GnRH is expressed in GnRH-deficient hypogonadal (hpg) mice after GnRH gene transfer with a herpes simplex virus (HSV) amplicon vector

Abstract

The challenge of gene therapy is to develop non-toxic vectors that can achieve stable transgene expression in target cells. The HSV amplicon is a unique vector that has essentially no toxicity, a large transgene capacity, and the potential to integrate the transgene into a specific site in the genome, thus making it a useful tool for gene therapy. Hpg mice are genetically deficient in GnRH production in the hypothalamus, resulting in many deficits in the reproductive system including infertility. Our goal was to determine neuronal expression of GnRH and the recovery of gonadal function after CNS delivery of an HSV amplicon vector containing the GnRH gene using hpg mice. An HSV amplicon containing the GnRH gene under its cognate promoter was prepared and packaged using a helper virus-free packaging system. This vector was injected into the preoptic area (POA) of the hypothalamus of hpg mice using a stereotaxic apparatus. Following injection, we determined GnRH neuronal expression by immunohistochemistry (IHC) and recovery of gonadal function using vaginal cytology and blood hormonal assays. Eight adult female hpg mice were studied. Five mice were injected with the HSV-GnRH vector in the POA, and 3 mice were injected with vehicle only as control. The HSV-GnRH vector (1× 108 transducing units/ml) was injected into the POA of hpg mice under anesthesia using a stereotaxic apparatus. Mice were sacrificed 10 days (n=4) or 50 days (n=1) after injection. Neuronal expression of GnRH was analyzed by IHC in brain tissue. Plasma FSH and LH levels were measured by radioimmunoassay. The weights of the ovary, uterus and oviduct were compared between the study and control groups. Vaginal cytology was also checked each day after HSV amplicon injection to evaluate estrous cyclicity. GnRH expression was present in neurons near the injection site, as determined by IHC 10 days and 50 days following the vector injections. However, there was no significant difference in plasma FSH (2.0 ± 0.7 ng/ml vs. 4.9 ± 0.9 ng/ml; study vs. control), LH (0.07 ± 0.01 ng/ml vs. 0.06 ± 0.02 ng/ml) levels, or in the weight of reproductive organs (24.2 ± 6.4 mg vs. 17.9 ± 2.7 mg) between the study and control groups. We observed only diestrus specific morphology in the vaginal epithelial cells. Expression of GnRH was observed in the hypothalamic neurons in the POA in GnRH-deficient hpg mice following GnRH gene transfer using an HSV amplicon vector. These data suggest that such HSV vectors may be useful in correcting the reproductive defect in these animals. Further studies are necessary to determine if normal hypothalamic-pituitary-gonadal axis function can be restored in hpg mice by gene therapy using this approach.