Cre Adeno-associated virus(AAV Serotype 9)

Adeno-associated virus (AAV) can penetrate the blood-brain barrier, but it is unknown whether AAV can penetrate other tight junctions. Genetic manipulation of the testis is hampered by the basement membrane of the seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and luminal zones. Here, researchers demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment by AAV1/9. AAV1/9 microinjected into the seminiferous tubules can penetrate the basement membrane and the BTB, thereby transducing not only Sertoli cells and SSCs, but also peritubular cells and Leydig cells. Furthermore, when the testes of congenitally infertile KitlSl/KitlSl-d mice (deficient in Sertoli cells) were treated with AAV expressing Kitl, spermatogenesis was rejuvenated and offspring were produced. All offspring did not contain the AAV genome. Therefore, AAV1/9 can achieve effective germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for developing gene therapies for reproductive defects.

Here, to study the transduction kinetics, the researchers used R26R-Eyfp mice, which allow for more sensitive identification of infected cells. They infected these mice with AAV9 expressing Cre. AAV9-Cre infection triggered the expression of EYFP, which was slightly stronger with tubular injection (Figure 1A). Immunostaining showed that WT1+ Sertoli cells were infected as early as 1 day after microinjection, while GFRA1+ spermatogonia showed no fluorescence (Figure 1B), indicating that Sertoli cell infection is a very rapid process.

Figure 1. Transduction Kinetics Analyzed by Microinjection of AAV9-Cre into R26R-Eyfp Mice. (Watanabe S, et al., 2018)

AAV infection does not seem to significantly affect the SSC microenvironment, as real-time PCR showed that neither AAV1 nor AAV9 transduction affected the expression levels of cytokines involved in spermatogonia self-renewal and differentiation (Figure 1C). Since AAV1 and AAV9 penetrate into the BTB, the researchers examined the distribution of CLDN11, a major component of the BTB. However, CLDN11 expression was not significantly altered by AAV1 or AAV9 infection (Figure 1D). Three days after tubular or interstitial injection of AAV1 or AAV9-mCherry, biotin (557 D) was microinjected into the interstitium of adult testes. Cldn11 knockout (KO) mouse testes, which lack the BTB, were used as a positive control. Although leakage into the tubule lumen was readily observed in Cldn11 KO mice (Figure 1E), AAV injection did not induce significant leakage of biotin into the tubule lumen. These results indicate that both AAV1 and AAV9 penetrated the basement membrane and the BTB and infected Sertoli cells and germ cells.