Single-injection Sterility via Lentiviral-Mediated Suppression of Androgen Receptor in Sertoli Cells

The development of a single-dose, injectable sterilant for cats and dogs would provide a significant refinement over current expensive neutering, and aid the drive to reduce euthanasia of unwanted animals. To address this unmet need we exploited the fundamental requirement for testosterone signaling through testicular Sertoli cells to produce functional sperm. We aimed to suppress androgen receptor (AR) gene expression through the delivery of two micro-RNAs to Sertoli cells using a modified virus that had itself been improved to target only this cell type. To demonstrate proof of principle we first delivered Cre recombinase enzyme to the Sertoli cells of male AR^flox/Y mice. Lentiviral particles contained both CMV-Cre recombinase and tRFP635 (red fluorescent protein) transgenes separated by an IRES, or CMV-tRFP635 alone. Shuttle vectors were packaged using a third-generation lentiviral vector pseudotyped for VSV-G, produced at a viral titer of >1 × 10⁹. Virus was introduced into the seminiferous tubules of adult male AR^flox/Y via injection into the efferent ducts, using 10 μl of Cre virus, tRFP635 control virus, or optiMEM (vehicle); an additional sham-operated, but not injected, control was also evaluated. To control for systemic effects, combinations of Cre/control Cre/optiMEM, Cre/sham, control/optiMEM, control/sham, were generated in testes from individual mice (one treatment per testis; n = 10 per group). We demonstrated that when injected into adult male mice carrying a floxed allele of AR, the virus entered the cells and delivered the transgenes, which successfully integrated into the cell DNA. Cre Recombinase was then expressed and the AR gene deleted. Importantly this resulted in a permanent block in sperm development only in mice lacking Sertoli cell AR. We next replaced the Cre enzyme construct with two synthetic micro-RNAs (designed after the scaffold of miRNA155, chained using the Invitrogen BLOCK-iT Pol II miR RNAi Expression vector kit). We had validated these as strong suppressors of AR gene expression and repeated the study, including the same controls, this time in wild-type (not transgenic) mice (n=12 per group). Again we observed correct targeting by the virus, integration of the miRNA genes into the genome of the cells, expression of the miRNAs and suppression of AR gene expression leading to an identical block in sperm development. Importantly, as suppression needed to be continuous (we had silenced the AR gene, but not deleted it), we were pleased to see that AR protein was still undetectable, and sterility remained, when mice were examined 5 months following this single injection. In the second part of the project, we tested and identified two peptide sequences that homed and bound the Follicle Stimulating Hormone (FSH)-receptor (only expressed in Sertoli and Granulosa cells). We genetically modified the human measles virus (which is not normally able to infect mouse cells) to express these targeting peptide sequences, and demonstrated that, when injected into the efferent ducts leading to the testes (n=8), this genetically modified virus facilitated delivery of genes to the Sertoli cells, whereas a control virus lacking these sequences was not able to gain entry into the cells. In summary, our successful induction of sterility means that this approach holds great promise in the continued search for such a universal single-dose injectable sterilant. Future work is now focussed upon transiting the technology to cat and dogs.