Retroviral Transduction Using Acoustic Waves

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，基于小鼠白血病病毒逆转录病毒载体的病毒是使用最频繁的，因为它能够稳定地将基因整合到靶细胞的染色体上。然而，逆转录病毒只能转导主动分裂的细胞。由于逆转录病毒颗粒的半衰期相对较短，传染性逆转录病毒与靶细胞相遇的概率很低，因为它们以随机的布朗运动运动。即使它们能够接近靶细胞，也存在带负电荷的逆转录病毒膜与细胞膜相互作用产生的排斥力。为了克服这些限制，人们研究了增加细胞与病毒接触的物理化学方法，如添加聚阳离子、含病毒介质的穿透、自旋聚集和磁场，并显示出改进的结果。然而，上述转导研究主要集中在锚定依赖的细胞上，不容易在大规模环境中进行。在本研究中，GFP编码的VSV-G伪型逆转录病毒载体比未修饰的逆转录病毒载体更不脆弱，将在由压电换能器和反射器产生的超声驻波场内感染细胞因子依赖的造血细胞株和原代CD34+造血细胞。将使用各种细胞因子鸡尾酒来刺激造血细胞进入细胞周期，从而增强逆转录病毒的转导。根据初生声辐射力和阻力，可以在1-2秒的时间尺度上估计悬浮细胞到达压力节面。我们推测，首先到达和聚集的细胞可能在100 nm大小的逆转录病毒在结平面的核聚集中发挥作用。提出了几种设计和操作方法，以减少不必要的声波和热流，这可能会防止直径约100纳米的颗粒聚集，如逆转录病毒。这项研究的意义在于，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传递到造血干/祖细胞的效率，而目前的医学方法很难将逆转录病毒转导到造血干/祖细胞。这项研究的成功将为基因治疗领域提供一种创新的方法。

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.