Gene Therapy for ALS with mutant SOD1 by ribozyme and catalytic DNA.

最近，短干扰RNA(SiRNA)被发现能有效地切割RNA，因此我们比较了它与核酶和催化DNA抑制靶基因表达的效率。在培养细胞中，针对野生型SOD1设计的siRNA比核酶和催化DNA更有效地抑制SOD1的表达。当单核苷酸替换位于siRNA正义序列中5‘侧第9-13位核苷酸时，它可以识别单核苷酸替换。利用这一原理，我们成功地制备了在不影响野生型SOD1表达的情况下，特异性抑制突变型G93A SOD1表达的siRNA。为了将siRNA有效地输送到神经元，可能需要开发病毒载体。首先，利用U6启动子和siRNA的茎环序列构建了高效表达siRNA的DNA载体。其次，利用该表达盒成功构建了siRNA表达的腺病毒载体和Adno-Associate病毒载体。因此，我们完成了突变型SOD1基因治疗ALS的第一步。

Recently, short interfering RNA (siRNA) has been found to cleave efficiently RNA, so that we compared its efficiency for the supprresion of expression of target gene with ribozyme and catalytic DNA. In culture cells the siRNA designed for wild-type SOD1 was much more efficient to suppress the expression of SOD1 than ribozyme and catalytic DNA. When single nucleotide alternation was positioned at 9-13th nucleotide from 5' side in the sense sequence of siRNA, it can recognize a single nucleotide alternation. Using this principle, we succeeded in making the siRNA which specifically suppress the expression of mutant G93A SOD1 without influencing that of the wild-type SOD1.For the efficient delivery of siRNA to neurons, development of the virus vectors might be necessary. First, excellent siRNA-expressing DNA vector was constructed when the U6 promotor and the stem-loop sequence of siRNA were used. Second, with this expression cassette, we successfully made siRNA-expressing adnovirus and adno-associate virus vectors. Therefore, we finish the first step of the gene therapy for ALS with mutant SOD1.