Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion

Abstract

Seizure disorders present an attractive gene therapy target, particularly because viral vectors such as adeno-associated virus (AAV) and lentivirus can stably transduce neurons. When we targeted the N-methyl-D-aspartic acid (NMDA) excitatory amino acid receptor with an AAV-delivered antisense oligonucleotide, however, the promoter determined whether focal seizure sensitivity was significantly attenuated or facilitated. One potential means to circumvent this liability would be to express an inhibitory neuroactive peptide and constitutively secrete the peptide from the transduced cell. The neuropeptide galanin can modulate seizure activity in vivo, and the laminar protein fibronectin is usually secreted through a constitutive pathway. Initially, inclusion of the fibronectin secretory signal sequence (FIB) in an AAV vector caused significant gene product secretion in vitro. More importantly, the combination of this secretory signal with the coding sequence for the active galanin peptide significantly attenuated in vivo focal seizure sensitivity, even with different promoters, and prevented kainic acid-induced hilar cell death. Thus, neuroactive peptide expression and local secretion provides a new gene therapy platform for the treatment of neurological disorders.

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

“…Thus, like findings with expression and secretion of galanin, NPY actions in the piriform cortex significantly attenuated seizure activity induced by the peripheral administration of the chemical convulsant kainic acid. As previously discussed, the constitutive secretion of these peptides renders immunohistochemical identification untenable, 3 but as previously shown for galanin, 1,3 the appropriate vectorderived NPY or NPY13-36 mRNA was present in the area of AAV infusion (see Figure 2). Thus, not only does this into the piriform cortex as described previously.…”

“…In contrast, expression of galanin without the secretory signal or expression and secretion of the reporter gene, GFP had no effect on seizure sensitivity. 1,3 In addition to galanin, rAAV delivery of NPY has also been shown to attenuate limbic seizures; 6 however, there is still some question as to which of the NPY receptors (Y1-Y5) mediates the antiseizure activity. Several studies have suggested a critical role for the Y2 receptor in mediating antiepileptic actions.…”

“…Gene Therapy (2007) Epilepsy is an attractive target for recombinant adenoassociated virus (rAAV) gene therapy, because the temporal lobe structures involved in seizure genesis and propagation have been shown to be permissive to AAV gene transfer. [1][2][3][4][5][6] Recently, galanin and neuropeptide Y (NPY) have been delivered as transgenes in rAAV vectors, and are shown to be effective in several epilepsy paradigms. 1,3,6,7 While previous approaches utilize prepro cDNA sequences, which rely on the cell to modulate release of the gene product, Haberman et al 3 were the first to use a novel secretion strategy whereby the secretion signal sequence from the constitutively secreted laminar protein fibronectin (FIB) is combined with the coding sequence for the active therapeutic peptide.…”

“…1,3,6,7 While previous approaches utilize prepro cDNA sequences, which rely on the cell to modulate release of the gene product, Haberman et al 3 were the first to use a novel secretion strategy whereby the secretion signal sequence from the constitutively secreted laminar protein fibronectin (FIB) is combined with the coding sequence for the active therapeutic peptide. Results from Haberman et al 3 and McCown 1 show that expression and constitutive secretion of galanin is not only achieved, but also effective in attenuating focal and limbic seizure activity. In contrast, expression of galanin without the secretory signal or expression and secretion of the reporter gene, GFP had no effect on seizure sensitivity.…”

“…1 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 seizure sensitivity, seizure behaviors or seizureinduced cell death. 1,3 One week later, the rats received a dose of 10 mg kg À1 intraperitoneal kainic acid, and subsequently the time to limbic seizure behaviors was recorded. As seen in Figure 1, both AAV treatment groups and the untreated control group developed wet dog shake behaviors with the same latency.…”