NEUROCHEMISTRY NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 12 No 2 12 February 2001 317 Intrathecal administration of PNA targeting galanin receptor reduces galanin-mediated inhibitory effect in the rat spinal cord Khadijeh Rezaei, 1 Isabella Shi Xu, 2 Wei-Ping Wu, 2 Tie-Jun Shi, 3 Ursel Soomets, 1,4 Tiit Land, 1 Xiao-Jun Xu, 2 Zsuzsanna Wiesenfeld-Hallin, 2 Tomas Ho È kfelt, 3 Tamas Bartfai 5 and U È lo Langel 1,5,CA 1 Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm University, Stockholm; 2 Department of Medical Laboratory Sciences and Technology, Section of Clinical Neurophysiology, Karolinska Institutet, Huddinge; 3 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; 4 Department of Biochemistry, Tartu University, Tartu, Estonia; 5 The Harold L. Dorris Neurological Research Center, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA CA,5 Corresponding Author and Address Received 3 October 2000; accepted 22 November 2000 Peptide nucleic acids (PNA) are nucleic acid analogues contain- ing neutral amide backbone, forming stable and tight complexes with complementary DNA/RNA. However, it is unclear whether unmodi®ed PNA can ef®ciently penetrate neuronal tissue in order to act as antisense reagent. Here we show that intrathecal (i.t.) injection of an unmodi®ed antisense PNA complementary to the rat galanin receptor type 1 (GalR1) mRNA is able to block the inhibitory effect of i.t. administered galanin on spinal nociceptive transmission. Autoradiographic ligand binding studies using [ 125 I]galanin show that the unmodi- ®ed PNA is able to reduce the density of galanin binding sites in the dorsal horn. Thus, unmodi®ed PNA applied i.t. appears to function as an effective antisense reagent in rat spinal cord in vivo. NeuroReport 12:317±320 & 2001 Lippincott Williams & Wilkins. Key words: Antisense; Drug delivery; Neuropeptides; Pain; Sensory neurons INTRODUCTION Oligonucleotides have been shown to function as effective antisense reagents in vivo and in vitro, and are proven valuable pharmacological tools [1±3]. Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the deoxyr- ibose phosphate backbone of DNA is replaced by a neutral polyamide backbone. The nucleobases allow complemen- tary hybridization to DNA or RNA but, in contrast to DNA and RNA, PNAs are stable against nucleases and proteases [4]. Moreover, because of the absence of charge repulsion in the PNA/DNA and PNA/RNA complexes, they are more stable than DNA/DNA and DNA/RNA complexes [5,6]. PNAs are also superior in selectivity since PNA/ DNA and PNA/RNA hybridization is less tolerant to base mismatches than DNA/DNA or DNA/RNA hybridization [7]. Thus, the ef®cient, selective and stable double stranded DNA invasion by PNAs [8±11] make them suitable for application as antisense or antigene reagents. In fact, PNAs have been used in several laboratories to arrest protein expression at the transcriptional and translational level [12±15]. Despite the above mentioned advantages, the use of PNAs has been limited due to their poor cellular uptake [14,16]. Efforts to improve cellular uptake of PNAs have been made by attachment of lipophilic or other helper groups such as transporter peptides (transportan or pene- tratin) to PNAs [17±20]. We have previously reported that intrathecal (i.t.) administration of PNA complementary to human galanin receptor subtype 1 (GalR1) mRNA coupled to the cell penetrating transporter peptides decreased the expression of rat GalR1 in the rat spinal cord and that such treatment effectively reduced the inhibitory effect of i.t. applied galanin on C-®ber stimulation-induced facilitation of the rat ¯exor re¯ex [19]. In the spinal cord, galanin, a 29±30 amino acid peptide [21], appears to have a complex, but important role in nociceptive transmission [22]. Since 1994 three G-protein coupled receptor subtypes of galanin (GalR1, GalR2 and GalR3) have been cloned and the identi®cation of the role played by these receptor subtypes in galaninergic transmis- sion still awaits solution. It is generally believed that the cellular uptake of unmodi®ed PNA is poor. However, Tyler et al. [12] have found that carrier-free PNAs, upon their direct injection into rat brain i.c.v., can enter neuronal cells and inhibit protein synthesis in a gene-speci®c manner. Recently, this group also showed that unmodi®ed PNA targeted to the neurotensin receptor-1 mRNA administered i.p. crossed the blood±brain barrier and speci®cally reduced gene expression in the CNS [23]. Therefore, we wanted to