Empty virus-like particles (eVLPs) as bio-compatible targeted drug-delivery vehicles

药理学中的一个主要挑战是设计药物可以特异性地递送到靶组织的方法。这在抗癌药物的情况下是一个特别的问题，抗癌药物通常通过癌细胞分裂更快的事实来区分癌细胞和正常细胞。然而，抗癌药物对所有细胞都是有毒的，因此通常具有严重的副作用。为了避免这种情况，显然需要将药物分子特异性靶向癌组织。实现这一点的一种潜在方法是将药物分子包装或封装在一种颗粒内，该颗粒被设计为仅与癌组织结合。这种包封将具有保护药物免于在血浆中分解的额外优点。为了实现这一点，有必要开发可以在其外表面上进行修饰以实现所需靶向并且可以包含药物分子的颗粒。这些颗粒需要足够小，以便能够在血液中移动，没有毒性，并且能够进入细胞。具有所有这些特征的一种类型的颗粒是植物病毒，豇豆花叶病毒（CPMV），其可以通过感染植物而大量产生。先前的研究表明，有可能将分子“粘”在CPMV颗粒的表面上，使它们能够靶向特定的细胞。尽管有这些优点，但迄今为止，CPMV颗粒还没有被用作药物递送载体。其原因是，感染植物产生的颗粒已经充满了病毒自身的遗传物质。这意味着在颗粒内部几乎没有空间放置其他任何东西，例如药物分子。即使这样的分子可以以某种方式被挤进去，仍然会有人担心含有病毒遗传物质的颗粒的管理，即使它是来自植物病毒，不能感染动物。该项目通过利用John Innes中心最近发现的一种在植物中产生大量CPMV纯空（缺乏遗传物质）病毒样颗粒（eVLP）的方法来解决病毒颗粒内遗传物质的问题。该方法包括使用最近开发的高效植物瞬时表达系统同时表达编码病毒外壳蛋白前体的基因和用于加工它的酶。应用这种方法，我们将产生经过修饰的颗粒，使它们特异性地结合癌细胞表面表达的蛋白质，而不是正常细胞。这种修饰将通过化学方法或与病毒外壳蛋白进行基因融合来完成。我们将研究用抗癌药物吉西他滨装载靶向颗粒的最佳方式。为了做到这一点，我们将利用病毒颗粒中的孔，这些孔允许小分子在一定条件下进入。靶向颗粒将药物递送到癌细胞的能力将通过测试它们在培养物中结合和杀死癌症的能力来研究。这将是潜在开发全新治疗药物以应对人类癌症的第一个重要步骤。

A major challenge in pharmacology is to devise methods whereby drugs can be delivered specifically to target tissues. This is a particular issue in the case of anti-cancer drugs which usually discriminate between cancerous and normal cells by the fact that the cancer cells are dividing more rapidly. However anti-cancer drugs are toxic to all cells and thus often have severe side-effects. To avoid this, it would clearly be desirable to target the drug molecule specifically to the cancerous tissue. A potential means of achieving this would be to package or encapsulate the drug molecules inside a particle which is designed to bind solely to the cancerous tissue. Such encapsulation would have the additional advantage of protecting the drug from breakdown in blood plasma. For this to become a reality it will be necessary to develop particles which can be modified on their outer surface to achieve the desired targeting and which can contain drug molecules. The particles need to be small enough to be able to move in the bloodstream, are not toxic and to be able to enter cells. One type of particle which has all these characteristics is the plant virus, cowpea mosaic virus (CPMV), which can be produced in large quantities by infecting plants. Previous research has shown that it is possible to 'stick' molecules on the surface of CPMV particles which enable them to be targeted to specific cells. Despite these advantages, CPMV particles have not, to date, been exploited as a drug-delivery vehicle. The reason for this is that particles produced by the infection of plants are already filled with the virus' own genetic material. This means that there is little or no room to put anything else, such as drug molecules, inside the particles. Even if such molecules could somehow be squeezed in, there would still be concerns about administering particles containing viral genetic material, even though it is from a plant virus that cannot infect animals. This project addresses the issue of the genetic material within the virus particles by exploiting the recent discovery at the John Innes Centre of a method of producing large quantities of pure empty (lacking genetic material) virus-like particles (eVLPs) of CPMV in plants. The method involves simultaneously expressing genes coding for a precursor of the viral coat proteins and the enzyme used to process it using a recently developed highly efficient plant transient expression system. Applying this approach we will produce particles which are modified so that they will specifically bind to proteins expressed on the surface of cancerous, but not normal, cells. This modification will be done either chemically or by making genetic fusions to the virus coat protein. We will investigate the best way of loading the targeted particles with the anti-cancer drug, gemcitabine. To do this we will make use of pores in the virus particles which allow small molecules to enter under certain conditions. The ability of the targeted particles to deliver the drug to cancer cells will be investigated by testing their ability to bind to and kill cancers in culture. This will be the first important step in potentially developing brand new therapeutic agents to tackle human cancer.