Science Editor

In the Jan. 12, 2009, online edition of the Proceedings of the National Academy of Sciences, researchers showed that morpholino oligomers can be used to transform red blood cells and reverse a form of thalassemia, a blood disorder caused by insufficient production of oxygen-carrying hemoglobin.

Oligomers - or, technically speaking, "splice switching oligonucleotides" or SSOs - are an RNA-based therapeutic. Like their better-known cousins antisense and short interfering RNAs, they work by targeting messenger RNA to change cellular processes, hopefully for the better.

But rather than preventing protein production from messenger RNA altogether, oligomers influence the process of splicing - the way different parts of messenger RNA are put together in the final protein.

Alternative splicing is a way for cells to make different forms of the same protein from the same starting DNA sequence. For example a protein might end up either membrane-bound or soluble depending on which exons of its messenger RNA sequence are strung, or spliced, together by the processing machinery.

Alternative splicing, Ryszard Kole told BioWorld Today, has been recognized increasingly over the past decade as a major way in which proteins diversify. "With the sequencing of the human genome, it became evident that alternative splicing is not an exception to the rule, but is the rule," he said. Kole is senior vice president of research and discovery at AVI BioPharma and senior author of the PNAS paper.

And with that recognition has come an understanding that splicing can be targeted to fight a number of diseases. Splicing errors are now thought to underlie the majority of inherited diseases. And the potential utility of targeting splicing is not limited to inherited disease, but could be used to influence metabolic and infectious disease as well.

"Splicing is not limited to genetic disease," Kole said, "so we can look at diseases that are not, strictly speaking, genomic."

Oligomers bind to splice sites and essentially make them invisible to the cell's processing machinery, which restores normal splicing if a mutation has caused an aberrant splice site in a cell's DNA sequence. AVI is in clinical trials with oligomers to treat muscular dystrophy.

But the work now published in PNAS showed that the technology has promise for treating other cell types that are more challenging than muscle fibers as well. Kole said that red blood cells "mature very rapidly" from stem cells to adult cells, and the beta-globin gene is expressed only for a very short time period.

Success, in the main, was due to two chemical features of morpholino oligomers.

Oligomers come with several backbones, and unlike most other types of backbones, the morpholino backbone is not negatively charged. That, Kole explained, has two consequences: First, it makes the morpholinos "very resistant to degradation by nucleases." Additionally, it gives them "different properties in terms of biodistribution and uptake."

Those properties make morpholinos more easily able to enter cells than other types of oligomers. But they still do not necessarily mean that the morpholinos can be delivered specifically. (See BioWorld Today, Nov 23, 2007.)

In the paper, the team from AVI BioPharma and the University of North Carolina achieved specific delivery through a second chemical modification. The researchers added a cell-penetrating peptide to the morpholinos that allowed them to specifically hone in on erythrocytes.

In general, Kole said, the morpholino backbone and the peptide tag work potentiate each other. When the two are combined, "the positive charge on the peptide is strongly accentuated. And that clearly improves uptake into erythrocytes - and, for that matter, a number of other cells that we have looked at."

In the PNAS paper, the authors reported that by injecting mice with morpholinos targeting the aberrant splice site for their particular form of thalassemia, they were able to improve beta-globin levels in knockout mice by about 10 percent - an increase that they argued is probably near the maximum observable effect, given a heterozygous mouse model and the life span of the animals.

AVI collaborated with academics from Thailand's Mahidol University and the University of North Carolina for the project, including 2007 Nobel Laureate Oliver Smithies, who has what Kole termed "a long-standing interest in beta-globin" and whose lab created the mouse model used by the scientists.

Kole said that the work, beyond its specific relevance to thalassemia, showed the general strengths of the combination of morpholinos and peptide tags.

AVI has investigated the approach in other indications as well and is considering a number of possibilities for which of them to advance, he said.

But basically, "we believe that [peptide-conjugated morpholinos] have clearly improved delivery to a number of tissues over existing chemistries . . . [the technology] opens up huge possibilities in genetic, metabolic and viral diseases," Kole noted.