Ljungberg, M. C. | Search bioRxiv

bioRxiv2024

Common AAV gene therapy vectors show indiscriminate transduction of living human brain cell types

The development of cell-type-specific gene therapy vectors for treating neurological diseases holds great promise, but has relied on animal models with limited translational utility. We have adapted an ex vivo organotypic model to evaluate adeno-associated virus (AAV) transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.

bioengineering↗

bioRxiv2024

A Comprehensive Atlas of AAV Tropism in the Mouse

Gene therapy with Adeno-Associated Viral (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of ten naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 and AAVrh74) following systemic delivery into male and female mice. A transgene expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.

molecular biology↗

bioRxiv2020

SOX7 deficiency causes ventricular sepal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2

SOX7 is located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with septal defects. Sox7-/- embryos die of heart failure around E11.5 due to defects in vascular remodeling. These embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. We also observed a ventricular septal defect in a rare Sox7flox/-;Tie2-Cre embryo that escaped early lethality. This led us to hypothesize that SOX7 plays a critical developmental role in the endocardium of the atrioventricular (AV) canal. We subsequently used AV explant studies to show that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). Since SOX7 is a transcription factor, we hypothesized that it functions in the endocardium by regulating the expression of EndMT-related genes. To identify these genes in an unbiased manner, we performed RNA-seq on pooled E9.5 hearts tubes harvested from Sox7-/- embryos and their wild-type littermates. We found that Wnt4 transcript levels were severely reduced, which we confirmed by RNA in situ hybridization. Previous studies have shown that WNT4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of BMP2 in the myocardium. Consistent with these findings, we found that Bmp2 transcript levels were diminished in Sox7-/- embryonic hearts. We conclude that SOX7 promotes EndMT in the developing AV canal by modulating the expression of Wnt4 and Bmp2. These data also provide additional evidence that haploinsufficiency of SOX7 contributes to the congenital heart defects seen in individuals with recurrent 8p23.1 microdeletions. SUMMARY STATEMENTIn the developing atrioventricular canal, SOX7 promotes endocardial-to-mesenchymal transition (EndMT) by positively regulating Wnt4 and Bmp2 expression. SOX7 deficiency leads to the development of hypocellular endothelial cushions and ventricular septal defects.

developmental biology↗