Retroviral Transduction Using Acoustic Waves

使用声波进行逆转录病毒转导

基本信息

批准号：
7047759
负责人：
Pin Wang
金额：
$ 19.05万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-06-01 至 2007-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7047759
关键词：
CD34 molecule Retroviridae Vesiculovirus bioengineering /biomedical engineering biotechnology cell line gene therapy genetic transduction green fluorescent proteins hematopoietic stem cells host organism interaction technology /technique development therapy design /development transfection /expression vector ultrasound virus protein

项目摘要

DESCRIPTION (provided by applicant): Among all types of viruses currently used in clinical trials, those based on the murine leukemia virus retroviral vectors are the most frequently used, because of its capability of stable gene integration to the chromosomes of target cells. However, retroviruses can transducer only actively dividing cells. With the relatively short half-life of retroviral particles, the probability of infectious retroviruses encountering target cells is very low because they move in a random Brownian motion. Even if they can get close to the target cells, there is the repulsion force generated from the interaction between negatively charged retrovirus envelope and cell membrane. To overcome these limitations, physicochemical approaches used to increase cell-virus contact such as addition of polycation, flow-through of virus-containing medium, spinoculation, and magnetic field have been studied and showed improved results. However, the aforementioned transduction studies were focused on anchorage-dependent cells and not easy for large-scale settings. In this study, GFP-encoding VSV-G pseudotyped retrovirus vector, which is less fragile than the un-modified retroviral vector, will be used to infect cytokine-dependent hematopoietic cell lines and primary CD34+ hematopoietic cells within ultrasonic standing wave fields generated by piezoelectric transducer and reflector. Various cytokine cocktails will be employed to stimulate the hematopoietic cells entering cell cycle and thus enhance the retroviral transduction. According to the primary acoustic radiation force and drag force, the suspended cells can be estimated to arrive at the pressure nodal planes on the 1 -2 second time scale. We speculate that the first arrived and agglomerated cells may play a role on nucleating collection of the 100 nm-sized retroviruses at the nodal planes. Several design and operating approaches are proposed to decrease the undesired acoustic and thermal streaming which might prevent the aggregation of particulates with diameter around 100 nanometer such as retroviruses. The significance of the proposed research is the engineering approach (i.e., ultrasonic standing waves fields) could be harnessed to enhance the retroviral gene delivery efficiency to hematopoietic stem/progenitor cells, which are not easy to be retrovirally transduced with current medical approaches. The success of this proposed study will provide an innovative method to the field of gene therapy.

描述（由申请人提供）：目前临床试验中使用的所有类型的病毒中，以鼠白血病病毒逆转录病毒载体为基础的病毒是最常用的，因为它能够将基因稳定整合到靶细胞的染色体上。然而，逆转录病毒只能转导活跃分裂的细胞。由于逆转录病毒颗粒的半衰期相对较短，感染性逆转录病毒遇到靶细胞的概率非常低，因为它们以随机布朗运动移动。即使它们能够接近靶细胞，带负电荷的逆转录病毒包膜与细胞膜相互作用也会产生排斥力。为了克服这些限制，人们研究了用于增加细胞-病毒接触的物理化学方法，例如添加聚阳离子、含病毒培养基的流过、旋接种和磁场，并显示出改善的结果。然而，上述转导研究主要集中在贴壁依赖性细胞上，并不容易进行大规模设置。在本研究中，GFP编码的VSV-G假型逆转录病毒载体比未修饰的逆转录病毒载体更脆弱，将用于在压电产生的超声驻波场内感染细胞因子依赖性造血细胞系和原代CD34+造血细胞换能器和反射器。将采用各种细胞因子混合物来刺激造血细胞进入细胞周期，从而增强逆转录病毒转导。根据初级声辐射力和阻力，可以估计悬浮细胞在1 -2 秒的时间尺度内到达压力节面。我们推测，首先到达并聚集的细胞可能在节点平面处 100 nm 大小的逆转录病毒的成核收集中发挥作用。提出了几种设计和操作方法来减少不需要的声流和热流，这可能会阻止直径在 100 纳米左右的颗粒（例如逆转录病毒）的聚集。该研究的意义在于，可以利用工程方法（即超声波驻波场）来提高逆转录病毒基因向造血干/祖细胞的传递效率，而目前的医学方法不易对造血干/祖细胞进行逆转录病毒转导。这项研究的成功将为基因治疗领域提供一种创新方法。