title: Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity.
abstract: The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 microl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracyclineoff promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy.
authors.affiliation: UNC Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Haberman
authors.forename: Hugh
authors.initials: H
authors.lastname: Criswell
authors.forename: Stephen
authors.initials: S
authors.lastname: Snowdy
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ming
authors.forename: George
authors.initials: G
authors.lastname: Breese
authors.forename: R
authors.initials: R
authors.lastname: Samulski
authors.forename: Thomas
authors.initials: T
authors.lastname: McCown
mesh_terms: D000818:Animals; D000229:Dependovirus; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000009:adverse effects; D009474:Neurons / Q000378:metabolism; D016372:RNA, Antisense; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism; D012640:Seizures / Q000235:genetics / Q000628:therapy*; D014161:Transduction, Genetic
chemical_list: D016372:RNA, Antisense; D016194:Receptors, N-Methyl-D-Aspartate