scAAV DJ-GFP

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder characterized by the formation of kidney cysts that originate from the epithelial tubules of the nephron and are primarily caused by mutations in polycystin-1 (PKD1) and polycystin-2 (PKD2). Metanephric organ culture (MOC) is an ex vivo system in which transplanted embryonic kidneys undergo tubular differentiation and renal development. Here, the researchers used a lentivirus and three serotypes of self-complementary adeno-associated virus (scAAV) plasmids expressing green fluorescent protein and found that scAAV serotype D/J transduced the epithelial compartment of MOCs with 68% efficiency. They used scAAV/DJ to deliver shRNA to knock down Pvt1, a long noncoding RNA that is upregulated in the kidneys of Pkd1 and Pkd2 mutant mice and humans with ADPKD. shRNA delivery by scAAV/DJ downregulated expression of Pvt1 by 45% and reduced the cyst index by 53% in wild-type MOCs and 32% in Pkd1-null MOCs. Knockdown of Pvt1 reduced c-MYC protein levels by 60% but did not affect Myc mRNA, suggesting that Pvt1 regulation of c-MYC is posttranscriptional. These results suggest that Pvt1 is a long noncoding RNA that regulates cyst progression in MOCs.

Embryonic day (E)14.5 kidneys from wild-type mice were incubated with viral particles for 4 h. Kidneys were then cultured on Transwell membranes and subjected to immunohistochemistry (IHC) analysis at 96 h. All viral constructs expressed green fluorescent protein (GFP) under the control of the cytomegalovirus promoter. GFP fluorescence signals were detected at 96 h in whole kidneys transduced with scAAV/DJ-GFP (Figure 1B), scAAV/9-GFP (Figure 1C), scAAV/2-GFP (Figure 1D), or VSV-G pseudotyped lentivirus-GFP (Figure 1E). GFP expression was detected in whole MOCs incubated with all viruses at 96 h post-transduction. When scAAV and lentivirus serotypes were compared, serotype DJ showed more than twice the RGFI compared to the other viruses tested (Figure 1F). To confirm the identity of the transduced cells, the metanephros were sectioned and stained with GFP antibodies. In MOCs transduced with lentivirus, GFP staining was minimal in tubular epithelial cells and higher in surrounding mesenchymal cells (Figure 1G and H). In contrast, GFP staining in metanephros transduced with scAAV/DJ was enriched in epithelial cells (Figure 1I and J). No GFP staining was detected in metanephros transduced with scAAV/DJ virus lacking GFP (Figure 1K).

Figure 1. Transduction of epithelial cells in the metanephric organ culture (MOC) by scAAV/DJ-GFP. (Eckberg K, et al., 2022)