Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-05-11
2001-03-13
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Vector, per se
C435S006120, C435S069100, C435S325000, C435S375000, C536S023100, C536S023500, C536S024100, C514S04400A
Reexamination Certificate
active
06200800
ABSTRACT:
The present invention relates to a sequence of natural or synthetic retroelements, in particular an LTR retroviral nucleotide sequence, more particularly retroviral DNA as well as a retroviral vector containing this sequence and which enables, on infection of a cell into which it is desired to integrate a gene of interest contained in this vector, a large part of the proviral sequences which are no longer necessary after integration of the recombinant provirus to be eliminated.
Recent progress in the use of retroviruses as gene vectors falls into three classes: (i) improvement of the packaging cell lines, (ii) manipulation of the tropism of the envelope proteins, (iii) expression of multiple genes. The modification of the structure of the integrated provirus has not yet been very much exploited because this latter contains certain cis-acting essential elements. These elements are the source of many problems.
From the point of view of the structure, the retroviral vector system is constructed on two elements: the transcomplementing genes (gag, pol and env) and the cis-acting sequences (U
3
, R, U
5
, the polypurine track (PPT), the primer binding site (PBS) and the packaging and dimerization signal).
The transcomplementing genes are incorporated in transcomplementing cells. They are not transferred to the target cells.
The cis-acting sequences are incorporated into retroviral vectors. Most of them are transferred to the infected target cells and are integrated into the recombinant provirus. The elimination of any one of these cis-acting sequences results in a non-functional retroviral system. The packaging signal is necessary for the packaging of the retroviral genome in viral particles. PBS, PPT and R are required for reverse transcription. The U
3
and U
5
sequences are essential for reverse transcription and for the integration of the retroviral product introduced into the cell.
Once the provirus is integrated, these sequences are no longer necessary for the expression of the gene. On the contrary, these sequences may even cause many problems. (i) A transcriptional interference may result from the presence of the strong promoter in the U
3
sequences. (ii) The PBS may function as a cis-acting inhibitory element for the internal promoters. For example, in the multipotent cells, the PBS is a recognition sequence for a strong repressor and in addition it acts as an inhibitory element. (iii) The U
3
sequence contains at least one of the negative regulatory elements situated in the direct repeated activators at a distance between −345 and −306. (iv) The 3′LTR (long terminal repeat) can activate genes flanking cells with sometimes deleterious consequences for the cell. (v) The RNA expressed from the promoter of the 5′LTR contains the packaging signal and can, consequently, be recovered in a retroviral particle. This RNA can recombine with a cellular RNA or the retroviral RNA of a phenotype, resulting in the recovery of a retrovirus endowed with novel properties (creating a potential biological danger). These problems have not yet been entirely overcome since all these elements are necessary for the replication and the insertion of the virus into the genome.
In the context of the present invention, the inventors have developed a sequence of natural or synthetic retroelements, in particular a retroviral nucleotide sequence, and in particular a retroviral vector containing this sequence, designed so as to permit the elimination of a large part of the proviral sequences which are no longer necessary after integration of the provirus into a cell host. More particularly, this retroviral sequence is a retroviral DNA capable of permitting the integration of a single LTR sequence, a retrotransposon or a sequence comprising a U
3
, R or U
5
region into the genome of a cell host.
The invention also relates to host cells, preferably eukaryotic cells, obtained after transfection by the retroelement sequence of the invention or a vector containing it.
The present invention hence concerns a retroelement sequence characterized in that it comprises an insertion sequence incorporated in a region capable of being transferred into a target cell and integrated into a recombinant provirus when this target cell is infected by a retrovirus containing this sequence. In particular, this insertion sequence is incorporated into a cis-acting region, more particularly in the 3′LTR or the 5′LTR region and preferably in the U
3
region of the 3′LTR or the U
5
region of the 5′LTR of this retroviral sequence. This insertion sequence comprises a nucleotide sequence of interest capable of being integrated into the genome of a cell host as well as a recognition sequence for a recombinase. Preferably, all of the retroviral sequence only contains a single recognition sequence situated with the sequence of interest, upstream or downstream for example, although this latter parameter is not absolutely necessary. The nucleotide sequence of interest may be situated downstream from the recognition sequence in as much as this latter is included in the region transferred into a target cell on infection of this target cell by a retrovirus containing the retroviral sequence. The maximal length of the insertion sequence that the retroviral vector of the invention contains is usually situated between 0.5 and 10 kb.
In particular, the sequence of the invention also comprises a DNA sequence coding for a recombinase capable of recognizing the sequence of its recognition site, this DNA sequence coding for a recombinase being advantageously situated between the 5′LTR and the 3′LTR regions of the vector. The invention also relates to a retroviral vector or recombinant retrovirus comprising the sequence described above. Preferably, the retroviral vector, which may take the form of a recombinant retrovirus, only contains a single recognition sequence incorporated into the region capable of being transferred to a target cell.
The DNA of the invention hence makes possible the insertion, through the intervention of a retroviral vector, of nucleotide sequences of interest in host cells, for example of the eukaryotic type, without the insertion of the proviral sequences which are no longer necessary after the integration of the sequences of interest in the provirus.
The term “nucleotide sequence of interest” used above refers to sequences to be inserted in the genome of host cells in order to allow these latter to produce molecules of interest, more particularly in therapy or for vaccination. These sequences of interest include, among others, genes, DNA or RNA sequences coding either for proteins (hormones, immunoglobulins, enzymes or others) when these retroviral vectors of the invention are used in gene therapy, or human or non-human proteins (such as viral proteins) when the retroviral vectors of the invention are to be used in the framework of vaccination protocols. The nucleotide sequences of interest may also be constituted in part of regulatory elements (i.e. promoter, enhancer) homologous or heterologous to the host cell on the one hand and, on the other hand, of sequences coding for all or part of one or more genes or complementary DNA. Furthermore, the nucleotide sequences of interest may also code for an antisense RNA or a ribozyme sequence.
The possible applications of the retroelement sequence of the present invention are manifold. The sequence of the invention is used either simply for the insertion of nucleotide sequences in host cells such as eukaryotic cells in an environment which promotes a better expression of this gene, or in gene therapy, or in vaccination. As an example, the sequence of interest described in Nature Medicine, Volume No. 7, July 1995 corresponding to the insert of the plasmids pMEPV
H
/pMEPV
L
or pMEPV
H
/pREPV
K
can be used and the in vivo expression product may be an element of a therapeutic composition.
The retroviral vector of the invention is obtained by the transfection of a transcomplementing viral cell line with a retroelement seque
Choulika Andre
Nicolas Jean-Francois
Elliott George C.
Epps Janet L.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
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