Abstracts

Rationale: In the context of adeno-associated virus (AAV) vectors, expression and constitutive secretion of the neuropeptide, galanin, has been shown to exert a significant anti-seizure action in vivo. An improvement to this approach would be the ability to express multiple gene products from a single AAV vector. One potential means to express multiple gene products from a single AAV vector would entail the insertion of a protease cleavage consensus site in between the coding sequences for the two gene products. Thus if the protease is active in the constitutive secretion pathway, the resulting fusion protein would be cleaved into the two peptides. Under such a condition, a single promoter could drive expression multiple peptides within the context of a single viral vector. Methods: Using an AAV vector that supports constitutive secretion of the gene product (AAV-FIB), the coding sequence for a furin cleavage consensus site (RKRRKR) was placed in between the coding sequence for galanin followed by the coding sequence for GFP or the reverse, GFP followed by galanin. HEK-293 cells were transfected with either of the plasmids and forty eight hours later, the media was collected. Western blots were conducted to determine if GFP existed in the media. Subsequently, recombinant AAV vectors were made for each of the above constructs and each individual vector was infused into the rat piriform cortex. One week later, kainic acid (10 mg/kg, i.p.) was administered to the rats, and the latency to limbic seizure behaviors was recorded. Results: When western blots conducted on the media of transfected HEK293 cells verified the presence of an appropriately cleaved GFP, regardless of whether the GFP preceded the RKRRKR or followed the RKRRKR cleavage site. One week after the infusion of AAV-FIB-GAL-RKRRKR-GFP or AAV-FIB-GFP-RKRRKR-GAL, both of the AAV vectors significantly delayed the appearance of limbic seizure behaviors following the administration of kainic acid. Conclusions: The in vitro results show that GFP is secreted from transfected HEK293 cells regardless of whether the GFP coding sequence occurs 3’ or 5’ to a furin cleavage consensus site. Moreover, when tested in vivo, sufficient galanin is secreted from transduced cells to significantly attenuate kainic acid-induced seizure activity, again regardless of whether the galanin sequence is 3’ or 5’ to the cleavage site. Thus, this approach appears to be an effective means to express and constitutively secrete multiple peptides from a single AAV vector. (Supported by NIH NS35633 to TJM)