We validate use of Anc80 for gene transfer by knocking out Gli2 from kidney mesenchyme using Anc80-Cre, confirming an antifibrotic effect after chronic obstruction
We validate use of Anc80 for gene transfer by knocking out Gli2 from kidney mesenchyme using Anc80-Cre, confirming an antifibrotic effect after chronic obstruction. proximal tubule.26,27 Thus, various AAVs have been shown to transduce kidney epithelia, but efficiency is generally low; they may require arterial or ureteral administration, and no protocol to date has demonstrated transduction of P005672 HCl (Sarecycline HCl) kidney mesenchymal cells. In this study, we systematically evaluated different AAV serotypes for their ability to deliver genetic material to kidney pericytes and fibroblasts, the myofibroblast progenitor populations.28 We report that the synthetic AAVAnc80 efficiently transduces kidney mesenchymal cells, including pericytes, fibroblasts, and mesangial cells. We validate use of Anc80 for gene transfer by knocking out Gli2 from kidney mesenchyme using Anc80-Cre, confirming an antifibrotic effect after chronic obstruction. We implicate the the retro-orbital venous plexus. Animal Experiments All mouse experiments were performed according to the animal experimental guidelines issued by the Animal Care and Use Committee at Washington University. Gli2-flox (JAX #007926), Rosa26tdTomato (JAX #007909), the left ventricle with 4C PBS for 1 minute. For histologic analyses, tissue sections were fixed in 10% formaldehyde for P005672 HCl (Sarecycline HCl) 1 hour, paraffin embedded, cut with a rotating microtome at a thickness of 3 tests. Paired tests were used for comparison of repeated measures in the same group. Statistical analyses were performed using GraphPad Prism 5.0c (GraphPad Software Inc., San Diego, CA). A value of 0.05 was considered significant. Results Kidney Transduction by AAV Variants The capsid protein is a major determinant of cellular tropism, and we generated a panel of hybrid AAV vectors using the genome of the approved AAV serotype 2 and capsid proteins from AAV Rabbit polyclonal to ZNF76.ZNF76, also known as ZNF523 or Zfp523, is a transcriptional repressor expressed in the testis. Itis the human homolog of the Xenopus Staf protein (selenocysteine tRNA genetranscription-activating factor) known to regulate the genes encoding small nuclear RNA andselenocysteine tRNA. ZNF76 localizes to the nucleus and exerts an inhibitory function onp53-mediated transactivation. ZNF76 specifically targets TFIID (TATA-binding protein). Theinteraction with TFIID occurs through both its N and C termini. The transcriptional repressionactivity of ZNF76 is predominantly regulated by lysine modifications, acetylation and sumoylation.ZNF76 is sumoylated by PIAS 1 and is acetylated by p300. Acetylation leads to the loss ofsumoylation and a weakened TFIID interaction. ZNF76 can be deacetylated by HDAC1. In additionto lysine modifications, ZNF76 activity is also controlled by splice variants. Two isoforms exist dueto alternative splicing. These isoforms vary in their ability to interact with TFIID serotypes 5, 6, 8, and 9. We generated an additional AAV using the synthetic capsid protein Anc80, which has been engineered to reduce antigenic epitopes and has been shown to have unique transduction characteristics.8,30C32 All AAV serotypes contained the same CMV.eGFP.WPRE transgene construct. We reasoned that systemic delivery would be most practical for future clinical application, and therefore, all studies were conducted with intravenous administration; unless noted otherwise, we evaluated transduction 3 weeks after injection. Using a dose of 1011 GCs per mouse, we confirmed efficient transduction by AAV2/8 and AAV2/Anc80 of liver, with lower levels in heart, lung, and kidney (Figure 1A, Supplemental Figure 1). AAV2/8 and AAV2/Anc80 were the only AAV serotypes to exhibit kidney transduction, with Anc80 achieving three- to fivefold higher transduction than AAV8. GFP expression was primarily interstitial and enriched in the outer medulla. Open in a separate window Figure 1. High efficiency transduction of kidney stroma by synthetic adeno-associated virus (AAV) Anc80. (A) Only AAV serotypes 2/2 and 2/Anc80 showed any kidney tropism with eGFP expression detected in glomeruli and interstitium. Scale bar, 50 positive, consistent with mesangium (Figure 1B). The only epithelial cells transduced were usually found in a tubule adjacent to the glomerulus in cells that communicate the Na+:K+:2ClC cotransporter Solute Carrier Family 12 Member 1, indicating the solid ascending limb. Intriguingly, only a subset of epithelia was transduced, however, and they were usually cells adjacent to renin and nitric oxide synthase 1Cexpressing cells that themselves were not transduced (Number 1C). These results indicate that AAV Anc80 efficiently transduces a subset of epithelia in the solid ascending limb adjacent to the juxtaglomerular apparatus (JGA). We next asked whether the synthetic CASI promoter, consisting of a truncated CMV enhancer, chicken reveals that Anc80 is definitely capable of transducing these kidney mesenchymal cells. (D) Quantitation of transduction effectiveness. DAPI, 4,6-diamidino-2-phenylindole; GC, genome copy. Discussion We statement three major findings. We have identified the 1st AAV serotype, namely Anc80, that efficiently transduces adult kidney mesenchymal cells and myofibroblast progenitors. We also display that Anc80-Cre P005672 HCl (Sarecycline HCl) mediates high-efficiency recombination of two independent floxed alleles, Gli2 and em /em -catenin, confirming important functions for these signaling pathways in mediating renal fibrosis and validating this approach in mice. Finally, we display that Anc80 also can also deliver genetic material to human being mesenchymal cells in tradition and in human being kidney organoids, providing a proof of principle for the use of AAV-based vectors in gene therapy approaches to treat kidney fibrosis. The mechanistic basis for the enhanced transduction properties of the mesenchymal lineage in adult murine kidney is the subject of future studies, but it may relate to the quick kinetics of manifestation after Anc80 illness that may enable transduction of less permissive cell focuses on.30,32 Our findings have applications for preclinical fibrosis models, because a major limitation of current investigations of stromal biology is the lack of high-efficiency Cre driver lines for the efficient manipulation of gene expression in adult kidney. Although FoxD1-Cre mediates recombination in nearly all kidney stroma, it is only P005672 HCl (Sarecycline HCl) active during nephrogenesis, and the FoxD1-CreERt2 driver is not indicated in adult kidney.36 We have used the Gli1-CreERt2 driver with.