siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme,☆☆

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Abstract

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.

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Materials and methods

siRNA and DNA-vector based expressing siRNA preparation. The targeted region of siRNA was the coding sequence of SOD1 cDNA. The 5 or 3 UTRs and regions nearby the start codon are avoided, as these may be richer in regulatory protein binding sites. siRNA sequences of the form AA N19–20 and CA N19–20 with GC content less than 70% were selected from this region [9]. Next, the nucleotide preference after AA/CA is G or A, because it is required for efficient RNA polymerase initiation. Selected 19-

siRNA and expressing siRNA for G93A SOD1

Two siRNAs targeting G93A SOD1 corresponding to regions 277–297 (siRNA G93A.1) and 275–294 (siRNA G93A.2) were designed (Fig. 1A). Transfection of both siRNAs into mammalian cells could effectively reduce G93A SOD1 expression. Western blot analysis revealed that both siRNA G93A.1 and 2 reduced the expression of G93A SOD1 protein by about 90% when expression efficiency was adjusted with co-transfected GFP (Fig. 1B). The suppression of G93A SOD1 protein by siRNA G93A.1 increased in a

Discussion

In autosomal dominant disease, such as familial ALS, the most effective therapeutic approach requires the reduction of the aberrant mutant protein leaving wild-type protein intact. Possible candidate methods include ribozyme, DNA enzyme, and siRNA to cleave mutant RNA selectively. Antisense RNA or DNA has been used to reduce the expression of target protein, but these do not discriminate between the wild-type and mutant mRNA of SOD1, most of which has a point mutation [15]. siRNA G93A.1 and 2,

Acknowledgements

The authors thank Alyson Peel, Ph.D., Dr. Li Yi, Dr. Hiroki Sasaguri. and Dr. Yuki Saito for their help. This study was supported by Grants from the National Institutes of Health (NS35155 to D.E.B.) and the ALS Association, the Ministry of Education, Science and Culture, Japan (A-130, T.Y.), from the Ministry of Health, Labor and Welfare, Japan (T.Y., H.M.), and from Japan Amyotrophic Lateral Sclerosis Association (T.Y.).

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    Abbreviations: ALS, amyotrophic lateral sclerosis; SOD, superoxide dismutase; siRNA, small interfering RNA; dsRNA, double-stranded RNA; rAAVs, recombinant adeno-associated viruses.

    ☆☆

    Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2003.12.098.

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