An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy.

The brain is generally considered immunoprivileged, although increasing examples of immunological responses to brain antigens, neuronal expression of major histocompatibility class I genes, and neurological autoimmunity have been recognized. An adeno-associated virus (AAV) vaccine generated autoantibodies that targeted a specific brain protein, the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor. After peroral administration of the AAV vaccine, transgene expression persisted for at least 5 months and was associated with a robust humoral response in the absence of a significant cell-mediated response. This single-dose vaccine was associated with strong anti-epileptic and neuroprotective activity in rats for both a kainate-induced seizure model and also a middle cerebral artery occlusion stroke model at 1 to 5 months following vaccination. Thus, a vaccination strategy targeting brain proteins is feasible and may have therapeutic potential for neurological disorders.

Insulators coupled to a minimal bidirectional tet cassette for tight regulation of rAAV-mediated gene transfer in the mammalian brain.

Recombinant AAV is increasingly becoming the vector of choice for many gene therapy applications in the CNS, due to its lack of toxicity and high level of sustained expression. With recent improvements in the generation of pure, high titer vector stocks, the regulation of gene expression is now a key issue for successful translation of gene therapy-based treatments to the clinic. The level of the transgene protein may need to be maintained within a narrow therapeutic window for the successful treatment of human disease. The doxycycline responsive system directs a dose-responsive, tightly regulated level of gene expression and has been used successfully in transgenic mouse models. Here, we have optimized an autoregulatory, bidirectional doxycyline responsive cassette specifically for use in rAAV. We minimized the size of the cassette and decreased the basal leakiness of the system, leading to tight regulation in the rat

Development of an insulated reporter system to search for cis-acting DNA sequences required for dosage compensation in Drosophila.

Dosage compensation (equalization of X-linked gene products) occurs in Drosophila melanogaster by a two-fold transcriptional increase of X-linked gene expression in the male. The cis-acting X-linked DNA sequences required for dosage compensation (called DCREs) remain elusive, despite numerous attempts to identify them. We have developed an insulated reporter system to minimise problems previously encountered with identifying these elements. The system consists of the constitutive autosomal armadillo promoter fused to the lacZ reporter gene (called arm-lacZ) which was flanked by SCS insulator elements to block potential repressive effects of an autosomal chromatin environment. Seven X-linked DNA fragments, totaling 62.7 kb, were each inserted between the SCS element and the armadillo promoter. If an X-linked fragment contains a DCRE, then transgenic males carrying an autosomal insert of the construct should produce twice the beta-galactosidase activity of females. However, in all cases, males and females expressed the same level of lacZ. Thus, it's likely that none of the X-linked fragments contained a DCRE, suggesting these elements may be rarer than previously thought. The insulated reporter system was also used to test the hypothesis that some genes may be dosage compensated due to repression by Sex lethal (Sxl) in females. A fragment from the runt gene containing three Sxl binding sites was inserted into the 3' untranslated region of arm-lacZ. Transgenic males carrying an autosomal insert of the construct had on average 1.31-1.46 times the level of beta-galactosidase than females, suggesting that some genes could be compensated, at least partially, by Sxl repression in females.

Adeno-associated viral glutamate decarboxylase expression in the lateral nucleus of the rat hypothalamus reduces feeding behavior.

In vivo gene transfer of glutamate decarboxylase (GAD) has been explored as a means of inducing or increasing the production of the inhibitory amino-acid neurotransmitter, GABA. This strategy has been applied to neuroprotection, seizure prevention, and neuromodulation. In the present experiment, AAV2 was used to transfer the genes for green fluorescence protein (GFP) and GAD65 into the lateral nucleus of the rat hypothalamus. Microinjection of 500 nl of AAV2 resulted in transduction of a 0.25+/-0.04 mm(3) with targeting errors of X=0.48 mm, Y=0.18 mm, Z=0.37 mm using standard stereotactic technique. Pre- and postinjection food and water consumption, urine and feces production, and weight were recorded. In comparison with rAAVCAGGFP- and PBS-injected animals, rats treated with rAAVCAGGAD65 demonstrated reduced weight gain (P<0.014) and transiently reduced daily food consumption (P<0.007) during the postoperative period. No changes in water consumption or waste production were recorded. Effective GAD65 gene transfer was confirmed with in situ hybridization using a probe to the woodchuck post-transcriptional regulatory element sequence included in the vector. These findings suggest that increased GABA production in lateral nucleus of the hypothalamus induced by GAD65 gene transfer may reduce weight gain through reduced feeding.