转载：荷兰进行的51号外显因子跳跃试验中4个孩子的肌肉中发现抗肌萎缩蛋白

荷兰进行的51号外显因子跳跃试验中，参加实验的4名儿童均发现了短的抗肌萎缩蛋白科学家正在为系统的全身递送试验，包括肺和心脏做准备。

请英文好的给翻译一下，谢谢。

Exon skipping, the first clinical trial with Duchenne boys.

The exon skipping technique tries to slow down the fast Du¬chenne dystrophy into the much milder Becker dystrophy. It does not alter the gene itself with its mutation, but affects how the defective gene is read and processed. Exon skipping will not be a cure for Duchenne dys¬trophy, it should only reduce the severity of its symptoms.
If a mutation - a deletion, duplication or point mutation - disturbs the reading frame of the messenger RNA, mRNA, and thus causes Du¬chenne dys¬trophy, the frame can be restored by artificially removing from the mRNA one or more exons with anti¬sense oligoribonucleotides, AONs. They are short pieces of RNA whose sequen¬ces are constructed in such a way that they attach themselves only to a complementary se¬quence of the pre-mRNA inside the exon to be removed or at its border regions. These AONs thus interfere with the splicing ma¬chinery so that the tar¬geted exon or exons are no longer included in the mRNA, they are skipped.
As this skipped mRNA is shorter than nor¬mal, the dystrophin protein is also shor¬ter, it con¬tains fewer amino acids. If the missing amino acids are part of non-essential regions, like the rod domain, the shorter protein can often still perform its stabi¬liz¬ing role for the muscle cell membrane. The result would be the change of the severe Du¬chenne symptoms into the much milder sym¬ptoms of Becker mus¬cular dys¬tro¬phy.
For exon skipping, mostly two kinds of che¬mically protected AONs are used. They have to be protected so that they are not or only slowly de¬stroyed in the muscle cells by nucleic acid destroying enzymes. The two AONs are the 2'O-methyl-phosphoro¬thioates, also called 2’O-methyls and the morpholinos.
As Judith van Deutekom, now head of research of Prosensa B.V., a biotechnology company in Leiden in the Netherlands, was unable to be present at the meeting in Philadel¬phia, Elizabeth Vroom , president of the inter¬national United Parent Project Muscular Dys¬trophy, UPPMD, reported on the first in-human trial with the exon skipping technique which was completed at the end of 2006. The following is a summary of what Drs. van Deutekom and Vroom said at the meetings in London and Philadelphia.
The aim of this trial was to prove that exon skipping is feasible in Duchenne patients. It was a local study on a small area of a single muscle, the tibialis anterior muscle of the shin, which was being treated with an 2’O-methyl AON against exon 51. The trial was designed to pro¬vide a proof of principle, and although dystro¬phin of the desired structure was generated, this was so local that the treated boys did not obtain a therapeutic benefit.
The Dutch researchers selected the 2'O-me¬thyl version of the anti-51 AON, because they have extensive experience with this type of che¬mically stabilized AONs, not only by success¬fully treating muscle fibers in cell cultures but also by local and systemic injection into indivi¬dual muscles and the blood circulation of living animals.
Exon 51 was selected as the first skipping target because successful skipping of this single exon would allow restoration of the protein reading frame for up to 25% of all Duchenne boys with deletions. Initially, six boys were selected for this trial, but as the results of the treatment of the first four boys were so signi¬ficant and positive, it was not necessary to treat the last two boys too. The four treated boys were 10 to 13 years old with deletions of the exons 50, 52, 48-50, and 49-50. The trial was open, mean¬ing that everybody concerned knew that all four boys were receiving the AON.
Because exon skipping is a new medical pro¬cedure, intensive clinical and molecular genetic tests were performed on each boy before the start of the trial. As the Dutch regulatory agency did not allow a muscle biopsy to be performed be¬fore the trial, a skin biopsy was taken from which cell cultures were prepared. In these labo¬ratory procedures, the particular deletion, pre¬viously determined on the DNA level, was con¬firmed in the mRNA, and the base sequences of the border regions before and after the deleted exons were determined also. In addition, the entire dystrophin gene was screened to make sure that there were no unexpected irregularities. Although it was already known that the skipping of exon 51 works well in living animals and in cell cultures from Duchenne patients, the skipp¬ing procedure was repeated on the muscle cell culture from the skin biopsies of each boy, in order to exclude any risk that it would not work in the living muscle during the trial. For further safety reasons, the boys were treated one after the other, meaning that only after the results for the first boy were positive and did not show any side effects, the second boy was treated, and so on.
Each of the four boys received one single injection under local anaesthesia into a small area of the tibialis anterior muscle of a solution containing 0.8 mg of the anti-51 2’O-methyl AON. Four weeks after the in¬jec¬tion, a muscle biopsy was performed at the injection site and the muscle tissue checked for the presence of the shortened, skipped, mRNA and its resulting dystrophin protein.
At the end of 2006, the trial was halted, the results were analyzed and prepared for publi¬cation. The mRNA and dystrophin analyses on the muscle tissue from the biopsies showed that new shortened dystrophin was present at signi¬ficant levels in the majority of muscle fibers in all four boys. It could also be shown that the dystrophin-positive fibers were not revertant fibers which could have been produced by spon¬taneous exon skipping without the help of an AON. And there were no side effects in all four treated patients. The full results will be published in the New England Journal of Medicine.
The Dutch researchers are now preparing the next clinical trial during which they will aim at full-body (systemic) administration of the exon 51 2’O-methyl AON so that the potential drug can reach all muscles including those of the lung and the heart. These studies will be short-term, one month, and long-term, six months, and will be done with different amounts of AON to deter¬mine the most effective dosage which possibly could already slow down the boys' Duchenne symptoms signifi¬cantly. How much the muscle function will be improved will also depend on the actual resulting functionality of the Becker-like proteins obtained after skipping.
Also, more systemic studies with mice will be performed to investigate the phamacodynamics of the AONs to find out what exactly happens with them inside the muscle fibers. In some of these animal studies, the AONs will be injected subcutaneously, under the skin, because this will later be the most practical type of application if repeated injections are needed. In similar studies, it has already been shown that the systemic ap¬pli¬cation works well, that even the heart muscles can be treated, and that there are no serious adverse effects on the liver enzymes or other blood values.
The full development of exon 51 skipping is just the start of Prosensa's research program to find an effective treatment for Duchenne mus¬cular dystrophy. The development of other AONs for other deletions will follow soon after. In addition to the 2’O-methyl AON for skipping exon 51, the company has already designed and produced in sufficiently large quantities of other AONs for skipping the exons 43, 44, 45, 46, 50, 52 and 53. These AONs together would allow the treatment of over 65% of all patients with deletions.
In the future, it will also be possible to use this technique to restore the reading frame for some duplications or when more than one exon has to be skipped. For instance, the multiexon skipping of the 11 exons 45 to 55 would produce a Becker dystrophin in up to 63% of Duchenne boys with deletions as described by C. Beroud et al. in Human Mutation 28:196-202 (2007). The present state of treating duplications was des¬cribed by A. Aartsma-Rus et al. in BMC Medi¬cal Genetics 8:43-47 (2007), and a review on the entire field of exon skipping was published by A. Aartsma-Rus and G-J. B. van Ommen in RNA 13:1-16 (2007). This first and successful trial and the very active research in universities and bio¬technology companies shows that, in the not too distant future, this single technique will lead to an effective therapy for the majority of Du¬chenne boys.