Abstract 4133040: Cardiac Favored Transcription of the Exogenous Gene in Mice after A Single Systemic Delivery of AAV9-cBIN1

Introduction: Heart failure (HF) is associated with high mortality and morbidity, and there are limited therapeutic options available to improve cardiac muscle function. Recent studies suggest that the impairment of cardiomyocyte T-tubule microdomains is a key factor in HF pathophysiology. AAV9-cBIN1 gene therapy effectively addresses this impairment. However, the kinetics and biodistribution of AAV9-cBIN1 have not been explored, limiting the knowledge of the transcription efficiency and organ specificity of cBIN1 gene therapy when delivered systemically. Aim: This study aims to elucidate the kinetics and biodistribution of AAV9-cBIN1 in a murine model. Method: Adult male C57BL/6J mice (n=20) were administered a single retro-orbital injection of AAV9-CMV-cBIN1-V5 at a dose (2x10^12 vg/kg) known to effectively transduce the majority of cardiomyocytes. Mice were sacrificed at pre-determined time points: prior to injection (Day 0, n=2) and post-injection at Days 3, 7, 14, 28, 56, and 84 (n=3/group). Tissues were harvested from the heart, lung, liver, kidney, skeletal muscle, and spleen. Genomic DNA was extracted and analyzed using qPCR to determine vector genome (vg) copies. For transcriptional analysis of the AAV9-transduced exogenous gene, mRNA was extracted and converted to cDNA for qRT-PCR quantification of cBin1-V5 , normalized to the housekeeping gene Hprt1 . Results: All mice survived to their designated sacrifice times. Kinetic analysis demonstrated a time-dependent expression of the exogenous cBin1-V5 gene, with peak expression observed at 4 weeks in both the heart and liver, with expression declining in the liver after this peak (Figure 1A). Genomic DNA analysis showed a gradual increase and plateau of viral genome distribution in the heart between 2-4 weeks, while in the liver, distribution peaked sharply between Days 3-7 and then decreased. When correcting the gene transcription of cBin1-V5 (ΔCq of V5/Hprt1 ) for vector DNA vg copies in the tissue, the transcription efficiency in the heart was higher than in the liver (Figure 1B). Outside of the liver and heart, cBin1-V5 mRNA expression was undetectable in all other tissues by Day 56. Conclusion: In adult male mice, a single systemic administration of AAV9-cBIN1 results in robust transduction and higher transcription efficiency in the heart compared to the liver. AAV9-cBIN1 holds promise for targeted gene therapy in cardiac tissues, with minimal off-target expression in other organs.