Development of In Vitro Potency Assays for Gene Therapy Products Targeting cGMP-Related Retinopathies.

The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited retinal diseases. 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 transgene expression and resulting restoration of vision 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. As such, there is need for rapid and reliable in vitro models to test whether therapeutic vectors can deliver their genetic payload and, more importantly, whether this leads to the intended biological function. We developed a cell-based system to evaluate AAV vectors designed to deliver a healthy CNGA3 gene copy into photoreceptors of patients with CNGA3-linked achromatopsia. Our system uses immortalized cells that express a rhodopsin guanylyl cyclase (RhGC) from Blastocladiella emersonii and a genetically encoded calcium indicator (GECI) in a tetracycline-inducible manner. When transduced cells express a functional AAV-borne CNG channel, the three proteins generate a fluorometric assay that relies on the interdependent activity of its components. 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. Finally, we show that this approach can be readily adapted for the assessment of AAV-borne phosphodiesterase function.