正價脂質奈米微粒與短鏈核苷酸複合體
在基因治療上的應用

基因治療是指利用適當的方法將基因送入細胞中，繼而穩定的表達其未能正常表現的蛋白質，而當疾病是由於蛋白質表現過量所造成的，就必須抑制其基因的表現。目前用來抑制表現的核酸物質有反意股核苷酸 (antisense deoxynucleotides)以及干擾型核醣核酸 (RNAi)，都可以使轉譯過程被停止而抑制不正常的蛋白質表現，而另外一種基因治療為基因修正，即改變基因上錯誤的鹼基而使基因表現正常。過去用來遞送核酸物質的載體主要可以分為兩種，病毒性載體以及非病毒性載體。由於病毒性載體的不穩定性，目前的遞送方式大多以非病毒性載體為主。 3β-[N-(2-guanidinoethyl)carbamoyl]cholesterol (GEC-Chol)為一以膽固醇為基礎的正價脂質，我們以莫耳數1：1的方式將GEC-Chol與膽固醇(Cholesterol)混合後，再以此GEC-Chol/Chol正價奈米微胞攜帶短鏈核苷酸並觀察其在基因治療上的應用。首先研究的是GEC-Chol/Chol正價奈米微胞攜帶短鏈核苷酸的最適條件，我們分別在不同的氮磷比 (N/P ratio)的情況下測量其大小、表面電荷、包覆率以及遞送效率等等，由實驗結果可以發現當氮磷比為3時可以有極高的遞送效率和包覆率。而在毒性測試中GEC-Chol/Chol正價奈米微胞也展現了低細胞毒性，而低細胞毒性即表示能以其遞送較大量的短鏈核苷酸以達到治療的目的。另外，以細胞存活率以及蛋白表現來觀察GEC-Chol/Chol正價奈米微胞遞送c-myc及bcl-2之反意股核苷酸的應用，也發現其為遞送反意股核苷酸的良好載體。在修正綠色螢光蛋白的實驗中，相較於市售正價微脂體Lipofectamine2000，我們發現使用GEC-Chol/Chol正價奈米微胞有2-3倍的修正機率。總結以上的實驗，GEC-Chol/Chol正價奈米微胞可以在活體外的實驗中有效的遞送短鏈核苷酸，並達到基因治療的目的。本論文提出GEC-Chol/Chol正價奈米微胞具有高度短鏈核苷酸遞送性以及低細胞毒性，相當適合當作一基因治療的載體，此正價奈米微胞未來也可以同時加入其他藥物或是專一性遞送的導向性分子增加其治療能力。而目前正著手進行初步的活體內實驗，以GEC-Chol/Chol正價奈米微胞攜帶反意股核苷酸直接打入皮下腫瘤中，觀察腫瘤的生長情形，然而此正價奈米微胞若要在活體內應用，其最適化條件可能需要微調才能有效應用在活體治療中，這也是未來可以努力的方向。

關鍵字

正價微脂體； GEC-Chol ；短鏈核苷酸；基因治療

並列摘要

Gene therapy is the procedure that a gene is delivered into cells, and subsequently expresses appropriately to compensate for the dysfunctional gene. And a gene needs to be repressed when its abnormal overexpression has resulted in a disease. Presently, the nucleic acids used to inhibit protein expression are antisense oligodeoxynucleotides (ODN) and RNAi, both of which could block translation to suppress abnormal gene-expression. Another type of gene therapy is gene correction which revises the wrong nucleotides to recover the coding sequence of the targeted protein. Viral and non-viral vector have been used to deliver nucleic acid materials. Considering to stability, the non-viral vectors is more prevalent. 3β-[N-(2-guanidinoethyl)carbamoyl]cholesterol (GEC-Chol) is a cationic lipid based on cholesterol (Chol). We mixed GEC-Chol and Chol with molar ratio 1：1 and investigated the applications of the GEC-Chol/Chol cationic nanomicelles carrying ODN in gene therapy. To obtain the most appropriate condition, we measured the size, zeta potential, encapsulation and delivery efficiency of GEC-Chol/Chol cationic nanomicelles with different N/P ratio. We found that the delivery efficiency and encapsulation of oligonucleotides was highest when the N/P ratio was 3. In addition, the cytotoxicity of GEC-Chol/Chol cationic nanomicelles was low which suggested that GEC-Chol/Chol cationic nanomicelles could be used to carry more oligonucleotides in gene therapy. Moreover, we found that GEC-Chol/Chol cationic nanomicelles are fine vectors for antisense ODN delivery by observing the cell viability and protein level in the cancer cells which antisense oligodeoxynucleotides of c-myc and bcl-2 were delivered to. Comparing to the commercial transfection reagent Lipofectamine2000, GEC-Chol/Chol cationic nanomicelles also showed higher efficiency to correct aberrant sequence of enhanced green fluorescent protein (EGFP). In summary, GEC-Chol/Chol cationic nanomicelle is a potential tool for gene therapy owing to its high efficiency of ODN delivery and low toxicity to cells in vitro. Drugs and specific targeting molecules can be capsulated or embedded into this vector to execute their therapeutic functions. However, the in vivo test of intratumor injection of antisense oligodeoxynucleotides carried by the GEC-Chol/Chol cationic nanomicelles is still at the initiation stage. In vivo applications of such nanomicelle needs further study.

並列關鍵字

Cationic liposmoes ； EC-Chol ； oligonucleotides ； gene therapy

參考文獻

Akhtar, S. (1998). Antisense technology: selection and delivery of optimally acting antisense oligonucleotides. J Drug Target 5, 225-234.

Akinc, A., and Langer, R. (2002). Measuring the pH environment of DNA delivered using nonviral vectors: implications for lysosomal trafficking. Biotechnol Bioeng 78, 503-508.

Alvarez, D., Harder, G., Fattouh, R., Sun, J., Goncharova, S., Stampfli, M.R., Coyle, A.J., Bramson, J.L., and Jordana, M. (2005). Cutaneous antigen priming via gene gun leads to skin-selective Th2 immune-inflammatory responses. J Immunol 174, 1664-1674.

Bao, S., Thrall, B.D., and Miller, D.L. (1997). Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 23, 953-959.

Biroccio, A., Leonetti, C., and Zupi, G. (2003). The future of antisense therapy: combination with anticancer treatments. Oncogene 22, 6579-6588.

國際替代計量

正價脂質奈米微粒與短鏈核苷酸複合體在基因治療上的應用

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