Advancing entirely virus-free CRISPR CAR T cells to clinic- Are they as good as lentiviral CAR?

我们已经利用大奥蒙德街医院的专业设施，将一些实验性的嵌合抗原受体(CAR)T细胞疗法投入临床试验。这些帮助治疗了患有某些类型白血病的儿童和成人，否则他们的疾病将没有进一步的选择。这些项目从实验室的概念和测试，到洁净室的制造和患者的临床使用。所有这些都依赖于使用被禁用的病毒将基因添加到T细胞中来重新编程。一些病毒的CAR T细胞通过基因组编辑--分子剪刀剪掉其他基因的片段--经历了额外的改变，这使得“通用”T细胞可以在没有任何匹配的情况下使用。现在，使用类似的技术来切割和插入新的基因已经成为可能，这意味着我们可以在不需要病毒添加CAR基因的情况下设计细胞。我们认为，这种方法可以消除供应瓶颈，特别是为每辆新车制造病毒库存，这是临床试验的一个主要阻碍问题。到目前为止，我们的实验室工作已经表明，使用CRISPR插入汽车的效率几乎与我们使用病毒时一样高，而且细胞在短期培养皿实验中的行为和功能也一样好。我们现在需要确保这个过程在更大的范围内工作，以一种可以在无尘室中使用的方式，并且当细胞在动物体内激活并在较长时间内对人类白血病做出反应时，它们的行为没有不同。这个应用程序将使用现有的慢病毒或新的无病毒策略来制造和比较CRISPR CAR CAR T细胞。这些测试的规模将适合未来的人体试验，并在可以转移到无尘室环境的条件下进行。这些细胞将在实验室和携带人类癌症的小鼠身上进行测试，并进行深入分析，以绘制不同细胞群体中哪些基因活跃的图谱。我们将包括测试，以找出哪种编辑组合效果最好，并包括根据CAR在基因组中插入位置来驱动CAR表达的版本。该项目将采用一种新的针对CD38的汽车，我们的实验室已经对其进行了测试。它是从抗多发性骨髓瘤和自身免疫性疾病的单抗Daratumab中衍生出来的，因此具有广泛的安全性和毒性。目前正在制定一种现成的通用CAR38疗法，以探索细胞介导的深度清除疾病的方法，该项目将有助于确定使用哪些策略来制造CAR T细胞库，以便在试验之前进行。如果CAR T细胞的生产能够转换到完全无病毒的平台上，这将节省时间和金钱，并从长远来看，允许更多的版本进入临床阶段。

We have delivered a number of experimental chimeric antigen receptor (CAR) T cell therapies into clinical trials using specialist facilities at Great Ormond Street Hospital. These have helped treat children & adults with certain types of leukaemia who would otherwise have no further options for their disease. These projects were taken from concept and testing in the lab, through to manufacturing in clean rooms and clinical use in patients. All have relied on using disabled viruses to add genes into T cells to reprogram them. Some viral CAR T cells have undergone extra changes using genome editing - molecular scissors to snip out bits of other genes - and this allowed 'universal' T cells to be used without any matching. Now its becoming possible to use similar technology to both cut genes and insert new ones, and this means we can engineer cells without the need for viruses to add the CAR gene. We think the approach could eliminate supply bottlenecks, especially for making virus stocks for each new CAR, a major hold up issue for clinical trials. We have shown in our lab work so far that the efficiency of CAR insertion using CRISPR is almost as high as when we use viruses, and the cells behave and function just as well in short-term dish experiments. We now need to make sure that the process works at a larger scale, in a way that can be used in clean rooms, and that the cells behave no differently when they activated and responding against human leukemia in animals over a longer period.This application will manufacture and compare CRISPR CAR T cells using existing lentiviral or new virus-free strategies to add the CAR. The tests will be on a scale suitable for future human trials and under conditions that can be transferred to a clean room setting. The cells will be tested in the lab and also in mice carrying human cancers, and analysed in depth to produce maps of which genes are active in the different populations of cells. We will include tests to find out which combinations of edits work best, and include versions where CAR expression is driven by where in the genome it has been inserted.The project will adopt a new CAR against CD38 which is already being tested in our lab. It was derived from the monoclonal antibody Daratumumab, which is being widely investigated against multiple myeloma and autoimmune disorders and so has an extensively mapped safety and toxicity profile. Plans for an 'off-the-shelf' universal CAR38 therapy to explore deep cell-mediated clearance of disease are being formulated, and this project will help determine which strategies to use to manufacture the CAR T cell banks ahead for a trial. If CAR T cell production can be switched to entirely virus-free platforms, it will save time and money, and in the long run allow many more versions to be advanced to a clinical stage.