'Gapmer Antisense' Targets MMD1 Defect for Destruction

Researchers at Baylor College of Medicine in Houston and Isis Pharmaceuticals in Carlsbad, Calif., have announced encouraging results for their antisense-based strategy in development for the treatment of type 1 myotonic muscular dystrophy (DM1, or MMD1).

Thomas Cooper
MDA research grantee Thomas Cooper at Baylor College of Medicine coordinated the research team that developed the new gapmer antisense compound.

The strategy, known as gapmer antisense, attracts a naturally occurring cellular enzyme called RNase H to the site of the genetic defect in MMD1-affected cells, where it destroys toxic excess genetic material. RNase H is located in the cell nucleus, which is where the MMD1 defect is located.

MDA research grantee Thomas Cooper, a professor in the Department of Pathology and Immunology at Baylor, coordinated the study team, which published its findings online Feb. 27, 2012, in Proceedings of the National Academy of Sciences-United States.

"There is a great deal of work ahead to develop this approach into a viable therapy," Cooper said, "but the results from this study and from other labs using antisense approaches provide a general direction for future research. An important component is the collaboration of biotech companies that have the expertise and facilities for the development and production of a variety of antisense compounds. One should always anticipate that there are biological roadblocks ahead, but indications are that this is a productive approach."

CUG repeat expansions cause trouble in MMD1

Blocking or destroying extra genetic material known as CUG repeats in MMD1 is the primary goal of many therapeutic strategies in development, including the one Cooper's group is developing.

It is believed that doing either or both of these would improve the functioning of nerve and muscle cells and thereby inprove the functioning of muscles and other organs in people with MMD1.

The underlying cause of MMD1, a complex, multisystem disease affecting skeletal muscles, as well as the heart, brain and other organs, is an expanded stretch of a repeated DNA sequence on chromosome 19 known as a DNA triplet repeat.

In MMD1, the triplet repeat is composed of three nucleotides (chemical components of DNA) — cytosine, thymine and guanine — which are abbreviated CTG.

Normally, there are between three and 37 CTG triplet repeats on chromosome 19. But in MMD1, there can be hundreds or even thousands of such repeats.

When the DNA is converted to RNA (a natural process), the triplet repeat becomes cytosine, uracil, guanine, or CUG, and it known as a CUG repeat.

CUG repeat expansions cause a lot of problems for MMD1-affected nerve and muscle cells. For instance, they:

  • cause clumps of RNA and protein, which may be harmful in and of themselves, to accumulate in cell nuclei;
  • cause a protein called MBNL1 to become ensnared in the extra CUG RNA repeats, keeping it from its usual, important role in cells;
  • signal a protein called CUGBP1 (also known as CELF1) to last longer than it should, a cell-damaging effect; and
  • prevent the genetic instructions (RNA) for a protein called DMPK from leaving cell nuclei, a step that's necessary for it to be used for protein synthesis.

    In MMD1-affected cells, extra DNA leads to the creation of extra RNA, which forms clumps, ensnares the MBNL1 protein and sets in motion a cascade of problems affecting many organs. The extra RNA contains sequences known as CUG repeats, which the experimental strategy called gapmer antisense targets and destroys.

CAG gapmers corrected several defects in cells, MMD1 mice

Cooper and his colleagues conducted several experiments with their gapmer antisense compounds in cells in a lab dish that contained large CUG repeat expansions in their nuclei; and in mice with this genetic defect that develop an MMD1-like disease.

The investigators injected the muscle of one hind leg of each mouse with the gapmer antisense compound and the opposite leg with a different substance, which they expected to be inactive, as a comparison (control).

They found that the gapmer antisense compounds, known in these experiments as CAG gapmers, had several desired effects. For example, they:

  • disrupted the formation of RNA-protein clumps in cells in a lab dish;
  • specifically targeted CUG repeat expansions, having no effect on stretches of RNA containing 12 CUG repeats, the average number found in non-MMD1-affected individuals;
  • destroyed genetic instructions (RNA) for the DMPK protein containing 960 CUG repeats in a mouse model of MMD1, reducing the number of expanded stretches of RNA by 50 percent compared with untreated (control-injected) muscles;
  • reduced RNA-protein clumps in cells in MMD1 mice by 40 percent in the treated muscles compared to the control muscles; and
  • partially restored normal RNA splicing, a cellular process needed for correct protein synthesis, in the mouse RNA for three genes in which splicing is disrupted by the CUG repeat expansion.

Some damage to muscles occurred in MMD1 mice

The CAG gapmers and the inactive control gapmers caused some abnormalities to appear in the injected mouse skeletal muscles. The researchers propose that this damage may have been caused by a process called electroporation, which they used in the mice following the gapmer injections.

Electroporation makes the cells more permeable to injected compounds, but it may have damaged them in the process and probably would not be used in humans.

Combining CAG gapmers and morpholino antisense was even more effective

Cooper and colleagues performed additional experiments in the MMD1 mice using a combination of the CAG gapmer and another type of antisense-based experimental treatment for MMD1 known as CAG25. This compound, known as a morpholino antisense, blocks the interaction of the expanded CUG repeat with an important muscle protein called MBNl1, freeing MBNL1 to perform its normal role in the cell.

CAG gapmer antisense by itself reduced the number of RNA stretches containing 960 CUG repeats by 50 percent, but when CAG25 was added, the reduction was 75 percent.

Future looks bright for antisense that attracts RNase H

"There are several systemically administered RNase H-based antisense drugs in clinical trials, suggesting bright prospects in the future," the investigators write in their Feb. 27 paper. "The growing interest in therapeutic [antisense] will accelerate development of novel nucleotide modifications and delivery methods addressing issues of toxicity and [distribution]."

To learn more

  • Disrupted Disease Process, discusses how a small molecule called pentamidine counteracts some of the effects of abnormal genetic instructions in MMD1, Quest News Online, Nov. 4, 2009
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