Nature Biotechnology
16, 857 - 861 (1998)
doi:10.1038/nbt0998-857
Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivoMargus Pooga1, 3, Ursel Soomets1, 4, Mattias Hällbrink1, Andres Valkna1, 4, Külliki Saar1, Khadijeh Rezaei1, Ulrika Kahl1, Jing-Xia Hao2, Xiao-Jun Xu2, Zsuzsanna Wiesenfeld-Hallin2, Tomas Hökfelt5, Tamas Bartfai1, 6
& Ülo Langel1, *
1Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden.
2Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institute, S-14186 Huddinge, Sweden.
3Estonian Biocentre, Riia 23, EE2400 Tartu, Estonia.
4Department of Biochemistry, Tartu University, Jakobi 2, EE2400 Tartu, Estonia.
5Department of Neuroscience, Karolinska Institute, S-17177 Stockholm, Sweden.
6F.Hoffmann-La Roche, Ltd., Dpt. PRPN, CH-4070 Basel, Switzerland.
*Corresponding author (e-mail: ulo@neurochem.su.se). Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43−58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.
REFERENCES
- Roush, W. 1997. Antisense aims for a renaissance. Science 276: 1192−1193. | Article | PubMed | ISI | ChemPort |
- Bennet, C.F. 1998. Antisense oligonucleotides: is the glass half full or half empty?Biochem. Pharmacol. 55: 9−19. | Article | PubMed | ISI | ChemPort |
- Troy, C.M., Derossi, D., Prochiantz, A., Greene, L.A., and Shelanski, M.L. 1996. Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci. 16: 253−261. | PubMed | ISI | ChemPort |
- Nielsen, P.E., Egholm, M., Berg, R.H., and Buchardt, O. 1991. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254: 1497−1500. | PubMed | ISI | ChemPort |
- Demidov, V.V., Potaman, V.N., Frank-Kamenetskii, M.D., Egholm, M., Buchard, O., Sönnichsen, S.H., and Nielsen, P.E. 1994. Stability of peptide nucleic acids in human serum and cellular extracts. Biochem. Pharmacol. 48: 1310−1313. | Article | PubMed | ISI | ChemPort |
- Knudsen, H. and Nielsen, P. 1996. Antisense properties of duplex-and triplex-forming PNAs. Nucleic Acids Res. 24: 494−500. | Article | PubMed | ISI | ChemPort |
- Peffer, N.J., Hanvey, J.C., Bisi, J.E., Thomson, S.A., Hassman, C.F., Noble, S.A. et al. 1993. Strand-invasion of duplex DNA by peptide nucleic acid oligomers. Proc. Natl. Acad. Sci. USA 90: 10648−10652. | PubMed | ChemPort |
- Hanvey, J.C., Peffer, N.J., Bisi, J.E., Thomson, S.A., Cadilla, R., Josey, J.A. et al. 1992. Antisense and antigene properties of peptide nucleic acids. Science 258: 1481−1485. | PubMed | ISI | ChemPort |
- Bonham, M.A., Brown, S., Boyd, A.L., Brown, P.H., Bruckenstein, D.A., Hanvey, J.C. et al. 1995. An assessment of the antisense properties of RNase H-compe-tent and steric-blocking oligomers. Nucleic Acids Res. 23: 1197−1203. | PubMed | ISI | ChemPort |
- Good, L. and Nielsen, P.E. 1998. Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA. Nat. Biotechnol. 16: 355−358. | Article | PubMed | ISI | ChemPort |
- Pooga, M., Hällbrink, M., Zorko, M., and Langel, Ü. 1998. Cell penetration of transportan. FASEB J. 12: 67−77. | PubMed | ISI | ChemPort |
- Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., and Prochiantz, A. 1996. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271: 18188−18193. | Article | PubMed | ISI | ChemPort |
- Allinquant, B., Hantraye, P., Mailleux, P., Moya, K., Bouillot, C., and Prochiantz, A. 1994. Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro. J. Cell Biol. 128: 919−927. | Article | ISI |
- Tatemoto, K., Rökaeus, Å., Jörnvall, H., McDonald, T.J., and Mutt, V. 1983. Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett. 164: 124−128. | Article | PubMed | ISI | ChemPort |
- Bartfai, T. 1995. Galanin: a neuropeptide with important central nervous system actions, pp. 563−571, in Psycheopharmacology: the fourth generation of progress. Bloom, F.E. and Kupfer, D.J. (eds.). Raven Press, New York.
- Habert-Ortoli, E., Amiranoff, B., Loquet, I., Laburthe, M., and Mayaux, J.F. 1994. Molecular cloning of a functional human galanin receptor. Proc. Natl. Acad. Sci. USA 91: 9780−9783. | PubMed | ChemPort |
- Burgevin, M.-C., Loquet, I., Quarteronet, D., and Habert-Ortoli, E. 1995. Cloning, pharmacological characterization, and anatomic distribution of a rat cDNA encoding for a galanin receptor. J. Mol. Neurosci. 6: 1−8. | Article | PubMed |
- Bartfai, T., Langel, Ü., Bedecs, K., Andell, S., Land, T., Gregersen, S. et al. 1993. Novel galanin receptor ligand M 40 distinguishes betweeen putative galanin receptor subtypes. Proc. Natl. Acad. Sci. USA 90: 11287−11291. | PubMed | ChemPort |
- Wynick, D., Smith, D.M., Ghatei, M., Akinsanya, K., Bhogal, R., Purkiss, P. et al. 1993. Characterization of a high-affinity galanin receptor in the rat anterior pituitary—absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor. Proc. Natl. Acad. Sci. USA 90: 4231−4235. | PubMed | ChemPort |
- Howard, A., Tan, C., Shiao, L., Palyha, O., McKee, K., Weinberg, D. et al. Molecular cloning and characterization of a new receptor for galanin. FEBS Lett. 405: 285−290. | Article | PubMed | ChemPort |
- Wang, S., He, C., Hashemi, T., and Bayne, M. 1997. Cloning and expressional characterisation of a novel galanin receptor. J. Biol. Chem. 272: 31949−31952. | Article | PubMed | ISI | ChemPort |
- Derossi, D., Joliot, A.H., Chassaing, G., and Prochiantz, A. 1994. The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269: 10444−10450. | PubMed | ISI | ChemPort |
- Wiesenfeld-Hallin, Z., Villar, M.J., and Hökfelt, T. 1989. The effects of intrathecal galanin and C-fiber stimulation on the flexor reflex in the rat. Brain Res. 486: 205−213. | Article | PubMed | ChemPort |
- Langel, Ü., Land, T., and Bartfai, T. 1992. Design of chimeric peptide ligands to galanin receptors and substance P receptors. Int. J. Pept. Protein Res. 39: 516−522. | PubMed | ISI |
- Egholm, M., Buchardt, O., Nielsen, P.E., and Berg, R.H. 1992. Peptide nucleic acids (PNA): Oligonucleotide analogues with an achiral peptide backbone. Journal of the American Chemical Society 114: 1895−1897. | ISI | ChemPort |
- Peterson, G.L. 1977. A simplification of the protein assay method of Lowry et al.which is more generally applicable. Anal. Biochem. 83: 346−356. | Article | PubMed | ISI | ChemPort |
- Harlow, E. and Lane, D. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
- Storkson, R.V., Kjorsvik, A., Tjolsen, A., and Hole, K. Lumbar catheteriza-tion of the spinal subarachnoid space in the rat. 1996. J. Neurosci. Methods 65: 167−172. | Article | PubMed | ISI | ChemPort |
- Wiesenfeld-Hallin, Z., Xu, X.J., Langel, Ü., Bedecs, K., Hökfelt, T., and Bartfai, T. 1992 Galanin-mediated control of pain: enhanced role after nerve injury. Proc. Natl. Acad. Sci. USA 89: 3334−3337. | PubMed | ChemPort |
- Young III, W. S. and Kuhar, M.J. 1988. A new method for receptor autoradiography: [SH]opioid receptors in rat brain. Brain Res. 179: 255−270.
- Melander, T., Köhler, C., Nilsson, S., Hökfelt, T., Brodin, E., Theodorsson, E. et al. 1988. Autoradiographic quantitation and anatomical mapping of 125I-galanin binding sites in the rat central nervous system. J. Chem. Neuroanat. 1: 213−233. | PubMed | ChemPort |
|
