基因疗法使用的载体病毒应该是对人无毒的，这样才可能保证治疗的安全性。Buffalo大学的研究人员报道说他们已经成功利用纳米颗粒完成了离体无毒基因传递。这项研究的相关文章刊登在最近一期的Proceedings of the National Academy of Sciences的网络版上。
研究人员利用一种新型的纳米颗粒作为DNA载体，成功地将一种荧光基因传递到细胞内。这个成功个案最终可能为基因疗法提供一个替代病毒的新型载体。
利用共焦点显微镜和荧光广谱法，研究人员对这个转染过程进行了可视化的实时追踪——包括将基因传递到细胞内、细胞核对基因的摄入以及基因的表达。当荧光蛋白在细胞中被表达时，研究人员就能够知道转染已经发生了。
这项研究为解决困扰人类基因治疗临床试验的一些问题给出了一线曙光，这些问题其中就包括病毒载体使用的安全性问题。靶标基因的有效传递以及在细胞内的充分释放是基因治疗的主要篱障。因为病毒的穿透细胞的能力而被用作传递的载体，但是这些病毒还是有可能恢复成野生型，从而引发疾病。虽然非病毒载体比较安全，但是却很难进入细胞并成功释放DNA。
Buffalo大学的这个研究组使用的方法与其它非病毒载体不同。他们使用的DNA纳米颗粒复合体能够在被细胞的防御系统摧毁前释放DNA，从而利于转染。研究人员还能够利用光子学方法展示转染的过程，因此研究人员能够跟踪基因传递的每一步以提高成功率。
这个研究组现在正进行活体研究，即利用这种新颖的纳米颗粒来转染小鼠大脑中的神经细胞。
美国的国家科学院的最新科研进展：发明了一种新型基因治疗方法，可取代有潜在危险的使用病毒为载体的传统基因导入方法。该方法已经完成了体外试验。使用纳米微粒作为DNA载体，已经成功地将荧光基因导入了细胞。这一实验的成功预示着纳米微粒可能最终取代病毒，成为新的基因导入载体。使用共焦显微镜和荧光分光镜，科学家们对基因转导过程进行了实时观察，包括基因如何进入细胞、基因如何被细胞核摄取及其表达过程。观察发现，使用纳米微粒，可监控基因的转导过程，追踪纳米微粒穿透细胞并将DNA释放进细胞核的过程。
这项发明有望解决近年来人类基因治疗中的难题，使用病毒作为基因载体，已经导致了数个病人不幸死亡。能否有效地释放目标基因并将其注入靶细胞是基因治疗的关键。由于病毒具有穿透细胞的能力，它们被用来作为基因载体。但某些情况下，病毒可逆转为“野生”型，对宿主产生危害。相比之下，使用非病毒载体要安全得多，但如何使它们穿透细胞并立即释放所携带的基因却困难得多。而纳米微粒载体在进入细胞后被细胞清除前，可有效地释放所携带的基因，引发基因转导过程。并且，通过影像学技术，还能观测整个过程，这在以前是无法实现的。
研究人员使用的纳米微粒是从混合有机硅(ORMOSIL)中制备的。这种材料有良好的生物兼容性，可用于不同生物组织的基因治疗。目前研究小组将进行小鼠神经细胞的基因转导实验。
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
