In vivo control of NMDA receptor transcript level in motoneurons by viral transduction of a short antisense gene.
Glutamate receptors play critical roles in normal and pathological processes. We developed an antisense gene delivery strategy to modulate the NMDA type of glutamate receptor. Using transient transfection in vitro and viral mediated gene transfer in vitro and in vivo, the effect of expression of an antisense gene fragment (60 bp) of the NR1 subunit was tested. Immunoblot analysis showed an antisense-concentration-dependent reduction in the NR1 subunit upon transient co-transfection of a plasmid expressing a sense NR1 gene and a plasmid expressing the antisense fragment into COS-7 cells. After recombination into an adenoviral vector, this antisense fragment reduced the amount of endogenous NR1 protein in PC12 cells. Finally, direct intraparenchymal injection of the viral vector into rat spinal cord resulted in diminished NR1 in motor neurons. Our results demonstrate the efficacy of this approach, which combines antisense with viral gene delivery to control the expression of specific genes in vivo. This approach may also be useful in reducing excitatory neurotransmission in vivo, with implications for the treatment of spinal disorders such as amyotrophic lateral sclerosis or chronic pain.
Finegold
A A
AA
Pain and Neurosensory Mechanisms Branch, NIH-NIDCR, Bldg 49, Rm 1A11, 49 Convent Dr. MSC 4410, Bethesda 20892, MD, USA. alan.finegold@perkinelmer
Perez
F M
FM
Iadarola
M J
MJ
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics*; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D007267:Injections; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009046:Motor Neurons / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D016716:PC12 Cells; D010957:Plasmids / Q000235:genetics; D020671:Posterior Horn Cells / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000235:genetics*; D013116:Spinal Cord; D014162:Transfection
D016373:DNA, Antisense; C409466:NR1 NMDA receptor; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate