Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-10-14
2002-08-13
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024100
Reexamination Certificate
active
06432701
ABSTRACT:
The present invention relates to a new system for gene expression. It also relates to the use of this system in gene or cell therapy, for increasing the expression of genes of interest or for producing recombinant proteins.
Gene and cell therapies consist in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) or in providing for the expression of a protein of therapeutic interest by introducing genetic information into the cell or organ affected. This genetic information may be introduced either ex vivo into a cell extracted from the organ, the modified cell then being reintroduced into the body (cell therapy), or directly in vivo into the appropriate tissue (gene therapy). Different techniques exist for performing gene transfer, including various techniques of transfection involving natural or synthetic chemical or biochemical vectors such as complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), cationic lipids, and the like. Another technique is based on the use of viruses as vectors for gene transfer. In this connection, different viruses have been tested for their capacity to infect certain cell populations, and especially retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses. One of the difficulties in developing these gene and cell therapies lies, however, in the efficacy of the treatment. In particular, it is important to be able to obtain a strong expression of the gene of interest in order to improve the therapeutic effect. The same type of problem arises in the case of methods for the production of recombinant proteins.
Different types of promoters have been described in the literature and are used to control the expression of genes of interest. However, the levels of expression obtained are sometimes insufficient for obtaining industrial amounts of recombinant protein in vitro, or for generating in vivo a substantial and/or lasting therapeutic effect. The present invention now describes a new, especially effective system for the expression of genes. The present invention is the outcome, in particular, of the identification of regions endowed with transcription enhancer activity, enabling the strength of the promoters, and thus the levels of gene expression, to be increased.
The present invention is the outcome, more especially, of the identification of regions of the first intron of the tyrosine hydroxylase gene which are capable of increasing the activity of transcription promoters. These regions are located more precisely in the microsatellite HUMTH01 present in the first intron of this gene.
Microsatellites represent an abundant class of DNA repeat sequences which display great polymorphism linked to variations in the number of repeat motifs and/or in the sequence of these motifs. For this reason, these microsatellites have been used as genetic markers for the construction of genetic maps and for the identification of loci involved in pathologies. The size of the different alleles of a microsatellite depends on variations in the number of repeat motifs. Sequencing experiments on repeat dimers have thus enabled a variation to be shown in the number of repeat motifs and in their sequence. These variations can correspond, in particular, to “perfect” repetitions, that is to say repetitions without interruption in the sequence of bases, or to “imperfect” repetitions containing one or more interruptions in the sequence of motifs, these interruptions entailing deletion(s) or insertion(s). In the same way, variations in length and/or in sequence have also been observed in trimeric or tetrameric repeat motifs. Thus, variations of this type have been observed in the microsatellites HUMHPRTB (Edwards et al., Genomics 12 (1992) 241) and HUMTH01 (Puers et al., Am. J. Hum. Genet. 53 (1993) 953).
The microsatellite HUMTH01 is located in the first intron of the gene for tyrosine hydroxylase (TH), which is the limiting enzyme of the pathway of catecholamine biosynthesis. A portion of the sequence of this intron is shown as SEQ ID No. 1 (from nucleotide 871). This sequence includes the microsatellite HUMTH01 (allele (TCAT)9, shown in bold characters), which is located at position 1170 (GenBank, accession # D00269). The microsatellite HUMTH01 consists of tetrameric TCAT repeat motifs. It displays a certain polymorphism, different alleles having been described possessing variable numbers of repeat motifs. The allele most often encountered (allele Ei) contains 10 repeat motifs and a deletion of one base pair in the fifth repeat motif, which contains the sequence CAT (SEQ ID No. 2). Other alleles have been identified carrying from 5 to 10 TCAT motifs.
The Applicant has now shown that, surprisingly, these sequences are specifically recognized by proteins present in different nuclear extracts (Example 1). Furthermore, the Applicant demonstrated that these sequences enabled gene transcription to be increased in different cell systems (Examples 3 and 4). Thus, in PC12 cells, an increase in transcriptional activity by a factor of 25 to 50 has been observed. In Hela cells, an exceptional increase by a factor of 100 to 350 times has been observed. Hence these sequences display very potent transcription activator properties. These properties are especially unexpected, and far superior to the effects observed with other transcription enhancers. Thus, an increase in transcriptional activity by a factor of 3 to 6 has been observed with HRAS1-VNTR sequences (Green et al., 1993, ref), and by a factor of 2 to 5 with INS-VNTR sequences (Catignani et al., 1995). The increase obtained with the sequences of the invention can exceed a factor of 300, and constitutes an enormous advantage over the previous systems. In addition, the results obtained show that the sequences of the invention are functional in different cell types and in cells of different organisms (PC12 rat cells, HeLa human cells), demonstrating a very broad potential of applications.
Hence, a first subject of the invention relates to a DNA fragment having transcription enhancer activity, consisting essentially of a portion of the first intron of the tyrosine hydroxylase gene. Advantageously, the fragment of the invention comprises less than 200 bp and includes an allele of the microsatellite HUMTH01. More preferably, the fragment comprises less than 100 bp. It is especially advantageous for the fragment to consist essentially of an allele of the microsatellite HUMTH01.
As mentioned above, the microsatellite HUMTH01 is located at position 1170 approximately of the human TH gene (GenBank accession #D00269) and consists mainly of tetrameric TCAT repeat motifs. Different alleles of this microsatellite have been described, possessing variable numbers of repeat motifs (from 5 to 10) and sequence variations, some alleles possessing base mutations. Thus, the allele most often encountered (allele Ei) contains 10 repeat motifs and a deletion of one base pair in the fifth repeat motif, which contains the sequence CAT (SEQ ID No. 2). Other alleles have been identified, carrying from 5 to 10 TCAT motifs (SEQ ID Nos 9-15). In addition, a DNA fragment carrying 3 TCAT motifs may be synthesized and tests as a transcription enhancer, according to the invention.
In this connection, a subject of the invention is also an isolated DNA fragment, characterized in that it possesses transcription enhancer activity and in that it possesses the sequence (TACT)
n
-(CAT)
o
-(TCAT)
p
, in which n is between 1 and 50, o is between 0 and 20 and p is between 0 and 50.
According to a particular variant of the invention, the transcription enhancer possesses a sequence (TCAT)
n
-(CAT)
o
-(TCAT)
p
in which n is between 2 and 20 inclusive and o and p equal 0.
According to another particular variant of the invention, the transcription enhancer possesses a sequence (TCA
Mallet Jacques
Meloni Rolando
Ravassard Philippe
Treilhou Fabienne
Aventis Pharma S.A.
Parkin Jeffrey S.
Scheiner Laurie
Wiley Rein & Fielding LLP
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