Meet the Scientist: Christian Vettermann, Ph.D.
When Assays Lead to More Questions
A型血友病的治疗通常旨在提高血浆中FVIII活性水平. To measure the effectiveness of these treatments,
scientists and clinicians have traditionally relied on two standard assays, or tests: the one-stage clot assay, and the chromogenic-substrate assay. For early hemophilia A treatments, such as human plasma-derived therapies, the two assays produced relatively comparable FVIII activity measurements.
然而，当你用这些检测方法来衡量当前的护理标准——重组FVIII治疗——时，这些测试可能会产生不同的结果. 对于正在研究的血友病A基因疗法，这些检测也产生了不同的结果, which we were the first to measure. Obviously, having measurements that don’t agree with each other is a problem. Which one do you trust? Researchers have been trying to solve this problem for years, and we were the first to propose and substantiate a plausible explanation.
Asking the Right Questions
十大正规平台调查的一个重要起点是十大正规平台进行的一项大型实地研究, 显示出世界各地实验室中不同的FVIII测量值的显著一致性. This phenomenon is clearly independent of assay kit reagents, gene therapy dose, FVIII expression level or time after gene therapy administration.
From there, 十大正规平台的问题是:是否有一种检测方法可以提供重组FVIII和血友病A基因治疗的可比测量值? When we compared FVIII activity as measured by the
one-stage clot (OS) assay and chromogenic-substrate (CS) assay, we saw noticeable differences between recombinant FVIII and gene therapy in the OS assay, 但两种处理的CS测试结果基本一致(当基于相同数量的FVIII蛋白进行比较时). You can see this in the graphic below.
该结果对在重组FVIII领域工作的研究人员有帮助，他们还不能完全回答使用哪种检测方法的问题, 因为十大正规平台表明CS测定确实是一种可以跨模式使用的方法，并且可能对环境因素的干扰不太敏感. 我应该注意的是，OS测定对血友病A基因治疗仍然具有临床意义:OS和CS测量与该治疗紧密相关，甚至可以通过使用简单的转换因子进行翻译. Nonetheless, 十大正规平台的发现可能会导致该领域重新考虑对新型血友病A治疗的常规临床测试的分析选择-特别是在美国.S., where most laboratories still prefer to use OS, since it’s cheaper and easier to implement in the lab.
Answers found along the way
十大正规平台还想知道为什么这些分析首先会产生不同的测量结果. For ongoing research in gene therapies in particular, the measurement was about 1.6 times higher with the OS than CS assay. So, I engaged with key biochemical experts in the coagulation field, Drs. Steffen Rosen and Stefan Tiefenbacher, to work with 十大正规平台 on this mechanistic puzzle. We found a two-part answer to this question.
第一部分涉及动力学，或在测定过程中发生的反应速率. When triggered adequately, 十大正规平台发现，基因治疗后产生的FVIII表现出最初的活性爆发，并比正常血浆中的天然FVIII更快地开始反应，导致凝血过程开始得更快. After this initial kick-start, 然后，基因治疗FVIII的动态速度开始趋于平稳，似乎与自然FVIII的速度一致.
The second part relates to the format and length of both assays. OS测定在很短的时间内进行，并在大约一分钟后读取FVIII活性. In contrast, the CS assay takes five minutes to read out. 十大正规平台意识到，当基因治疗FVIII经历其最初的活动爆发时，OS分析正在读出, while the CS assay was reading out later, when the pace of activity had become similar between gene therapy and natural FVIII. 总之，这些发现有助于解释基因治疗FVIII的高OS测量. 值得注意的是，目前还没有针对血友病A的基因疗法被批准使用或被确定为安全有效，这些结果与正在进行的血友病A研究有关.
I also want to share one important, related observation: despite the faster onset of coagulation with gene therapy FVIII, 十大正规平台还发现，凝血酶(一种直接介导血凝块形成的酶)的总体水平在最后没有受到影响.
Industry reaction
We’ve now published our work and presented it at several scientific conferences, 人们一致认为CS检测是研究血友病a基因疗法的可靠方法.
虽然卫生当局要求十大正规平台解决不同的测量结果，以使FVIII活性合法化，作为临床试验的主要终点, we are still assessing the physiological and clinical relevance of this difference. Nonetheless, 选择更保守的CS测量使用理性的科学论据作为十大正规平台的研究计划的一部分，为十大正规平台带来了与十大正规平台的利益相关者的信誉.
Ultimately, 十大正规平台的研究成果发表在《十大正规平台》杂志上，对血友病患者的临床医生来说是一个巨大的成就——帮助他们更好地了解AAV基因治疗后的FVIII测量结果.
目前还没有针对A型血友病的基因疗法被批准使用或被确定为安全有效.