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

Area of Science:

• Biotechnology
• Gene Therapy
• Ophthalmology

Background:

• Adeno-associated virus (AAV) vectors are crucial for developing gene therapies for inherited retinal diseases.
• Current preclinical testing relies on animal models, which are not suitable for manufacturing batch release or later-stage validation.
• There is a need for rapid, reliable in vitro models to assess therapeutic vector function.

Purpose of the Study:

• To develop and validate a novel cell-based system for evaluating adeno-associated virus (AAV) vectors.
• To assess the potency and biological function of AAV vectors designed for treating CNGA3-linked achromatopsia.
• To establish a reliable in vitro method for gene therapy vector assessment.

Main Methods:

• Developed an immortalized cell line expressing rhodopsin guanylyl cyclase (RhGC) and a genetically encoded calcium indicator (GECI) under tetracycline-inducible control.
• Utilized the cell-based system to evaluate AAV vectors delivering a functional CNGA3 gene.
• Employed a fluorometric assay based on the interdependent activity of expressed proteins to measure ion channel function.

Main Results:

• Successfully confirmed and quantified the function of the ion channel encoded by AAV/CNGA3.
• Demonstrated the ability to differentiate between the potencies of various AAV vector preparations.
• Showed the adaptability of the system for assessing other AAV-borne functions, such as phosphodiesterase activity.

Conclusions:

• The developed cell-based system provides a rapid and reliable in vitro method for evaluating AAV vector potency and biological function.
• This assay system is valuable for the preclinical development and quality control of gene therapies for inherited retinal diseases.
• The approach is versatile and can be adapted for assessing a range of AAV-mediated gene therapies.