(485g) Robust Biomanufacturing of Recombinant AAV for Cardiac-Specific Gene Delivery

Gene therapy using recombinant adeno-associated virus (rAAV) shows promise in treating heart diseases due to its low immunogenicity, stable gene expression, and minimal adverse effects. However, several challenges must be overcome before a viable clinical practice, including lack of targeted delivery of genes of interest to cardiomyocytes and a robust scalable biomanufacturing of rAAV. This study aimed to develop and optimize an advanced bioproduction and purification process to generate high-yield, high-purity, high-quality cardiomyocyte-specific rAAV.

To achieve this goal, an AAV expression vector was synthesized by cloning the mitochondrial optogenetic (mOpto) gene into the AAV-DJ Helper-Free Promoterless Expression System. The cTnT promoter was used to enhance cardiomyocyte-specific gene expression. High-titer AAV was produced by engineered HEK293 cells in shaker flasks and 2-L stirred-tank bioreactors. The AAV was then purified using Next Generation Chromatography (NGC) equipped with a Foresight Nuvia HPQ column and desalted using a HiTrap Desalting column. After we confirmed the expression and quality of AAV produced by shaker flasks in small scale, scaled-up bioreactor was performed for in vivo studies. Our results showed that the average titer of crude virus was 5-10x10¹⁰ vg/mL from bioreactor productions. The SDS-PAGE with silver staining and Western Blotting confirmed the presence of three AAV capsid proteins, and transmission electron microscopy (TEM) verified the presence of AAV particles.

The in vitro characterizations showed that the produced rAAV effectively transduced AC16 cardiomyocytes with varying multiplicity of infection (MOI). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) indicated MOI-dependent mOpto gene expression. Blue LED illumination elicited light intensity-dependent mitochondrial depolarization in the transduced cells, indicating functional mOpto protein expression. The rAAV was further concentrated using a Savant Speed Vacuum concentrator to 2x10¹² vg/mL and injected subcutaneously into 10-day-old mice. Three to four weeks later, the mice were sacrificed, and major organs were harvested and subjected to biochemical analysis. qRT-PCR showed high mOpto gene expression in cardiac tissue, but non-detectable in any other organs such as the brain and liver. Moreover, LED illumination caused mitochondrial depolarization only in isolated cardiomyocytes but not in isolated fibroblasts, demonstrating cardiomyocyte-specific mOpto expression.

In conclusion, this study describes a robust, scalable, high-yield biomanufacturing process of rAAV with high quality (high in vivo transduction efficiency and cardiac specificity). The use of a cardiomyocyte-specific promoter, cTnT, and the AAV-DJ capsid, allows for targeted and efficient gene expression in cardiac tissue.