Retroviral Transduction Using Acoustic Waves

使用声波进行逆转录病毒转导

• 批准号: 6922983
• 负责人: CHING-AN PENG
• 金额: $ 21.8万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 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/6922983
• 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

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）： 在目前用于临床试验的所有类型的病毒中，基于鼠白血病病毒逆转录病毒载体的那些病毒是最常用的，因为其稳定基因整合到靶细胞染色体的能力。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，也会有带负电荷的逆转录病毒包膜与细胞膜相互作用产生的排斥力。为了克服这些限制，用于增加细胞-病毒接触的物理化学方法，如添加聚阳离子、含病毒培养基的流通、旋转接种和磁场，已经进行了研究，并显示出改进的结果。然而，上述转导研究集中于贴壁依赖性细胞，并且不容易进行大规模设置。在这项研究中，GFP编码的VSV-G假型逆转录病毒载体，这是不太脆弱的比未修饰的逆转录病毒载体，将被用来感染依赖于精氨酸的造血细胞系和原代CD 34+造血细胞内的超声驻波场由压电换能器和反射器。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拖曳力，可以估计悬浮细胞在1 - 2秒的时间尺度上到达压力节面。我们推测，最先到达的细胞和聚集的细胞可能在结平面上的100 nm大小的逆转录病毒的成核收集中起作用。提出了几种设计和操作方法来减少不期望的声流和热流，这可能会防止直径约100纳米的颗粒（如逆转录病毒）的聚集。拟议研究的意义在于工程方法（即，超声驻波场）可用于增强逆转录病毒基因向造血干/祖细胞的递送效率，而造血干/祖细胞不容易用当前的医学方法进行逆转录病毒转导。本研究的成功将为基因治疗领域提供一种新的方法。