Duchenne muscular dystrophy (DMD) is a horrific disease characterized by relentless, progressive muscle loss. The effects of this disease are insidious and it leads to a terrible wasting away that ends in a slow, painful death. This muscle loss is very difficult to halt or reverse, and even though there have been tremendous advances in the cloning, sequencing and manipulation of the DMD gene, a variety of attempts to treat this disease have failed.
In 1986, Dr. A.P. Monaco et al. of Dr. L. Kunkel’s group isolated the DMD gene (Monaco et al., Nature 1986 22;323(6089):646-50). The DMD gene is extremely long (it is 2,500 kb long and consists of 79 exons that cover 1% of the x-chromosome, and it transcribed to form a transcript that is 14 kilobases long). In 1987, Dr. E.P. Hoffman and co-workers identified a 427 kilodalton protein encoded by the DMD gene. He named this protein “dystrophin”, which is absent from the skeletal muscle of most DMD patients (Hoffman et al., Cell 1987 ;51(6):919-28).
Success in cloning the DMD gene led to gene therapy trials to correct the genetic defect in the DMD gene. “Anti-sense Oligonucleotides” or AOs are short, easy to produce stretches of nucleic acid that specifically bind to the messenger RNA (mRNA) made from the DMD gene and influence the processing of that RNA. By changing the manner in which the messenger RNA is processed, a new mRNA is made that encodes a normal dystrophin protein rather than an abnormal dystrophin that does not work (Wu B, et al., Proc Natl Acad Sci U S A. 2008 105(39):14814-9; also see Wells KE, et al., FEBS Lett. 2003 552(2-3):145-9). The second strategy is to use modified viruses of deliver a normal copy of the DMD gene into the muscle cells (Goyenvalle A, et al., Science. 2004 306(5702):1796-9). While these techniques showed hopeful results in laboratory animals (mdx mice and DMD dogs), trials in human patients have been less successful and lead researchers to be less sanguine about gene therapy to treat DMD. As it turns out, the immune system of DMD patients views introduced dystrophin as foreign and mounts an immune response to it. Therefore, a new strategy is required.
Now, work by Francesco Saverio Tedesco and colleagues at the Division of Regenerative Medicine, Stem Cells and Gene Therapy at the San Raffaele Scientific Institute in Milan, Italy combines stem cell and gene therapy to deliver an artificial human chromosome into implanted stem cells to overcome these challenges in the mdx mouse model of DMD.
Previously, this research team had identified a blood vessel stem cell called a ”mesoangioblast.” Mesangioblasts have the ability to form blood vessels, but they can also cross blood vessel walls and differentiate into a many different types of mesodermal cell types, which includes muscle cells. Since the discovery of this stem cell, researchers in this lab constantly wondered if mesangioblasts could deliver a replacement dystrophin gene to abnormal muscles in mdx mice? Tedesco and his colleagues used a human artificial chromosome vector that was engineered to carry the entire normal human DMD gene (including the large regulatory regions). Then they transferred this vector into cultured mesangioblast from mdx mice, and injected the corrected mesoangioblasts directly into the skeletal muscles of recipient mdx mice that had a poorly functioning immune system. The use of mdx mice with compromised immune systems is an important step in preventing the immune system from rejecting the implanted mesangioblasts. The authors showed that the transplanted mesoangioblasts effectively engrafted into the muscles of mdx mice and expressed normal dystrophin protein. The muscle fibers from mdx mice transplanted with the modified mesangioblasts did not show any signs of muscular dystrophy, but, instead, were functional muscle fibers that lacked all DMD pathology. They also found that the transplanted mesoangioblasts differentiated into muscle satellite cells. Muscle satellite cells are the working muscle stem cell pool that produces new muscle cells under normal conditions.
However, in order to treat DM patients, all the muscles must be treated and not just injected muscles. To address this concern, the authors injected corrected mesoangioblasts into the arterial circulation of mdx mice. They found that the cells were able to not only able to cross blood vessel walls and home to dystrophic muscles, but they could contribute to the formation of new dystrophin-expressing muscle fibers. In further tests, mice that had received the mesoangioblast transplants showed reduced fiber fragility, increased force, and greater motor capacity on treadmill and freewheel tests.
There are still technical and regulatory hurdles that must be addressed before this strategy is used in DMD patients. For one, the immune response against dystrophin must be addressed. Nevertheless, stem cell-mediated transfer of a normal DMD gene by means of a human artificial can chromosome does show promise as a potential treatment for this tragic and ultimately fatal disease.