基因療法使用的載體病毒應該是對人無毒的，這樣才可能保證治療的安全性,。Buffalo大學的研究人員報道說他們已經(jīng)成功利用納米顆粒完成了離體無毒基因傳遞,。這項研究的相關(guān)文章刊登在最近一期的Proceedings of the National Academy of Sciences的網(wǎng)絡版上。
研究人員利用一種新型的納米顆粒作為DNA載體,，成功地將一種熒光基因傳遞到細胞內(nèi),。這個成功個案最終可能為基因療法提供一個替代病毒的新型載體。
利用共焦點顯微鏡和熒光廣譜法,，研究人員對這個轉(zhuǎn)染過程進行了可視化的實時追蹤——包括將基因傳遞到細胞內(nèi)、細胞核對基因的攝入以及基因的表達,。當熒光蛋白在細胞中被表達時,，研究人員就能夠知道轉(zhuǎn)染已經(jīng)發(fā)生了。
這項研究為解決困擾人類基因治療臨床試驗的一些問題給出了一線曙光,，這些問題其中就包括病毒載體使用的安全性問題,。靶標基因的有效傳遞以及在細胞內(nèi)的充分釋放是基因治療的主要籬障。因為病毒的穿透細胞的能力而被用作傳遞的載體,，但是這些病毒還是有可能恢復成野生型,，從而引發(fā)疾病。雖然非病毒載體比較安全,，但是卻很難進入細胞并成功釋放DNA,。
Buffalo大學的這個研究組使用的方法與其它非病毒載體不同。他們使用的DNA納米顆粒復合體能夠在被細胞的防御系統(tǒng)摧毀前釋放DNA,，從而利于轉(zhuǎn)染,。研究人員還能夠利用光子學方法展示轉(zhuǎn)染的過程，因此研究人員能夠跟蹤基因傳遞的每一步以提高成功率,。
這個研究組現(xiàn)在正進行活體研究,，即利用這種新穎的納米顆粒來轉(zhuǎn)染小鼠大腦中的神經(jīng)細胞。
美國的國家科學院的最新科研進展：發(fā)明了一種新型基因治療方法,，可取代有潛在危險的使用病毒為載體的傳統(tǒng)基因?qū)敕椒?。該方法已?jīng)完成了體外試驗。使用納米微粒作為DNA載體,，已經(jīng)成功地將熒光基因?qū)肓思毎?。這一實驗的成功預示著納米微粒可能最終取代病毒,，成為新的基因?qū)胼d體,。使用共焦顯微鏡和熒光分光鏡，科學家們對基因轉(zhuǎn)導過程進行了實時觀察,，包括基因如何進入細胞,、基因如何被細胞核攝取及其表達過程。觀察發(fā)現(xiàn),，使用納米微粒,，可監(jiān)控基因的轉(zhuǎn)導過程,，追蹤納米微粒穿透細胞并將DNA釋放進細胞核的過程。
這項發(fā)明有望解決近年來人類基因治療中的難題,，使用病毒作為基因載體,，已經(jīng)導致了數(shù)個病人不幸死亡。能否有效地釋放目標基因并將其注入靶細胞是基因治療的關(guān)鍵,。由于病毒具有穿透細胞的能力,，它們被用來作為基因載體。但某些情況下,，病毒可逆轉(zhuǎn)為“野生”型,，對宿主產(chǎn)生危害。相比之下,，使用非病毒載體要安全得多,，但如何使它們穿透細胞并立即釋放所攜帶的基因卻困難得多。而納米微粒載體在進入細胞后被細胞清除前,，可有效地釋放所攜帶的基因,，引發(fā)基因轉(zhuǎn)導過程。并且,，通過影像學技術(shù),，還能觀測整個過程，這在以前是無法實現(xiàn)的,。
研究人員使用的納米微粒是從混合有機硅(ORMOSIL)中制備的,。這種材料有良好的生物兼容性，可用于不同生物組織的基因治療,。目前研究小組將進行小鼠神經(jīng)細胞的基因轉(zhuǎn)導實驗,。
Nanoparticles used to successfully deliver gene therapy
A gene therapy method that doesn't rely on potentially toxic viruses as vectors may be growing closer as the result of in vitro research results reported by University at Buffalo scientists in the current online issue of the Proceedings of the National Academy of Sciences. The paper, which describes the successful uptake of a fluorescent gene by cells using novel nanoparticles developed as DNA carriers at UB, demonstrates that the nanoparticles ultimately may prove an efficient and desirable alternative vector to viruses.
Using confocal microscopy and fluorescent spectroscopy, the UB scientists tracked optically in real-time the process known as transfection, including the delivery of genes into cells, the uptake of genes by the nucleus and their expression.
"We have shown that using photonics, the gene-therapy transfer can be monitored, tracking how the nanoparticle penetrates the cell and releases its DNA in the nucleus," explained Paras N. Prasad, Ph.D., executive director of the UB Institute for Lasers, Photonics and Biophotonics, SUNY Distinguished Professor in the Department of Chemistry in the University at Buffalo's College of Arts and Sciences, and a co-author of the paper.
"When the fluorescent protein was produced in the cell, we knew transfection had occurred," he said.
The work is important in light of the difficulties that have plagued gene-therapy human trials in recent years, including some fatalities that may have resulted from the use of viral vectors.
"Efficient delivery of the desired gene and substantial release inside the cell is the major hurdle in gene therapy," explained Dhruba J. Bharali, Ph.D., a co-author and postdoctoral researcher in the UB Department of Chemistry and UB's Institute for Lasers, Photonics and Biophotonics, where the work was done.
"Viruses have been used as efficient delivery vectors due to their ability to penetrate cells, but there is the chance they can revert back to 'wild' type," he said.
While non-viral vectors are safer, he noted that it is much more difficult to get them into cells and then to achieve the release of DNA once they do penetrate cells.
The advantage of the UB team's approach, he explained, is that unlike most other nonviral vectors, the DNA-nanoparticle complex releases its DNA before it can be destroyed by the cell's defense system, boosting transfection significantly.
The UB scientists also were able to use photonic methods to provide an unprecedented look at how transfection occurs, from the efficient uptake of nanoparticles in the cytoplasm to their delivery of DNA to the nucleus.
"No gene-delivery vehicle -- either viral or non-viral -- has never been tracked in the cell before," explained Tymish Y. Ohulchanskyy, Ph.D., the third co-author and post-doctoral research scholar at the institute. "By using our photonics approach, we can track gene delivery step by step to optimize efficiency," he said.
The research team makes its nanoparticles from a new class of materials: hybrid, organically modified silicas (ORMOSIL).
"The structure and composition of these hybrid ORMOSILs yield the flexibility to build an extensive library of tailored nanoparticles for efficiently targeting gene therapy into different tissues and cell types," said Prasad.
The UB researchers now are collaborating on in vivo studies with colleagues from the UB School of Medicine and Biomedical Sciences to use their novel nanoparticles to transfect neuronal cells in the brains of mice.
This research was supported by the U.S. Air Force through its Defense University Research Initiative on Nanotechnology (DURINT) grant.
From University at Buffalo: "Using Customized Nanoparticles, UB Scientists Achieve Non-Viral Gene Delivery In Vitro and Track it in Real-Time"
By BJS at 12/28/2004 - 07:06
