Optimization of U7snRNA Cassettes for Expression of Therapeutic Antisense Sequences

Pre-mRNA maturation can be modulated using antisense oligonudeotides. These approaches have been recently used in clinical trials where exon-skipping is induced on a mutated DMD pre-mRNA in order to reestablish the translational reading frame. Stable delivery of antisense sequences can be obtained with modified snRNAs such as U1 or U7, expressed from viral vectors. The main limitation remains the amount of snRNA expressing vector which can be produced and administered to patients with Duchenne Muscular Dystrophy. One way to increase the therapeutic index of these vectors is to optimize the expression level of the snRNA shuttle. In this aim, we have replaced the RNA polymerase (RNAP) II promoter of U7 with RNAPHI promoters such as those used for the transcription of U6 and 7SK genes which are considered the most active for small RNA expression. A U7 shuttle containing antisense sequences known to efficiently mediate skipping of exon 51 on the human DMD pre-mRNA was placed under the control of these promoters and the new cassettes were introduced into lentiviral vectors. No snRNA expression (nor exon 51 skipping) could be detected with these new cassettes. Further modifications have therefore been introduced in the U7 sequence, primarily in order to avoid premature termination of RNAPIII caused by runs of Ts in the sequence. In a second approach, we have introduced a strong muscle specific enhancer (MHCK7, a synthetic fusion between the MCK a-MHC enhancers) upstream of U7 shuttles driven by their cognate RNAPII promoter. These new designs have been applied to U7 carrying antisenses sequences for the skipping of human exon 51 or murine exon 23, and both lentiviral or AAV2/1 vectors are currently tested in human myoblasts or following direct injection into the muscle of mdx mice.