CAG-GFP Adeno-associated virus(AAV Serotype 8)

Gene therapy can treat a variety of kidney diseases. However, the efficiency of gene delivery to kidney cells is low. This is partly due to the kidney's rejection of molecules larger than 50 kDa and the fact that most gene delivery vectors are megadaltons in mass. Here, researchers compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentivirus (LV) vectors to deliver genes to kidney cells. When vectors were delivered to mice via the intravenous (IV) route, weak luciferase activity was observed in the kidneys, while significant activity was observed in the liver. When gene delivery was observed in the kidneys, expression was primarily in the glomeruli. To circumvent these limitations, vectors were injected directly into the kidneys of mice via retrograde ureteral (RU) and subcapsular (SC) injections. Small AAV vectors transduce the kidneys, but can also leak out of the organ and mediate higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection showed that AAV6.2 and 8 had the highest delivery rates. Larger Ad and LV vectors transduce renal cells and mediate less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier.

To assess renal transduction of a more typical reporter gene, AAV8-CAG-GFP at 2e11 GC was injected into normal FVB mice by IV, RU, and SC routes. Mice were sacrificed 4 weeks later and their kidneys and livers were analyzed for GFP by immunofluorescence staining (Figure 1). IV injection of AAV8-CAG-GFP resulted in strong, nearly ubiquitous GFP expression in the liver, but no observable GFP expression in the kidney. RU and SC injected kidney tissues showed fewer GFP-positive cells than AAV-Cre injected mT/mG mice. Interestingly, the most abundant and consistent GFP expression mediated by RU and SC injections was in the injected glomeruli and, surprisingly, in the uninjected kidneys as well. The SC-injected right kidneys also contained GFP-positive cells of tubular morphology, some of which colocalized with proximal tubule (PT) marker, lotus lectin (Figure 1). The livers of the RU- and SC-injected mice also showed significant leakage of the AAV8-CAG-GFP vector into the liver.

Figure 1. AAV8-CAG-GFP reporter vector mediates weaker expression than AAV8-Cre reporter vector. (Rubin J D, et al., 2019)