Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-11-03
2001-11-06
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C435S235100, C435S440000, C435S455000, C435S456000, C536S024100
Reexamination Certificate
active
06312948
ABSTRACT:
The object of the invention is novel retroviral vectors, particularly for the transfer and expression of genes in eukaryotic cells. In this respect the invention proposes vectors particularly suitable for use in the transfer of genes for clinical therapeutic, prophylactic or diagnostic purposes.
The rapid development of molecular genetics has led to the identification of an increasing number of molecular abnormalities responsible for human diseases. Within the function unit constituted by the gene regions responsible for the expression of a biological signal and its regulation lie side by side. Each of these regions is liable to be the seat of pathological changes leading to a qualitative or quantitative abnormality of synthesis. The detection of these abnormalities allows screening for them but the major objective remains therapeutic.
The transfer of genes for therapeutic purposes or somatic “gene therapy” consists of inserting a “repairer” gene in the somatic cells of a constituted organism in order to compensate for the dysfunction of an endogenous gene; or even to add a novel function for a therapeutic purpose. The resulting genetic change is likely to be transmitted to the daughter cells of the manipulated cell but it will not be inherited. The normal counterpart of impaired DNA sequences is thus transformed into a medicine.
The field of gene therapy is today being very actively developed and combines clinical assays (for still very small patient populations) with very fundamental research work in to matters such as the modes of gene expression or the vectorization of the therapeutic nucleic acid sequences. The vectors presently used are derived either from inactivated viruses, such as retroviruses or adenoviruses, or macromolecular complexes. The retroviruses are more suitable for use in a target tissue comprising a contingent of stem cells capable of being manipulated ex vivo on the other hand, when the target tissue is constituted of terminally differentiated cells or intimately enmeshed in an organ whose architectural constraints have major functional consequences, such as the lung, the transfer of genes must be performed in vivo, for example by means of adenoviruses. Gene therapy finds applications in diseases as diverse as hereditary diseases due to the alteration of a single gene, such as Duchenne's myopathy, lysosomal diseases, mucoviscidosis or acquired diseases such as AIDS, cancers, thrombo-embolic disease or degenerative neurological diseases, constitutional hematological diseases.
Nonetheless, although the potential applications of gene transfer are extraordinarily large, the therapeutic developments of this approach and its appropriateness still come up against technological difficulties.
In this connection, the development of retroviral vectors more efficacious than the existing tools constitute a major objective. In fact, the retroviral vectors have demonstrated their efficacy in systems in which the target cells of the transfer are classically the subject of mitoses and ideally involve a contingent of stem cells; but the limitations are linked essentially to inadequate infectivity of the viruses used and/or a too moderate level of transcription. For this purpose useful vectors may be selected by considering in particular their infective titer.
The object of the invention is to propose more efficient vectors than those existing, most of which are presently derived from the backbone of the Moloney murine leukemia virus.
The invention is based on work performed starting from a particularly virulent strain of the Friend virus. The isolate I-5 of the ecotropic Friend murine leukemia virus was obtained from long-term bone marrow cultures infected by the Friend virus complex which induces polycythemia (FV-P) (Mathieu-Mahul et al., 1982). The FB29 strain of F-MuLV derived from the isolate I-5 (Sitbon et al., 1986) is responsible for cytolytic and leukemogenic effects on erythroid cells, leading to severe early hemolytic anemia followed by late erythroleukemia in susceptible mice inoculated at birth. The regions responsible for the erythroleukemia were localized in the U3 region of the viral LTR (Sitbon et al., 1986; Sitbon et al., 1991). The principal determinant of the hemolytic anemia seems to depend on specific envelop sequences of the FB29 strain; its severity may be affected by three distinct regions, including a structural segment of the envelope, enhancer sequences of transcription localized in the U3 region and, finally, sequences of the U5-gag-pol regions (Sitbon et al., 1990).
Furthermore, electron microscopical analyses of the viral particles have confirmed a significantly higher packaging capacity (1.5 to 2 log).
The inventors were interested in the specific properties of this strain FB29 and have used it to define retroviral vectors.
According to a first embodiment, the object of the invention is a recombinant retroviral vector for the cloning and/or expression and/or transfer of an exogenous nucleotide sequence, characterized in that it consists of any sequence contained in the ClaI-PvuII fragment situated approximately between nucleotides 7702 and 1527 (SEQ ID NO:11) of the sequence given in FIG.
1
and comprising the LTR sequence included between nucleotides 7842 and 144, the PBS site starting at nucleotide 145, the packaging sequence included in the sequence of 250 nucleotides following the end of the LTR sequence, the said sequence being capable of controlling the cloning and/or expression and/or transfer of the exogenous sequence.
According to another embodiment of the invention, the recombinant vector is characterized in that it consists of any sequence contained in the ClaI-BamHI fragment comprising the nucleotides 7702 to 310 (SEQ ID NO:13) of the sequence shown in
FIG. 1
, and comprising the LTR sequence included between the nucleotides 7842 and 144, the PBS site starting at nucleotide 145, the packaging sequence included in the sequence of 250 nucleotides following the end of the LTR sequence, the said sequence being capable of controlling the cloning and/or expression and/or transfer of the exogenous sequence, whatever its transcriptional orientation with respect to the transcriptional orientation of the virus.
According to this second embodiment of the invention, the vector is thus a retroviral vector for the cloning and/or expression and/or transfer of an exogenous nucleotide sequence consisting of any sequence contained in the ClaI-BamHI fragment situated approximately between nucleotides 7702 and 310 (SEQ ID NO:13) of the sequence given in
FIG. 1
, the said sequence having the capacity to control the cloning and/or expression and/or transfer of the exogenous sequence.
The ClaI and BamHI sites referred to above have their origin in the FB29 strain.
During the construction of the retroviral vectors or vectors destined for the production of packaging lines, these sites may be modified and in particular replaced, optionally creating distinct enzymatic cleavage sites.
A vector comprising the ClaI-BamHI fragment thus contains two LTR sequences, 5′ LTR and 3′ LTR having the same viral origin. This vector may be modified by deletion of all or part of the viral envelope sequence present upstream and/or downstream from the sequences 5′ LTR and 3′ LTR A vector of this type is for example pFOCH29—PL described in FIG.
7
.
These LTR sequences may be separated in the vector by the presence of the gag sequence referred to above and/or by the exogenous nucleotide sequence which it is desired to transfer, clone or express.
According to an attractive embodiment of the invention, the recombinant vector is characterized in that it consists of all of the Cla—PuvII fragment, comprising nucleotides 7702 to 1527 (SEQ ID NO:11) of the sequence shown in FIG.
1
.
Another preferred retroviral vector consists of all of the
Cla
I—BamHI fragment (7702 to 310 (SEQ ID NO:13)).
A retroviral vector of the invention can be used for therapeutic or diagnostic purposes in order to introduce into the patient a nucleotide sequence of clinic
Clark Deborah J. R.
Sorbello Eleanor
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