Biodistribution of a supplementary gene therapy approach for phospholamban deficiency in a genetically modified pig model

Introduction Phospholamban (PLN) is a small protein that regulates calcium cycling in heart muscle cells by binding to sarcoplasmic Ca²⁺-ATPase (SERCA), controlling contraction, relaxation, and heart rate. The monoallelic Arg(R)14del mutation in the PLN gene disrupts calcium homeostasis and leads to dilated or arrhythmogenic cardiomyopathies in over 1000 patients, mainly of Dutch origin. Founding on the hypothesis that decreasing the proportion of mutated mRNA transcripts by wild-type (WT) overexpression would alleviate its pathogenic effect, we here aimed at gene therapy in a genetically modified (GM) pig model. Methods A pig model was created to express either human WT PLN (huWT-PLN) or the human R14del variant of PLN from one of the endogenous PLN sites. AAV6 vectors expressing human WT PLN, a crimson reporter gene, or combinations of both were delivered via catheter-based retrograde infusion into the main coronary veins under short term venous and arterial occlusion. Different doses were tested. After 1-4 months, hearts were segmented into 60 defined pieces across left ventricle (LV) and right ventricle (RV). First, twenty segments per heart were analyzed by quantitative PCR (qPCR) to gain an overview of AAV transduction and transgene expression across the heart. Specific qPCR assays measured endogenous humanized R14del, humanized WT, porcine WT, and AAV6-delivered huWT-PLN. All assays and cDNA samples were tested for quality threshold of slope and linearity. Histological analyses were performed on samples transduced with the crimson reporter gene. Results A total of 8 pigs underwent retrograde venous infusion. Endogenous PLN expression was consistent across heart regions, differing only by a factor of 2 across different areas in LV and RV and reaching up to 50% of GAPDH transcript levels. Porcine WT transcripts were about 50% of humanized alleles, likely due to 3′-UTR differences. Following AAV6-mediated huWT-PLN delivery, transgene expression was heterogeneous. At a high dose of 1 × 10¹³ vector genomes, transgene expression was comparable to or exceeded that of endogenous PLN in some areas of the heart, with higher levels observed in apical regions and around large vessels. High expression correlated with increased AAV6 copy numbers. Histology showed preferential cardiomyocyte transduction with AAV6, some endothelial transduction, and patchy distribution of AAV6 transduction throughout the tissue. Conclusions / Outlook These findings confirm successful gene delivery and high AAV6 transcriptional activity but highlight the challenge of achieving uniform myocardial transduction. Ongoing experiments explore different strategies to enhance local vector uptake. A 3D reconstruction of vascular anatomy is planned for more detailed biodistribution analysis.