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Upgrading U7snRNA to complete efficient restoration of dystrophin by exon-skipping in DMD patients
Most cases of Duchenne muscular dystrophy (DMD) are caused by mutations that disrupt the dystrophin mRNA reading frame. In many cases, skipping of a single exon could restore the reading frame, giving rise to a shorter but still functional quasi-dystrophin protein. It has previously been proposed to use antisense oligoribonucleotide (AON) to induce removal of appropriate exons during pre-mRNA processing. Indeed, this promising approach is being tested in clinical trials in the Netherlands and the UK with antisense sequences against exon 51. However, since the AO have a limited life span in the muscle, long term correction will require repeated administration. To overcome this limitation, we proposed to use small nuclear RNAs (snRNA) as carriers and to vectorize them into adeno-associated virus (AAV) vectors. The use of AAV as vectors has some important advantages since they are highly effective in muscle and heart, and offer a long term correction. We previously demonstrated that a single treatment with an AAV2/1 vector containing appropriately modified U7 snRNA induces widespread exon-skipping of the dystrophin pre-mRNA and results in the sustained correction of muscular dystrophy in both the mdx mouse (Goyenvalle et al., 2004) and the GRMD dog.
A phase I clinical trial concerning the exon 51 using AAV-U7 is currently planned by Genethon in France, but considering the diversity of mutations among DMD patients, treatment of DMD will need a specific antisense U7snRNA adapted to each subset of patient. As with the Antisense Oligonucleotide approach, choice of the most efficient sequence will require optimization and depending on the exon, this sequence will not necessary target the same sites. To avoid the lengthy optimization necessary to target each individual exon, we proposed the development of an almost “universal” U7snRNA carrying a complementary sequence to the exon and a free tail harbouring canonical binding sites for the heterogeneous nuclear ribonucleoproteins A1/A2 (hnRNP) that are powerful splicing repressors. Therefore, this tail is thought to induce skipping of any targeted exon via recruitment of these proteins in its near vicinity. Thus, the presence of this generic strong silencer tail could indeed circumvent the complex and time-consuming specific optimization required for each new exon-target.
We first focused on exon 51 of the dystrophin gene and therefore designed new tailed U7snRNA constructs. Each construct was inserted into lentiviral vectors for in vitro analysis on myoblasts from DMD patients. After transduction of these cells with lentiviral vectors encoding the tailed U7-ex51, we confirmed the skipping of the exon 51 by nested RT-PCR and dystrophin restoration by western blot. By comparison with controls encoding a mutated tail, we showed that the skipping efficiency of these constructs was due to the tail carrying silencer motifs, therefore confirming its splicing repressor action, and not the annealing sequence to the exon.
These very encouraging results for exon 51 provide evidence that bifunctional U7snRNA can achieve efficient exon-skipping in myoblasts from DMD patients. These tailed U7 could be very useful to develop the exon-skipping strategy for all other dystrophin exons. Thus, the validation of their efficiency shown in this study offers very promising tools for clinical treatment of DMD.
We are currently evaluating the efficiency of these constructs in vivo in transgenic mice carrying a human dystrophin gene, using AAV vectors. Several companies now propose clinical grade AAV vector manufacturing worldwide, and these vectors are actually the object of several clinical trials in The United States and in Europe to prove their safety and efficiency in humans.
2 Comments
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- Mark Silverman
- January 31st 2008
- 22:33pm
Aurelie This looks very interesting, particularly given that you are looking for a universal solution for all exons. Do you have a timescale? regards Mark
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Hello! thank you for your post! But I have a question. Does not the AAV viral vector cause an immune response and so you can only use it once? thanks, Marie, GenMed Lab Mom