GFP Adeno-associated virus(AAV Serotype 8)

Despite the growing popularity of deliverable transgenes, a robust and well-validated method for targeting Leydig cells that can deliver long-term transgene expression has not been identified. Here, researchers delivered lentiviral, adenoviral, and adeno-associated (AAV) viral particles to the interstitial region of adult mouse testes. Immunolocalization and stereological studies were performed to characterize the ability of the vectors to target and deliver transgenes to Leydig cells. Transgene expression in lentiviral-targeted Leydig cells was detected within 7 days post-injection, followed by apoptosis of the Leydig cells. Adenoviral-delivered transgene expression was detected within 10 days post-injection, with no evidence of targeted cell apoptosis. Researchers found serotype differences in AAV-injected testes, with AAV serotype 9 targeting a significant fraction of Leydig cells. Addition of neuraminidase during injection increased targeting efficiency to an average of 59.63% (with a maximum of 80%). Transgene expression was detectable in sections of AAV-injected testes up to 50 days post-injection.

The viral titers of AAV particles delivered to adult testes were much higher than those of lentiviral and adenoviral vectors. To ensure that the viral load was not toxic and that AAV particles did not affect Leydig cell survival, researchers performed co-localization studies of GFP and Cl.Casp3 in sections of testes injected with AAV8-GFP and AAV9-GFP at each time point, including sections injected simultaneously with neuraminidase (Figure 1A, B). In all sections observed, there was no evidence of Cl.Casp3 indicating that the targeted Leydig cells did not undergo apoptosis. This suggests that Leydig cell survival was not impacted by targeting with AAV particles despite their high titer.

Figure 1. Delivery of transgenes using AAV does not result in apoptosis of the targeted Leydig cells. (Darbey A, et al., 2021)