Improvement in reproductive parameters in hypogonadal female mice by regulated gene replacement therapy in the central nervous system.

One of the challenges of gene targeting is to achieve regulated transgene expression in specific target cells. The hypogonadal (hpg) mice are genetically deficient in hypothalamic gonadotropin-releasing hormone (GnRH) production due to a deletion in the GnRH gene, resulting in hypogonadotropic hypogonadism. Here we show an improvement in reproductive parameters of adult female homozygous hpg mice by direct infusion into the hypothalamic preoptic area (POA) of a herpes simplex virus (HSV)-based amplicon vector containing a 13.5 kb genomic fragment encoding the GnRH gene together with its cognate promoter and regulatory elements. Following vector injection, GnRH-expressing neurons were detected in the POA, and pituitary and plasma gonadotropin levels as well as ovarian and uterine weights increased. In addition, a subset of injected hpg mice demonstrated cyclic estrous changes, consistent with regulated control of GnRH production. Administration of kisspeptin-10 resulted in an increase in plasma luteinizing hormone levels, further supporting appropriate regulation of the introduced GnRH transgene. These findings indicate that delivery of the GnRH gene resulted in selective neuronal expression of GnRH and regulated hypothalamic GnRH release. To our knowledge, this is the first example of the correct targeting of a gene under its cognate promoter to neurons resulting in selective and regulated synthesis of a biologically active peptide, and thus may have a wide range of applications in the treatment of human disorders.

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

Herpes simplex virus type 1/adeno-associated virus rep(+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration.

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep(-) HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep(+) HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep(-) vector 3' AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep(+), but not the rep(-), hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep(+) hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by "deconcatenating" the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury.

Fibroblast growth factor-2 (FGF-2) promotes proliferation of neuroprogenitor cells in culture and is up-regulated within brain after injury. Using mice genetically deficient in FGF-2 (FGF-2(-/-) mice), we addressed the importance of endogenously generated FGF-2 on neurogenesis within the hippocampus, a structure involved in spatial, declarative, and contextual memory, after seizures or ischemic injury. BrdUrd incorporation was used to mark dividing neuroprogenitor cells and NeuN expression to monitor their differentiation into neurons. In the wild-type strain, hippocampal FGF-2 increased after either kainic acid injection or middle cerebral artery occlusion, and the numbers of BrdUrd/NeuN-positive cells significantly increased on days 9 and 16 as compared with the controls. In FGF-2(-/-) mice, BrdUrd labeling was attenuated after kainic acid or middle cerebral artery occlusion, as was the number of neural cells colabeled with both BrdUrd and NeuN. After FGF-2(-/-) mice were injected intraventricularly with a herpes simplex virus-1 amplicon vector carrying FGF-2 gene, the number of BrdUrd-labeled cells increased significantly to values equivalent to wild-type littermates after kainate seizures. These results indicate that endogenously synthesized FGF-2 is necessary and sufficient to stimulate proliferation and differentiation of neuroprogenitor cells in the adult hippocampus after brain insult.

Gene therapy in the CNS.

Gene therapy for neurological disorder is currently an experimental concept. The goals for clinical utilization are the relief of symptoms, slowing of disease progression, and correction of genetic abnormalities. Experimental studies are realizing these goals in the development of gene therapies in animal models. Discoveries of the molecular basis of neurological disease and advances in gene transfer systems have allowed focal and global delivery of therapeutic genes for a wide variety of CNS disorders. Limitations are still apparent, such as stability and regulation of transgene expression, and safety of both vector and expressed transgene. In addition, the brain adds several challenges not seen in peripheral gene therapy paradigms, such as post-mitotic cells, heterogeneity of cell types and circuits, and limited access. Moreover, it is likely that several modes of gene delivery will be necessary for successful gene therapies of the CNS. Collaborative efforts between clinicians and basic researchers will likely yield effective gene therapy in the CNS.

Atlas of the neurons that express mRNA for the long form of the prolactin receptor in the forebrain of the female rat.

Prolactin has a variety of important physiological effects on peripheral tissue and on the brain. The behavioral effects of prolactin include the induction of maternal behavior and increased food intake. Prolactin acts via its cognate receptors which have two forms, a short and a long form. The long form of the receptor is predominant in the preoptic area-hypothalamus and is positioned to support maternal behavior since this form is regulated across pregnancy and lactation (Nagano and Kelly [1994] J. Biol. Chem. 269:13337-13345; Sugiyama et al. [1994] J. Endocrinol. 141:325-333). By using in situ hybridization with [33P] labelled cRNA probe specific for the long form of the receptor mRNA(L-PRL mRNA) we have mapped, in brains from 2- and 21-day-old pregnant females, the neuroanatomical distribution of neurons expressing the long form of the receptor. Many neurons with high expression of L-PRL mRNA were located in the anteroventral periventricular nucleus, the medial preoptic area (MPO), specific subdivisions of the paraventricular and supraoptic nuclei, and in the arcuate and ventromedial nuclei. Labelled neurons were also found in limbic system structures such as the bed nucleus of stria terminalis (BST) and the medial nucleus of the amygdala, in a few thalamic nuclei, and in the central gray. All cells throughout the choroid plexus expressed high levels of L-PRL mRNA. The levels of L-PRL mRNA were higher in females on day 21 of pregnancy in the MPO and in the choroid plexus, than in females on day 2 of pregnancy; levels in the ventromedial nucleus of the hypothalamus (VMH) were unchanged across pregnancy. The neuroanatomical distribution of neurons expressing L-PRL mRNA may have special relevance for the mediation of maternal behavior, lactation, sexual behavior, and feeding.

Quantitative autoradiographic analysis of D1 and D2 dopamine receptors in rat brain in early and late pregnancy.

This study examined the levels of D1 and D2 dopamine receptors in the rat brain during pregnancy, a physiologically unique and important naturally occurring state. We are particularly interested in changes in the dopamine receptor complement of the brain during pregnancy because these receptors might support some components of the immediate postpartum onset of normal maternal behavior. Quantitative in vitro receptor autoradiography was applied particularly focusing on brain areas that control maternal behavior. The D1 dopamine receptor selective antagonist -3H-SCH23390 and the D2 dopamine receptor selective antagonist [3H]spiperone were used as the ligands. We examined the levels of binding to D1 and D2 dopamine receptors in brains in females on day 2 (early pregnancy) and day 21 (late, but prepartum pregnancy) of pregnancy. In addition, brains from females on diestrus-1 and from males provided reference points to the existing literature. Late in pregnancy females had significantly 18-27% lower levels of binding to D1 dopamine receptors in the lateral striatum, the medial striatum, and the nucleus accumbens when compared to all other groups. Late in pregnancy, females had also significantly 11-25% lower levels of binding to D2 dopamine receptors in the lateral striatum, the anterior striatum, the nucleus accumbens and the olfactory tubercle compared to all other experimental groups. We examined all of the brain regions already established to be important for maternal behavior, and found that dopamine receptor binding changed across pregnancy only in one such region, the nucleus accumbens. Thus pregnancy, perhaps the hormones of pregnancy, reduces the levels of D1 and D2 dopamine receptors in the striatum, and the nucleus accumbens, but not in other brain regions.