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標題: | 芳香族L-胺基酸脫羧酵素缺乏症之小鼠模式 A murine model of Aromatic L- amino acid decarboxylase deficiency |
作者: | Ni-Chung Lee 李妮鍾 |
指導教授: | 余家利,胡務亮 |
關鍵字: | 芳香族L-胺基酸脫羧酵素,小鼠模式,基因治療,腺相關病毒,神經傳導物質, Aromatic L-amino Acid Decarboxylase,mouse model,gene therapy,adeno-associated virus,neurotransmitter, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 背景:芳香族L-胺基酸脫羧酵素(Aromatic L-amino Acid Decarboxylase; AADC)是合成神經傳導物質多巴胺(dopamine)以及血清素(serotonin)過程中最後一個步驟的酵素。AADC缺乏症是一種非常罕見的遺傳疾病,但是在台灣的盛行率較國外為高。患者的臨床表現為發展遲緩,尤其是以動作功能的障礙最為明顯。此症的患者對於藥物治療反應不佳,因此需要開發其他的治療方式。但是由於沒有AADC缺乏症之動物模式,此症的治療開發僅能藉助於其他的動物模式,例如依循巴金森氏症之基因治療之方法,以AAV2-hAADC病毒載體直接注射大腦殼核(Putamen)等。為了能夠發展AADC缺乏症之治療,我們亟需建立AADC缺乏症之小鼠模式。
研究主題:(1)建立AADC缺乏症的小鼠模式,了解其臨床表現及特色。(2)進行基因治療相關實驗,並評估治療後AADC缺乏症小鼠臨床的改善狀況。 方法與結果:過去已經有人嘗試以基因剔除(knock out)方式製造小鼠但是失敗,因此我們採取knock-in (KI)台灣病人常見的AADC基因突變點位: IVS6+4A>T突變及保留neomycin-resistance gene之策略後,順利獲得活產小鼠。Homozygous IVS6+4 KI小鼠(簡稱DdcKI; DdcIVS6/IVS6小鼠) 的腦部AADC mRNA缺少exon 6,AADC酵素活性濃度為wild-type mice的0.3%以下。約一半的病鼠可以活產,剛出生時與正常老鼠大小並無沒有差別,但是在一星期內可以觀察要病鼠生長遲緩,並有明顯的動作障礙及hindlimb clasping。約三分之二的病鼠可以活到離乳,離乳之後的生長及動作功能開始改善。然而這些病鼠仍有因血清素缺乏導致之心血管功能障礙以及行為異常。病鼠兩周大時腦部多巴胺含量為wild-type mice的9.39%,而在8周大時上升為37.86%。成年之後的病鼠注射apomorphine後有過度興奮的反應,且紋狀體c-Fos表現亦增加,顯示突觸後的神經細胞的變化。由於多巴胺及血清素在腦部是由兩群不同的細胞所製造,目前直接注射殼核的治療並未矯正腦部其他區域的神經傳導物質缺乏。因此我們嘗試在小鼠模式進行基因治療,在新生小鼠以腦室內注射(Intracerebral ventricular; ICV)的方式注入AAV9-hAADC病毒載體,讓AADC基因能夠在腦部其他地方表現。4周大未治療的病鼠其腦部多巴胺及血清素含量為wild-type mice的25%及15%。接受治療的病鼠則為100%及40%。這樣的治療亦改善了病鼠的生長速度及存活率,並降低了hindlimb clasping的程度及心血管功能障礙。而在行為異常方面的效果則是部分改善,接受治療的病鼠較wild-type mice有輕微過動的現象。此外,接受治療的病鼠沒有觀察到明顯的免疫反應。 結論:我們建立了AADC缺乏症的小鼠模式,經由小鼠的代償性調節機制,這樣的小鼠可以存活至成年。我們也證明了新生病鼠接受腦室內注射之基因治療是安全且有效的。這個小鼠模式也將成為日後基因治療方式的改進以及其他神經傳導物質治療的研發的工具。 Background: Aromatic L-amino Acid Decarboxylase (AADC) is responsible for the last step of the synthesis of neurotransmitter dopamine and serotonin. AADC deficiency is a very rare inherited disease, but the disease is with a relatively higher incidence in Taiwan due to the IVS6+4 founder mutation. Children with AADC deficiency exhibit compromised development, particularly with regard to their motor functions. The motor delay does not response to medical treatment well. Therefoere, a need for alternate treatment emerged. However, because there is no animal model for AADC deficiency, the design of AADC deficiency gene therapy can only follow the gene therapy for Parkinson disease: Intraputaminal injection of AAV2-hAADC. In order to further develop treatment for AADC deficiency, having a mouse model of AADC deficiency is mandatory. Thesis: (1) To create an AADC deficiency mouce model and characterize it (2) Application of gene therapy on this model and evaluate the treatment effect Methods and Results: We inserted an AADC gene mutation (IVS6+4A>T) and a neomycin-resistance gene into intron 6 of the mouse AADC (Ddc) gene to create the AADC deficiency mouse model. In the brains of homozygous knock-in mice (DdcKI mice; DdcIVS6/IVS6), AADC mRNA lacked exon 6, and AADC activity was < 0.3% of that in wild-type mice. Half of the DdcKI mice were born alive but grew poorly and exhibited severe dyskinesia and hindlimb clasping after birth. Two-thirds of the live-born DdcKI mice survived the weaning period, with subsequent improvements in their growth and motor functions; however, these mice still displayed cardiovascular dysfunction and behavioral problems due to serotonin deficiencies. The brain dopamine levels in the DdcKI mice increased from 9.39% of the levels in wild-type mice at 2 weeks of age to 37.86% of the levels in wild-type mice at 8 weeks of age. Adult DdcKI mice also exhibited an exaggerated response to apomorphine and an elevation of striatal c-Fos expression, suggesting post-synaptic adaptations. Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat DdcKI mice. Intracerebral ventricular (ICV) injection of neonatal mice with an adeno-associated virus (AAV) vector serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, four-week-old DdcKI mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of DdcKI mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the DdcKI mice were partially corrected, and the treated DdcKI mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Conclusions: We generated an AADC deficient mouse model, in which compensatory regulation allowed the mice to survive to adulthood. We also demonstrated this congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice. This mouse model will be useful both for developing gene therapies for AADC deficiency and for designing treatments for diseases associated with neurotransmitter deficiency. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58565 |
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顯示於系所單位: | 臨床醫學研究所 |
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