Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-04-21
2001-05-22
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C536S023100, C536S023500, C536S024100
Reexamination Certificate
active
06235497
ABSTRACT:
The present invention relates to a nucleic acid sequence coding for a protein involved in the vesicular transport of acetylcholine and to the corresponding protein. It also relates to expression vectors integrating the said sequence and to the use of this sequence or the said vectors for therapeutic purposes.
Acetylcholine, ACh, is a neurotransmitter synthesized by the enzyme choline acetyltransferase, ChAT. At the ends of the cholinergic neurons, the majority of the ACh produced is transported from the cytoplasm to the inside of the synaptic vesicles. The accumulation of ACh in these vesicles is mediated by the activity of an ATPase which pumps H
+
ions, generating an electrochemical gradient (Anderson D. C. et al., (1982) Biochemistry 21, 3037-3043). This gradient is utilized by a transporter to take up ACh via an exchange of protons (Parsons S. M. et al., (1993), Int. Rev. neurobiol. 35, 279-390). This type of mechanism is comparable to the one involved in the transport of biogenic amines into the synaptic vesicles.
An understanding of the different mechanisms of regulation participating in the expression of ACh would be especially valuable from a therapeutic standpoint.
Recently, cDNAs coding for vesicular ACh transporters in Caenohabditis elegans and Torpedo have been cloned and sequenced (Alfonso A. et al., (1993) Science 261, 617-619; Varoqui H. et al., FEBS Letters 342, 97-102). A study of the sequences of the corresponding proteins revealed the existence of structural similarities between these two proteins, and likewise with respect to two vesicular transporters of biogenic amines in mammals, VMAT1 and VMAT2 (Liu et al., (1992) Cell 70, 539-551). These common structural features are, in particular, (i) 12 transmembrane domains (TM), (ii) the presence in these transmembrane domains of charged amino acid residues which probably participate in the transport of substrate, (iii) a glycosylated loop localized between TM1 and TM2, and (iv) cytoplasmic C- and N-terminal ends which display fewer similarities than the remainder of the protein.
The subject of the present invention is, more especially, the isolation, sequencing and characterization of a region of the gene coding for ChAT capable of expressing a vesicular ACh transporter, as well as the identification of promoter sequences involved in the expression of this transporter. It also describes cassettes for expression of this gene, vectors containing it and their use for directing the expression of this transporter.
More specifically, the present invention relates to a nucleic acid sequence coding for a protein involved in the vesicular transport of ACh, characterized in that it is localized within the first intron of the gene coding for ChAT, in the same transcriptional orientation.
From the 5′ region of the gene coding for rat ChAT, and more precisely from a restriction map of this region, the inventors isolated a HindIII-BamHI fragment carrying the first intron of this gene and, in part, the sequences of the first two corresponding R and N exons, respectively, at the 5′ and 3′ ends of this fragment. Unexpectedly, the cloning and sequencing of this fragment revealed the presence of a 1590-bp open reading frame coding for a protein of the order of 530 amino acids, equivalent to a mass of the order of 56.5 kDa, and displaying similarities in respect of its sequence with proteins of the transporter type. This protein was identified as a rat vesicular ACh transporter and is designated hereinafter rVAT.
This DNA sequence is, more specifically, localized within the first intron of the gene coding for ChAT, downstream of the R-type promoter of the ChAT gene and in the same transcription orientation as the ChAT gene. It is not interrupted by any intron.
More especially, the present invention relates to a nucleic acid sequence coding for a vesicular ACh transporter, characterized in that it comprises all or part of SEQ ID No. 1 or one of its derivatives.
Preferably, the sequence comprises all or part of the sequence SEQ ID No.2 or one of its derivatives.
For the purposes of the present invention, the term derivative denotes any sequence differing from the sequence under consideration as a result of the degeneracy of the genetic code, obtained by one or more modifications of a genetic and/or chemical nature, as well as any sequence hybridizing with these sequences or fragments of the latter and whose product possesses the stated activity. Modification of a genetic and/or chemical nature may be understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as, in particular, that of increasing the affinity-of the corresponding polypeptide for its ligand(s), that of improving its levels of production, that of increasing its resistance to proteases, that of increasing and/or modifying its activity or that of endowing it with new pharmacokinetic and/or biological properties. Among derivatives resulting from an addition, chimeric nucleic acid sequences containing an additional heterologous portion linked to one end may, for example, be mentioned. The term derivative also comprises sequences homologous to the sequence under consideration, originating from other cellular sources, and in particular from cells of human origin, or from other organisms, and possessing an activity of the same type. Such homologous sequences may be obtained by hybridization experiments. The hybridizations may be carried out using nucleic acid libraries, employing as probe the native sequence or a fragment of the latter, under conventional stringency conditions (Maniatis et al., see general techniques of molecular biology), or preferably under high stringency conditions.
The present invention shall also be understood to cover the corresponding antisense sequences whose expression enables transcription of cellular mRNAs to be controlled. Such sequences can consist of all or part of the nucleic acid sequence under consideration, transcribed in the reverse orientation.
More preferably, the sequence in question is the nucleic acid sequence coding for the rat vesicular ACh transporter, rVAT.
Bearing in mind the localization of the gene according to the invention, namely in the first intron of the gene coding for ChAT, downstream of the R-type ChAT promoter and in the same transcriptional orientation, the inventors sought to find out whether the ChAT and VAT mRNAs could be expressed from the same promoter.
This hypothesis was verified experimentally. Two forms of mRNA coding for the VAT transporter were identified. They contain all or part of the sequence of the R1- or R2-type R exon (of ChAT mRNA) (Kengaku et al. Mol. Brain Res. 18: 71-76 (1993)) (FIG.
1
), immediately followed by the sequence of a second exon beginning 308 bp upstream of the translation initiation codon (of VAT). Besides these two mRNAs, there are three other forms of VAT mRNA which are apparently more abundant than the two forms mentioned above, coding for the polypeptide which is the subject of the invention. One form of VAT mRNA contains all or part of the R exon mentioned above. The 5′ ends of the other two forms are localized downstream of the R exon. The form which we designate V1, of approximately 2.6 kb, has two 5′ ends located 426 bp and 402 bp upstream of the translation initiation codon of VAT (positions 949 and 972, respectively, on SEQ ID No. 1). The form of VAT mRNA which we designate V2, of approximately 3 kb, has several 5′ ends located between 863 bp and 888 bp upstream of the translation initiation codon of VAT (positions 486 to 511 of SEQ ID No. 1).
The subject of the present invention is also promoter regions involved in the expression of VAT and localized in the gene coding for ChAT. Such regions are, more especially, the promoter region comprising all or part of SEQ ID No.4 or one of its derivatives, and attached promoter regions localized in SEQ ID No. 1.
For the purposes of the present invention, promoter region denote
Bejanin Stéphane
Berrard Sylvie
Cervini Riccardo
Mallet Jacques
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Priebe Scott D.
Rhone-Poulenc Rorer S.A.
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