Adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo.

Neuropeptide Y (NPY) is a 36-amino-acid peptide that attenuates seizure activity following direct infusion or adeno-associated virus (AAV)-mediated expression in the central nervous system. However, NPY activates all NPY receptor subtypes, potentially causing unwanted side effects. NPY13-36 is a C-terminal peptide fragment of NPY that primarily activates the NPY Y2 receptor, thought to mediate the antiseizure activity. Therefore, we investigated if recombinant adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic seizure sensitivity. Rats received bilateral piriform cortex infusions of AAV vectors that express and constitutively secrete full-length NPY (AAV-FIB-NPY) or NPY13-36 (AAV-FIB-NPY13-36). Control rats received no infusion, as we have previously shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect on seizures. One week later, all animals received kainic acid (10 mg kg(-1), intraperitoneally), and the latencies to wet dog shakes and limbic seizure behaviors were determined. Although both control and vector-treated rats developed wet dog shake behaviors with similar latencies, the latencies to class III and class IV limbic seizures were significantly prolonged in both NPY- and NPY13-36-treated groups. Thus, AAV-mediated expression and constitutive secretion of NPY and NPY13-36 is effective in attenuating limbic seizures, and provides a platform for delivering therapeutic peptide fragments with increased receptor selectivity.

GABAB receptor antagonist SGS742 improves spatial memory and reduces protein binding to the cAMP response element (CRE) in the hippocampus.

Memory storage in the brain requires protein synthesis initiated through signaling pathways that control transcription. Such mechanisms are under active investigation for therapies in disorders involving cognitive dysfunction. Long-term memory can be improved by inhibiting activation or reducing expression of transcription factors such as ATF4/CREB2 and some C/EBP family members which appear to serve as memory suppressors. Here, we provide evidence that GABAB receptor antagonists may enhance cognition, at least in part, by this mechanism. We tested a GABAB receptor antagonist, SGS742 (CGP36742), on hippocampal-dependent memory and hippocampal nuclear CRE-binding activity in rats. As a result, acute in vivo administration of SGS742 both improved memory and reduced total hippocampal CRE-binding activity of which a large proportion in the basal state could be immunoneutralized with CREB2 antibodies. Consistent with its activity on information storage mechanisms, acute SGS742 effectively improved long-term memory in retrograde protocols, in which drug was given at times when memory formation can be interrupted by blocking new protein production. In conclusion, GABAB antagonists may provide a pharmacological therapy for cognitive impairment, sharing mechanistic features with genetic approaches to reduce CREB2 activity and to augment long-term memory.

Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element.

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604-1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the "off" state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the A/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.

Inducible long-term gene expression in brain with adeno-associated virus gene transfer.

Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo.

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.