Internal ribosome entry site and vector containing same

Details Internal ribosome entry site and vector containing same Internal ribosome entry site and vector containing same

1999-03-17

2004-08-31

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S069100, C435S455000, C536S023100, C536S024100

Reexamination Certificate

active

06783977

ABSTRACT:

The present invention relates to the use of a nucleotide sequence derived from the 5′ end of the genomic RNA or of the proviral DNA of a reticuloendotheliosis virus as internal ribosome entry site (IRES) and/or for improving retroviral encapsidation. More particularly, it relates to expression vectors comprising this sequence and in particular polycistronic vectors allowing the effective and stable expression of several genes of interest under the control of the same promoter. The present invention finds an advantageous application in the field of vectors for gene therapy.
BACKGROUND OF THE INVENTION
The feasibility of gene therapy applied to humans no longer needs to be demonstrated, and this relates to numerous therapeutic applications such as genetic diseases, infectious diseases and cancers. Numerous prior art documents describe the means using gene therapy, in particular by means of viral vectors. The vectors are generally obtained by deletion of at least part of the viral genes which are replaced by the therapeutic genes of interest. Such vectors can be propagated in a complementation line which provides in trans the viral functions deleted in order to generate a viral particle defective for replication but capable of infecting a host cell. To date, retroviral vectors are among the most widely used but there may also be mentioned vectors derived from adenoviruses, adeno-associated viruses, pox viruses and herpes viruses. This type of vectors, their organization and their mode of infection are widely described in the literature accessible to persons skilled in the art.
As a guide, the retroviral genome consists of a single-stranded linear RNA of positive polarity. In addition to the regulatory sequences R and U5 and U3 and R present at the 5′ and 3′ ends respectively, it carries three genes: gag encoding the capsid proteins, pol encoding reverse transcriptase and integrase and env encoding the envelope proteins. The encapsidation signals, situated upstream of the U5 sequences up to the beginning of the coding region of the gag gene, participate in the dimerization and the encapsidation of the viral RNA into the viral particles. The 5′ end of the genome comprises a cap and the 3′ end is polyadenylated. During the infectious cycle, the viral RNA is converted to a double-stranded linear proviral DNA provided at each end with inverted repeat sequences LTR (for Long Terminal Repeat) which are necessary for the initiation of transcription. The latter, which is carried out by the cellular machinery, allows the production of genomic and subgenomic RNAs from which the viral proteins are synthesized. Retroviruses may be classified into 4 subgroups A to D, on the basis of their morphology. Type C groups together the majority of retroviruses including the MLV (Murine Leukemia Virus) and MSV (Murine Sarcoma Virus) viruses used in most of the gene therapy vectors and the REV viruses (Reticuloendotheliosis Virus) from which the nucleotide sequence of the present invention is derived.
It may be advantageous to have gene therapy vectors which are more effective and capable in particular of efficiently producing several proteins of interest. However, the presence of several promoters within the same vector very often results in a reduction or even in a loss of expression over time. This is due to a well known phenomenon of interference between the promoter sequences. In this context, the publication of international application WO 93/03143 provides a solution to this problem which consists in using an internal ribosome entry site (IRES). It describes a dicistronic retroviral vector for the expression of two genes of interest placed under the control of the same promoter. The presence of a picornavirus IRES site between them allows the production of the product of expression derived from the second gene of interest by internal initiation of the translation of the dicistronic mRNA.
Normally, the entry of the ribosomes at the level of the messenger RNA (mRNA) occurs via the cap situated at the 5′ end of all eukaryotic mRNAs. The 40S ribosomal subunits move along the RNA until an appropriate AUG codon is encountered in order to start the protein synthesis. Generally, the initiation takes place at the level of the first AUG codon. However, if the latter is in a context which is not very favorable, the 40S subunits continue up to a subsequent AUG codon situated in a better translational context (Kozak, 1984, Nucleic Acid Res. 12, 3873-3893; Kozak, 1991, J. Biol. Chem. 266, 19867-19870; Pain, 1996, Eur. J. Biochem. 236, 747-771).
However, there are exceptions to this universal rule. The absence of a cap in certain viral mRNAs suggests the existence of alternative structures allowing the entry of the ribosomes at an internal site of these RNAs. To date, a number of these structures, called IRESs because of their function, have been identified in the noncoding 5′ region of noncapped viral mRNAs such as that in particular of the picornaviruses such as the poliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol. 8, 1103-1112) and EMCV (Encephalomyocarditis virus (Jang et al., 1988, J. Virol. 62, 2636-2643). Cellular mRNAs possessing IRES elements have also been described. There may be mentioned those encoding the BIP protein (for Immunoglobulin heavy chain binding protein; Macejak and Sarnow, 1991, Nature 353, 90-94), certain growth factors (Teerink et al., 1995, Biochem. Biophy. Acts 1264, 403-408; Vagner et al., 1995, Mol. Cell. Biol. 15, 35-44), the translational initiation factor eIF4G (Gan and Rhoads, 1996, J. Biol. Chem. 271, 623-626) and two yeast transcriptional factors TFIID and HAP4 (Iizuka et al., 1994, Mol. Cell. Biol., 14, 7322-7330). IRES sites have also been demonstrated in the VL30-type murine retrotransposons (Berlioz et al., 1995, J. Virol. 69, 6400-6407) and, more recently in the mRNAs encoding the gag precursor of the Friend (FMLV) and Moloney (MoMLV) murine leukemia viruses (Berlioz and Darlix, 1995, J. Virol. 69, 2214-2222; Vagner et al., 1995, J. Biol. Chem. 270, 20376-20383).
DETAILED DESCRIPTION OF THE INVENTION
A new internal ribosome entry site has now been found in the noncoding 5′ region of the RNA of the avian reticuloendotheliosis virus (REV) type A (REV-A) and its efficiency for initiating the translation of coding sequences placed after it in a monocistronic or dicistronic manner has been shown.
The IRES site of the present invention is particularly advantageous compared with those already described in the literature. In the first place, it allows a high level of expression of the cistron which it controls. In addition and, unexpectedly, it can also, within the framework of a retroviral vector, contribute or improve, in association with an appropriate encapsidation region, the dimerization or encapsidation functions, allowing an increase in the viral titer. And finally, because of its weak homology with the murine retrovirus sequences used in most of the gene therapy vectors intended for human use, its use considerably reduces the risk of production of replication-competent viruses.
Most of the gene therapy protocols approved by the RAC (Recombinant DNA Advisory Committee) in the United States use vectors derived from the MoMLV virus. Currently, the choice of a specific retroviral vector for a given therapeutic application remains empirical and the factors influencing the viral titer and the expression of the genes have not yet been clearly elucidated. The study of the cis-acting sequences which control the encapsidation and the establishment of the relative strengths of the various IRES elements can make it possible to optimize the gene therapy vectors in terms of titer and gene expression. One of the aims of the present invention is to provide new retroviral vectors capable of being propagated at a high titer and of allowing optimal expression of one or more genes of interest.
Accordingly, the subject of the present invention is the use of a nucleotide sequence derived from all or part of the 5′ end of the

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