摘要：所謂基因治療是對細胞提供外來的核酸以改變其蛋白質表現，是治療先天或後天疾病的一種可能方法，目前病毒是最常使用的基因轉殖載體，但從安全觀點考量，非病毒載體如帶正價電之高分子帶便是一種選擇，一般認為，因為帶正價電之粒子相較於不帶電或是帶陰電性之粒子較易吸附於細胞膜上被細胞所吞噬，因此帶正價電之高分子亦常用於與核酸行成複合體(polycation/DNA complex)，然而，在我們的初步研究中意外發現帶負電的單股核酸(oligonucleotide)塗在polycation/DNA complex上使其整體成為帶陰電性的三成份奈米粒子，仍能進入細胞展現高的基因表達，因此，是否塗佈單股核酸在polycation/DNA complex上的技術能使其成為安全又有效率的基因轉殖載體，將在本計畫深入探討。本計畫可分成下列四個目標：(目標一) 形成polycation/DNA/oligonucleotide complexes的最佳化條件。包括考慮粒子大小，表面電位，細胞毒性，吞噬與轉殖效力與血清相容性等。(目標二) 分析不同的組成的oligonucleotides對三成份奈米粒子基因轉殖效力之影響。(目標三) 分析三成份polycation/DNA/oligonucleotide complexes 之吞噬機制與路徑。(目標四) 建立具細胞瞄準特異性之基因傳送平台。藉由先在互補之單股核酸接上特異抗體，再與polycation/DNA/oligonucleotide complexes交合，如此三成份奈米粒子表面便可擁有不同的生物分子。據我們所知，這是第一個研究欲證明細胞可由塗佈單股核酸在polycation/DNA complex上形成帶負電的奈米粒子所轉染。本計畫研究結果將發表於國際著名的期刊，並訓練參與人員使其具有實作能力。
Abstract: Gene therapy offers a potential method to cure inherited or acquired diseases by transferring exogenous nucleic acids into cells to alter protein expression profiles. Although recombinant viruses are currently used for this purpose because of their high transfection activity, the use for non-viral vectors such as cationic polymers is still desired from the viewpoint of safety. Generally, the uptake of polycation/DNA complexes is mediated by electrostatic interactions of positively charged complexes with negatively charged cellular surface. Accidentally, in our preliminary study, we discovered that the negatively charged oligonucleotide-coated polycation/DNA complexes could enter cells to exhibit high gene expression. Therefore, whether the ternary polycation/DNA/oligonucleotide nanoparticles can facilitate the use of safe and efficient gene delivery will be studied in this project. The objectives in this project will be achieved through the following specific aims.Specific Aim 1: Optimization of conditions of the polycation/DNA/oligonucleotide complex formation for gene delivery. Particle size, surface potential, cytotoxicity, transfection efficiency, cellular uptake, and in vitro serum compatibly of polycation/DNA/oligonucleotide complexes at various oligonucleotide/polycation molar ratios will be investigated.Specific Aim 2: To analyze the effect of different constituent oligonucleotides coated on polycation/DNA complexes on transfection efficiency.Specific Aim 3: To analyze uptake mechanisms and trafficking pathways of ternary polycation/DNA/oligonucleotide.Specific Aim 4: To establish a gene delivery platform with cell targeting specificity. The polycation/DNA/oligonucleotide complexes can be targeted by hybridization with complementary oligonucleotides conjugated with specific ligand or antibody, which allows for the surface conjugation of different biomolecules to polycation/DNA/oligonucleotide complexes to mediate internalization in a cell-specific manner.To our knowledge, this is the first study to show cells can be effectively transfected with the anionic nanoparticles by coating oligonucleotides on polycation/DNA complexes. At the end of this project, the outcome will be published in famous journals, and the entire people join this project will gain good training.
negatively-charged gene delivery vector