High Katherine A, Le Blond Dave, Doucette Karen, Liu Dezhong, Farjo Rafal, Ignatova Irena, Buchlis George, Chung Daniel, Couto Linda B
Spark Therapeutics, Philadelphia, PA 19103, USA.
Robert Singer Consulting, Sonora, CA 95370, USA.
Mol Ther Methods Clin Dev. 2025 Jan 25;33(1):101423. doi: 10.1016/j.omtm.2025.101423. eCollection 2025 Mar 13.
Voretigene neparvovec-rzyl (Luxturna) is an AAV2 vector (AAV2-hRPE65v2) that expresses a cDNA encoding the human retinal pigment epithelium-specific 65 kDa protein (RPE65). It has been approved for the treatment of visual deficits associated with biallelic mutations in human RPE65 in the US, European Union (EU), and multiple other countries. To achieve regulatory approval, it was necessary to validate an assay demonstrating its biological activity or potency. The assay measures AAV2-hv2 transduction in HEK293 cells and the subsequent biological activity of the vector-encoded RPE65 protein in cell lysates. RPE65 converts all--retinol to 11--retinol, which is quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The assay was validated for seven characteristics, namely system and sample suitability, specificity, linearity, precision, relative accuracy, range, and robustness. The validated assay can be used to confirm the relative potency levels of different lots of Luxturna in the range of 50%-150% of a reference standard (defined as 100% potent). This represents the first report of validation studies supporting an cell-based relative potency assay for an AAV vector, which was used to evaluate lot-to-lot consistency, stability, and comparability following manufacturing changes and to successfully launch Luxturna, the first gene therapy approved in the US for a genetic disease.
维替吉恩帕罗维c-rzyl（Luxturna）是一种AAV2载体（AAV2-hRPE65v2），可表达编码人视网膜色素上皮特异性65 kDa蛋白（RPE65）的cDNA。它已在美国、欧盟（EU）和其他多个国家被批准用于治疗与人类RPE65双等位基因突变相关的视力缺陷。为获得监管批准，有必要验证一种能证明其生物活性或效力的检测方法。该检测方法可测量HEK293细胞中AAV2-hv2的转导情况以及细胞裂解物中载体编码的RPE65蛋白的后续生物活性。RPE65可将全反式视黄醇转化为11-顺式视黄醇，这可通过液相色谱-串联质谱法（LC-MS/MS）进行定量。该检测方法针对系统和样品适用性、特异性、线性、精密度、相对准确度、范围和稳健性这七个特性进行了验证。经过验证的检测方法可用于确认不同批次的Luxturna在参考标准（定义为100%效力）的50%-150%范围内的相对效力水平。这是支持基于细胞的AAV载体相对效力检测的验证研究的首份报告，该检测方法用于评估生产变更后的批次间一致性、稳定性和可比性，并成功推出了Luxturna，这是美国首个获批用于治疗遗传病的基因疗法。
