2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658148摘要：Aromatic L-amino acid decarboxylase (AADC)是合成dopamine及serotonin所必需的一個酵素。缺乏AADC的病童在動作的發展上有嚴重的缺陷。藥物治療或許可以減輕一些症狀，但無法延長存活。目前我們正在發展AADC缺乏症的基因治療。台灣是全世界AADC缺乏症發生率最高的地方。這是因為我們有一個祖先留下來的AADC基因突變IVS6+4A>T；這個突變造成了AADC mRNA多了37 bp。最近我們製造了一個AADC缺乏症的小鼠。這個小鼠同樣帶有IVS6+4A>T的突變，但是活產的AADC缺乏小鼠卻和正常小鼠有著同樣的存活率。我們發現AADC缺乏小鼠主要的AADC mRNA是缺乏exon 6，但是全長且序列正常的mRNA也可以被看到。我們推測小鼠病情較輕的原因是因為老鼠沒有intron 6在+38位置的一個cryptic splice donor。因此在這個研究計畫中，我們想要來校正AADC缺乏症的mRNA剪接異常。我們將用幾個方法來達到這個目的。我們目前已經有了體外測試AADC mRNA剪接的系統，我們也將為小鼠的序列建立相同的系統來測試治療。我們將設計反義核苷(antisense oligonucleotide，ASO)來壓抑+38 cryptic splice donor，然後觀察全長的AADC mRNA的量是否會增加。我們也將利用exon 6上的bifunctional RNA來增加突變splice donor的效率；如果效率好我們就可以利用AADC缺乏小鼠來測試。最後我們希望能建立具有人類序列的AADC缺乏小鼠，提供未來轉譯研究使用。當然使用藥物來影響AADC缺乏小鼠mRNA的剪接，是直接而且簡單的工作，我們將用valproic acid及amiloride來測試，觀察突變mRNA剪接的變化。這個研究將支持下世代AADC缺乏症治療的開發，也將繼續讓台灣站在轉譯醫學研究之先端。<br> Abstract: Aromatic L-amino acid decarboxylase (AADC) is required for the synthesis of dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly with regard to their motor functions. Drug therapy has only marginal effects on some of the symptoms and does not change the early childhood mortality. Currently we are working on a gene therapy for this condition.Taiwan has the highest prevalence of AADC deficiency in the word, and it is because of the founder mutation IVS6+4A>T which causes an aberrant splicing 37 bp downstream. Recently, we created a mouse model of AADC deficiency by knocking in this splicing mutation, and live born mice had normal survival. Interestingly, the product of the mutated mouse AADC gene was deletion exon 6 (△6), and a full-length splicing product could be identified. We suspect that it is because the +38 cryptic splice donor site is absent in mouse. Therefore, we would like to propose a novel treatment for AADC deficiency.We would like to try a few different strategies. We have an in vitro splicing assay for the human AADC sequences, and will make the mouse gene splicing cassette. We will suppress the human +38 cryptic splice donor with an antisense oligonucleotide, and check if the full-length splicing product could appear. We will also use bifunctional RNA to target exon 6 to enhance the mutated splice donor. If the bifunctional RNA treatment is promising, we could test it in the AADC deficiency mouse model. Lastly, we will create a humanized AADC deficiency mouse model for future preclinical study. Certainly, it is easy and straightforward that we can treat our AADC deficiency mice with drugs that may modify splicing, including valproic acid and amiloride, and then monitor the brain AADC gene splicing.The studies will support the next generation therapy for AADC deficiency, and will keep us on the front of translational medicine.