Department of Biological Structure, University of Washington, Seattle, WA 98195, USA.
Department of Ophthalmology, University Hospital, LMU Munich, 80336 Munich, Germany.
Int J Mol Sci. 2022 Apr 20;23(9):4538. doi: 10.3390/ijms23094538.
The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited monogenic diseases, such as retinal dystrophies. To evaluate the potency of new gene therapy vectors in a preclinical context, it is common to use animal models, such as gene-deficient or mutant animal models of a given human disease, and then assess vision restoration with functional or behavioral assays. While such animal models are invaluable to the preclinical testing process, they cannot be readily used as batch release tests during manufacturing or to validate biological activity at later stages of development. There is therefore a need for rapid and reliable in vitro models that can determine whether therapeutic vectors have delivered their cargo gene, and more importantly, whether this has resulted in the intended biological activity. Given our previous experience, we chose CNGA3-linked achromatopsia to develop a cell-based system to verify biological activity of AAV vectors designed to deliver a healthy gene copy into human cone photoreceptors. Our system is based on an immortalized cell line with high susceptibility to AAV transduction, i.e., HeLa cells, which we engineered to express a fungal rhodopsin guanylyl cyclase (RhGC) from and a sensitive genetically encoded calcium indicator (GECI) under the control of a tetracycline operator. Using this system, we were able to confirm and quantify the function of the ion channel encoded by AAV/CNGA3 and differentiate between AAV vector potencies with a simple fluorometric assay. Finally, we show that this approach can be readily adapted for the assessment of phosphodiesterase function.
高效的腺相关病毒（AAV）衍生病毒载体的出现促使许多研究团队开发针对遗传性单基因疾病（如视网膜营养不良）的基因疗法。为了在临床前背景下评估新基因治疗载体的效力，通常使用动物模型，例如特定人类疾病的基因缺失或突变动物模型，然后使用功能或行为测定来评估视力恢复情况。虽然这些动物模型对于临床前测试过程非常宝贵，但它们不能在制造过程中作为批量放行测试或在开发的后期阶段验证生物活性使用。因此，需要快速可靠的体外模型来确定治疗性载体是否已递送其 cargo 基因，更重要的是，是否已产生预期的生物活性。鉴于我们之前的经验，我们选择与 CNGA3 相关的色盲来开发基于细胞的系统，以验证旨在将健康基因拷贝递送至人锥体光感受器的 AAV 载体的生物活性。我们的系统基于对 AAV 转导高度敏感的永生化细胞系，即 HeLa 细胞，我们通过该细胞表达来自 的真菌视蛋白鸟苷酸环化酶（RhGC）和受四环素操纵子控制的敏感遗传编码钙指示剂（GECI）。使用该系统，我们能够确认和量化 AAV/CNGA3 编码的离子通道的功能，并通过简单的荧光测定法区分 AAV 载体效力。最后，我们表明这种方法可以很容易地适应磷酸二酯酶功能的评估。
