Introduction
Phospholamban (PLN) is a small protein that interferes with calcium cycling in heart muscle cells by binding to sarcoplasmic Ca²⁺-ATPase (SERCA) and thereby controls contraction, relaxation and frequency of the heart. The mono-allelic Arg(R)14del mutation in the PLN gene disturbs calcium homeostasis and leads to dilated and/or arrhythmogenic cardiomyopathies in > 1000 patients, mainly of Dutch origin. Founding on the hypothesis that decreasing the proportion of mRNA transcripts by WT overexpression would alleviate its pathogenic effect, we here aimed at gene therapy in a genetically modified (GM) pig model.
Methods
A pig model has been created to express the human WT PLN or the human R14del variant of PLN from one of the endogenous PLN sites. AAV6 vectors expressing either human WT PLN, a Crimson reporter genes or combinations of both were delivered by catheter-based retrograde infusion to GM pigs. Different dosages were tested.
After 1-4 months, the hearts were segmented in 60 defined tissue pieces across the left (LV) and right (RV) ventricles. 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 were designed for endogenous humanized R14del, the humanized WT, and the porcine WT alleles, as well as the AAV6-delivered huWT-PLN transgene. All assays and each cDNA was tested for quality threshold of slope and linearity control included integrity assessment of cDNA and gDNA to confirm assay sensitivity and linearity. Second, histological evaluation was performed on samples that were transduced with the crimson reporter gene
Results
A total of 8 pigs underwent retrograde infusion. Endogenous PLN expression was found to be very consistent across examined heart regions, differing only by a factor 2 across different areas in LV and RV, and considerably high, reaching almost 50% of GAPDH transcript levels. The porcine WT transcripts were approximately 50% of the GM human alleles, presumably due to different genetic constitution in the 3´-UTR. Transgene expression of the human PLN WT after AAV6 gene transfer led to a heterogeneous distribution in the myocardium. At high doses of 1x 10^13 vp, transgene expression levels were similar to or higher than the endogenous PLN in some areas of the heart in some areas of the heart. However, transgene expression was not equally distributed across the heart. Generally it was higher in apical regions than at the basal areas. In more detail, expression was highest in samples localized around large blood vessels. The levels of high transgene expression correlated to increased copy numbers of the AAV6 vector at genomic level. Complementary histological analysis of tissue areas that showed high transgene expression, revealed a clear preference for cardiomyocyte transduction with AAV6, with some bystanding endothelial cell transduction and a patchy distribution of AAV6 transduction throughout the tissue.
Conclusions / Outlook
These findings confirm successful gene delivery and high transcriptional activity of the AAV6-vectors and also point at the difficulty of achieving ubiquitous myocardial transduction.
Future experiments will focus on improving distribution by testing different administration routes, occlusion times, or vector shaping.
