genes in viral infections and cancer. Now,antisense is coming on as a hired healer, deployed to correct aberrantgene sequences, rather than suppressing them.
This softer, gentler role for antisense molecules made its bow intoday's Proceedings of the National Academy of Sciences (PNAS)under the title: "Repair of thalassemic human b-globin mRNA inmammalian cells by antisense oligonucleotides." Molecular biologistRyszard Kole at the University of North Carolina in Chapel Hill isthe article's senior author.
Thalassemia is a many-faceted hereditary disease that afflicts largepopulations in southern Europe, North Africa and parts of SouthAsia. (See BioWorld Today, Dec. 6, 1995, p. 1.)
It results from a deficiency in the globin protein of hemoglobin, thepayload of red blood cells, which ferry oxygen around the body.Normal adult globin consists of four polypeptide chains, produced bytwo separate genes _ alpha and beta. Mutant genes in either parent,or both, give rise to about a score of different degrees of thalassemicseverity, from no symptoms at all to a range of systemic ailmentsthroughout a miserable, shortened life, to fetal death in utero.
Treatment, such as it is, involves frequent blood transfusions.
Of nearly 100 mutations identified to date, no more than 10 areculpable of some 90 percent of cases worldwide.
In one form of the b-thalassemia gene sequence, the mutation causesan error in the splicing of its messenger-RNA sequences, in theirprocess of discarding the gene's now-redundant introns. (SeeBioWorld Today, Oct. 7, 1996, p. 1.)
"In such a mutation," Kole told BioWorld Today, "the splicing isincomplete, and retains in the splice array an additional 73nucleotides, which should have been removed. Because of this, nocorrect globin is translated from that mRNA."
"The main problem in thalassemia," biochemist Sudhir Agrawal toldBioWorld Today, "is that current therapeutic efforts haven't worked.Initially we used antisense as a tool to see if it is indeed the splicingthat causes the disease." Agrawal, a co-author of the PNAS paper, ischief scientist at Hybridon Inc. in Worcester, Mass., a pioneer firm inantisense research and development.
Antisense Forces Mis-Splice To Repent
"I hypothesized," Kole recalled, "that blocking the aberrant splicesites with antisense oligonucleotides may force the splicingmachinery to reselect the correct splice sites, and restore genefunction."
"After we confirmed that causality," Agrawal continued, "the nextquestion was: Can we correct it with antisense?" The affirmativeemerged from today's PNAS paper, which reports joint experimentsconducted by Agrawal at Hybridon and Kole in Chapel Hill.
"The validation is that the antisense oligonucleotides enter the cellnuclei, bind to the target and correct the globin-gene splicing in asequence-specific manner. That led us to this stage, where we arethinking of using it as a therapeutic approach for treatingthalassemia," Agrawal said.
In the experiments described, they incubated human (HeLa) cellsexpressing the mutant human b-globin gene with antisense oligostargeted at the aberrant splice sites. This generated correct mRNAand its globin product, both of which persisted for up to 72 hours.
"This novel approach," the paper concluded, "in which antisenseoligonucleotides are used to restore rather than to down-regulate theactivity of the target gene, is applicable to other splicing mutants, andis of potential clinical interest."
At the time the group began this research last year, Agrawalobserved, "appropriate animal models of thalassemic splice mutantswere not available." But in a neighboring laboratory, right at thesame University of North Carolina, molecular geneticist OliverSmithies was creating transgenic mouse models of thalassemia. (SeeBioWorld Today, Dec. 6, 1995).
Agrawal and Kole began using these animals about three months ago,to extend their validation from cells to entire mammals. "In the livinganimal," the Hybridon scientist said, "we inject the oligosintravenously or subcutaneously. They will be distributed throughoutthe body, and should get into the thalassemic cells, which are floatingaround in the bone marrow."
Looking Ahead To Human Hemotherapy
He expects these transgenic mouse trials to take 12 to 18 months."Now in animals," Agrawal observed, "we can perfect the oligos fortheir pharmacology and toxicology, while we look at their efficacy.Then we'll be ready to move toward the clinic, and see if thisapproach is applicable to human hemotherapy."
Based on the tissue-culture experiments, in which the corrected RNAand protein persisted for two to three days, Agrawal surmised that"clinically, we can implant them subcutaneously, with a slow-releasesystem for perhaps once-a-week administration." This would be lessonerous, he suggested, than the daily injections of insulin thatdiabetics must put up with.
He pointed out: "This is a new application of antisense, really on theborder of gene therapy. For the first time, it is being applied tocorrect a gene function, not suppress it _ as so far in antiviral andanticancer uses. In that familiar antisense strategy, the oligo cleavesthe target with RNA enzymes, and destroys it." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.