Anticonvulsant effects and behavioural outcomes of rAAV serotype 1 vector-mediated neuropeptide Y overexpression in rat hippocampus.

Neuropeptide Y (NPY) is an endogenous peptide with powerful anticonvulsant properties. Its overexpression in the rat hippocampus, mediated by the local application of recombinant adeno-associated viral (rAAV) vectors carrying the human NPY gene, results in significant reduction of seizures in acute and chronic seizure models. In this study, we characterized a more efficient rAAV-NPY vector to improve cell transfection in the injected area. The changes included pseudotyping with the AAV vector serotype 1 (rAAV1), and using the strong constitutive hybrid CBA promoter, which contains a cytomegalovirus enhancer and chicken beta-actin promoter sequences. We compared NPY expression and the associated anticonvulsant effects of this new vector, with those mediated by the former rAAV vector with chimeric serotype 1/2 (rAAV1/2). In addition, we investigated whether rAAV serotype 1 vector-mediated chronic NPY overexpression causes behavioural deficits that may detract from the clinical utility of this therapeutic approach. We report that rAAV-NPY serotype 1 vector has significantly improved anticonvulsant activity when compared with serotype 1/2 vector, as assessed by measuring EEG seizure activity in kainic acid treated rats. rAAV1-mediated NPY overexpression in naive rats did not result in alterations of physiological functions such as learning and memory, anxiety and locomotor activity. In addition, we did not observe glia activation, or humoral immune responses against serotype 1 vector, which could inactivate gene expression. Our findings show that rAAV1-NPY vector with the CBA promoter mediates powerful anticonvulsant effects and seems to be safe in rodents, thus it may be considered a vector of choice for possible clinical applications.

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.

Soluble adhesion molecules (E-selectin, ICAM-1 and VCAM-1) in breast carcinoma.

Adhesion molecules are important in cell-cell and cell-basement membrane interactions. They are intimately involved in inflammatory reactions and a role in tumour progression has been postulated. E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) play a role in cell adhesion to the vascular endothelium, and may have a role in tumour cell dissemination. Soluble forms of these molecules have been described and this study was established to examine these adhesion molecules in patients with breast carcinoma. Serum was obtained from 92 patients with breast carcinoma and 31 age-matched patients with benign breast disease. All samples were obtained prior to surgery. Soluble levels of E-selectin, ICAM-1, and VCAM-1 were significantly elevated in patients with Stage 4 disease compared with controls. (E-selectin 88.6 (47.9) versus 51.4 (18.4) ng/ml; P<0.001: ICAM-1 447 (249) versus 244 (79) ng/ml; P<0.001: VCAM-1 779 (159) versus 552 (135) ng/ml; P<0.001 results expressed on mean (SEM) SD placed above this.). The prognostic value of the adhesion molecules was examined. In patients with Stage 2 disease, elevated VCAM-1 was predictive of decreased survival, even when corrected for T and N status. Adhesion molecules are elevated in patients with advanced disease and elevation in VCAM-1 has prognostic significance in patients with breast carcinoma.

Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette.

Combined injection of rAAV with mannitol enhances gene expression in the rat brain.

Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene transfer into the central nervous system (CNS). However, a major hurdle for gene delivery to the mammalian brain is to achieve high-level transduction in target cells beyond the immediate injection site. Therefore, in addition to improvements in expression cassettes and viral titers, optimal injection parameters need to be defined. Here, we show that previous studies of somatic cell gene transfer to the mammalian brain have used suboptimal injection parameters, with even the lowest reported perfusion rates still excessively fast. Moreover, we evaluated the effect of local administration of mannitol to further enhance transgene expression and vector spread. Ultraslow microperfusion of rAAV, i.e., <33 nl/min, resulted in significantly higher gene expression and less injury of surrounding tissue than the previously reported rates of 100 nl/min or faster. Co-infusion of mannitol facilitated gene transfer to neurons, increasing both the total number and the distribution of transduced cells by 200-300%. Gene transfer studies in the CNS using rAAV should use very slow infusion rates and combined injection with mannitol to maximize transduction efficiency and spread.

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

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.

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.