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(12) Patent Application Publication (10) Pub. No.: US 2003/0203870 A1 Blatt Et Al US 2003O2O387OA1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0203870 A1 Blatt et al. (43) Pub. Date: Oct. 30, 2003 (54) METHOD AND REAGENT FOR THE Continuation-in-part of application No. PCT/US01/ INHIBITION OF NOGO AND NOGO 04273, filed on Feb. 9, 2001. RECEPTOR GENES Continuation-in-part of application No. PCT/US02/ 10512, filed on Apr. 3, 2002, which is a continuation (75) Inventors: Lawrence Blatt, San Francisco, CA in-part of application No. 09/827,395, filed on Apr. 5, (US); James McSwiggen, Boulder, CO 2001, now abandoned, which is a continuation-in-part (US); Bharat M. Chowrira, Louisville, of application No. 09/780,533, filed on Feb. 9, 2001, CO (US); Peter Haeberli, Berthoud, now abandoned. CO (US) (60) Provisional application No. 60/181,797, filed on Feb. Correspondence Address: 11, 2000. Provisional application No. 60/181,797, McDonnell Boehnen Hulbert & Berghoff filed on Feb. 11, 2000. Provisional application No. 32nd Floor 60/181,797, filed on Feb. 11, 2000. 300 S. Wacker Drive Chicago, IL 60606 (US) Publication Classification (73) Assignee: Ribozyme Pharmaceuticals, Inc. (51) Int. Cl." ........................... A61K 48/00; CO7H 21/04 (52) U.S. Cl. ........................... 514/44; 536/23.2; 536/23.5 (21) Appl. No.: 10/430,882 (22) Filed: May 6, 2003 (57) ABSTRACT Related U.S. Application Data The present invention relates to nucleic acid molecules, (63) Continuation of application No. 09/827,395, filed on including antisense and enzymatic nucleic acid molecules, Apr. 5, 2001, now abandoned, which is a continua Such as hammerhead ribozymes, DNAZymes, and antisense, tion-in-part of application No. 09/780,533, filed on which modulate the expression of NOGO and NOGO recep Feb. 9, 2001, now abandoned. tor genes. Patent Application Publication Oct. 30, 2003 Sheet 1 of 5 US 2003/0203870 A1 Figure 1: Ribozyme Motifs Group intron RNAse P (M1 RNA) . Y. T. i EGSRN 5 s Group Illntron WS RNA i C E -- A. - Domains ; : IBS BS ss 3's'-----' 5'- Exor2. ' t 3-Exor Hairpin Ribozyme (SEQSEOID NO 2606 ) (N) HOf Royme s' N- Felix5RN/ \s hieli VN", G - 3. Hammerhead Ribozyme S. NNN s d SN - s' N N *AGN.E.), E 3' ' 3. N(N) : Helix 1 A NN -- G 8 E: 3' Helix3 N-N CUC | A -la. f NN se- ( i.S. T iii. AC N *GG . N, C K NN (SEQ ID NO 2605) Helix 4 INN N) (N) Patent Application Publication Patent Application Publication Patent Application Publication US 2003/0203870 A1 ZMIJ??ou-V?uuÁZu|Z Patent Application Publication Oct. 30, 2003 Sheet 5 of 5 US 2003/0203870 A1 105.IeLVNH„S 18 UU34SV?IIIUU34S ? |O JL J?OWNQUÁZVNGI:ÇQ.In??H V(LI9,ONCIIÒ?S) !OJOÎn=Å 5)JOV=>. US 2003/0203870 A1 Oct. 30, 2003 METHOD AND REAGENT FOR THE INHIBITION (Weibel et al., 1994, Brain Res., 642, 259-266). Addition OF NOGO AND NOGO RECEPTOR GENES ally, IN-1 treatment in vivo has resulted in the recovery of Specific refleX and locomotor functions after Spinal cord BACKGROUND OF THE INVENTION injury in adult rats (Bregman et al., 1995, Nature, 378, 0001. This patent application is a continuation-in-part of 498-501). Blatt, U.S. Ser. No. 09/780,533 filed Feb. 9, 2001, entitled 0007 Recently, the cloning of NOGO-A (Genbank “METHOD AND REAGENT FOR THE INHIBITION OF Accession No AJ242961), the rat complementary DNA NOGO GENE” which claims priority from Blatt, U.S.S No. encoding NI-220/250 has been reported (Chen et al., 2000, 60/181,797, filed Feb. 11, 2000, entitled “METHOD AND Nature, 403, 434-439). The NOGO gene encodes at least REAGENT FOR THE INHIBITION OF NOGO GENE. three major protein products (NOGO-A, NOGO-B, and This application is hereby incorporated by reference herein NOGO-C) resulting from both alternative promoter usage in its entirety including the drawings. and alternative splicing. Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the Spreading 0002 The Sequence Listing file named “MBHB00 of 3T3 firboblasts. Monoclonal antibody IN-1 recognizes 878-C Sequence Listing” submitted on Compact Disc-Re NOGO-A and neutralizes. NOGO-A inhibition of neuronal cordable (CD-R) medium (“0104.04- 1540”) submitted in growth in vitro. Evidence Supports the proposal that duplicate is in compliance with 37 C.F.R. S.1.52(e) is incor NOGO-A is the previously described rat NI-250 since porated herein by reference. NOGO-A contains all six peptide sequences obtained from 0003. The present invention provides compounds, com purified bNI-220, the bovine equivalent of rat NI-250 (Chen positions, and methods for the Study, diagnosis, and treat et al Supra). ment of conditions relating to the expression of NOGO and 0008 Prinjha et al., 2000, Nature, 403, 383-384, report NOGO receptor genes. In particular, the invention provides the cloning of the human NOGO gene which encodes three nucleic acid molecules that are used to modulate the expres different NOGO isoforms that are potent inhibitors of neu sion of NOGO and NOGO receptor gene products. rite outgrowth. Using oligonucleotide primers to amplify 0004. The following is a brief description of the current and clone overlapping regions of the open reading frame of understanding of NOGO and NOGO receptors. The discus NOGO cDNA, Phrinjha et al., Supra identified three forms Sion is not meant to be complete and is provided only to of cDNA clone corresponding to the three protein isoforms. assist understanding the invention that follows. The Sum The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No. AJ251383). An intermediate mary is not an admission that any of the work described length splice variant that lacks residues 186-1,004 corre below is prior art to the claimed invention. sponds to NOGO-B (Accession No. AJ251384). The short 0005 The ceased growth of neurons following develop est splice variant, NOGO-C (Accession No. AJ251385), ment has Severe implications for lesions of the central appears to be the previously described rat Vp20 (Accession nervous System (CNS) caused by neurodegenerative disor No. AF051335) and foocen-s (Accession No. AF132048), derS and traumatic accidents. Although CNS neurons have and also lacks residues 186-1,004. According to Prinjha et the capacity to rearrange their axonal and dendritic foci in al., Supra, the NOGO amino-terminal region shows no the developed brain, the regeneration of severed CNS axons Significant homology to any known protein, while the car Spanning distance does not exist. AXonal growth following boxy-terminal tail shares homology with neuroendocrine CNS injury is limited by the local tissue environment rather Specific proteins and other members of the reticulon gene than intrinsic factors, as indicated by transplantation experi family. In addition, the carboxy-terminal tail contains a ments (Richardson et al., 1980, Nature, 284, 264-265). consensus Sequence that may serve as an endoplasmic Non-neuronal glial cells of the CNS, including oligoden reticulum retention region. Based on the NOGO protein drocytes and astrocytes, have been shown to inhibit the Sequence, Prinjha et al., Supra, postulate NOGO to be a aXonal growth of dorsal root ganglion neurons in culture membrane associated protein comprising a putative large (Schwab and Thoenen, 1985, J. Neurosci., 5, 2415-2423). extracellular domain of 1,024 residues with seven predicted Cultured dorsal root ganglion cells can extend their axons N-linked glycosylation sites, two or three transmembrane across glial cells from the peripheral nervous system, (ie, domains, and a short carboxy-terminal region of 43 residues. Schwann cells), but are inhibited by oligodendrocytes and 0009 Grandpre et al., 2000, Nature, also report the myelin of the CNS (Schwab and Caroni, 1988, J. Neurosci, identification of NOGO as a potent inhibitor of axon regen 8, 2381-2393). eration. The 4.1 kilobase NOGO human cDNA clone iden 0006 The non-conducive properties of CNS tissue in tified by Grandpre et al., Supra, KIAA0886, is homologous adult vertebrates is thought to result from the existence of to a cDNA derived from a previous effort to sequence inhibitory factors rather than the lack of growth factors. The random high molecular-weight brain derived cDNAS identification of proteins with neurite outgrowth inhibitory (Nagase et al., 1998, DNA Res., 31, 355-364). This cDNA or repulsive properties include NI-35, NI-250 (Caroni and clone encodes a protein that matches all Six of the peptide Schwab, 1988, Neuron, 1,85-96), myelin-associated glyco sequences derived from bovine NOGO. Grandpre et al., protein (Genbank Accession No M29273), tenascin-R (Gen Supra demonstrate that NOGO expression is predominantly bank Accession No X98085), and NG-2 (Genbank Acces associated with the CNS and not the peripheral nervous sion No X61945). Monoclonal antibodies (mAb IN-1) raised system (PNS). Cellular localization of NOGO protein against NI-35/250 have been shown to partially neutralize appears to be predominantly reticluar in origin, however, the growth inhibitory effect of CNS myelin and oligoden NOGO is found on the surface of some oligodentrocytes. An drocytes. IN-1 treatment in Vivo has resulted in long distance active domain of NOGO has been identified, defined as fiber regeneration in lesioned adult mammalian CNS tissue residues 31-55 of a hydrophilic 66-residue lumenal/extra US 2003/0203870 A1 Oct. 30, 2003 cellular domain. A Synthetic fragment corresponding to this Fournier et al., 2001, Nature, 409,341-346), NI-35, NI-220, Sequence exhibits growth-cone collapsing and outgrowth and/or NI-250, myelin-associated glycoprotein (Genbank inhibiting activities (Grandpre et al., Supra). Accession No M29273), tenascin-R (Genbank Accession No 0010) A receptor for the NOGO-A extracellular domain X98085), and NG-2 (Genbank Accession No X61945).
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