Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1998-06-18
2001-05-08
Bui, Phuong T. (Department: 1638)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S184100, C424S188100, C435S069100, C435S069300, C435S235100, C435S325000, C435S320100, C530S300000, C536S023720
Reexamination Certificate
active
06228369
ABSTRACT:
The present invention relates to a composition resulting from the combination of trans-dominant variants of two viral proteins originating from the same virus. The combination of these two variants enables a resistance to infection by or propagation of the virus in question to be conferred. The present invention finds an advantageous application in the treatment or prevention of infections caused by HIV (human immunodeficiency virus) responsible for AIDS (acquired immunodeficiency syndrome).
In the context of the search for and preparation of medicinal products that inhibit viral infections, somatic gene therapy by intracellular immunization constitutes one of the promising approaches for the future. This concept, defined by Baltimore (1988, Nature, 335, 395-396), consists in genetically modifying cells so as to cause them to synthesize a nucleic acid or a heterologous protein endowing them with resistance against the viral infection. One of the possible approaches for acquiring this resistance is to cause the host cell to synthesize genes that inhibit a step of the viral cycle. Although all the steps of the viral cycle may be targeted in order to implement such a therapy, the genes participating in an early step constitute a potentially advantageous target. Such is the case with the tat and rev genes of the HIV virus, which play an essential part in initiation of the viral replication.
The tat gene codes for a TAT protein which trans-activates the expression of all of the genes of HIV (Arya et al., 1985, Science, 229, 69-73). It appears that TAT participates at both transcriptional and post-transcriptional level. It interacts specifically with a short nucleotide sequence localized at the 5′ end of the genome and of the viral transcripts and designated TAR (standing for trans-activation responsive region) sequence (Rosen et al., 1985, Cell, 41, 813-823).
The REV protein, the expression product of the rev gene, promotes the transport of large-sized messenger RNA (mRNA) to the cytoplasm, to be translated therein. For its part, REV specifically recognizes an RRE (standing for REV responsive element) sequence on the mRNA. This sequence is localized in proximity to another sequence termed CRS (cis-acting repression sequence), involved in the nuclear retention of the mRNA carrying it. It is assumed that binding of the REV protein to its RRE target sequence has the effect of counterbalancing the inhibitory effect of CRS on the passage of large viral mRNA into the cytoplasm.
Although their expression products are very different from a molecular mass, sequence and mechanism of action standpoint, regulator genes exerting a function similar to TAT and/or REV have been identified in the genome of other infectious viruses, such as HTLV-I and -II (human T-cell leukemia virus) retroviruses responsible for severe forms of leukemia and herpesviruses (chicken pox, shingles and Epstein-Barr viruses).
For some years, negative and dominant (trans-dominant) mutants of these various viral proteins have been described in the literature. In particular, several teams have generated trans-dominant variants of the TAT and REV proteins capable of binding to their target sequence but incapable of exerting the function of the native protein. While their inhibitory effect with respect to the action of the native protein has been demonstrated, their efficacy in HIV-infected cells nevertheless remains to be determined.
It has now been found that the expression in an HIV-infected cell of at least two genes coding for trans-dominant variants of the TAT and REV proteins, respectively, of the HIV virus enables propagation of the virus to be blocked. The authors have demonstrated that the inhibition of viral infection resulting from the joint expression of the two genes is markedly greater than that observed on expression of either one of these genes. This synergistic effect constitutes an unexpected advantage.
The objective of the present invention is to make available to the public an especially effective pharmaceutical composition, and which is hence safer for the purpose of use in humans, which enables the replication of a virus to be inhibited at different points of the viral cycle.
Thus, the subject of the present invention is a composition comprising at least:
(a) a first trans-dominant variant of a viral protein; and
(b) a second trans-dominant variant of another viral protein originating from the same virus as the viral protein in (a).
In the context of the invention, a first and a second trans-dominant variant are preferably derived from proteins originating from the viruses HTLV-I and -II (human T-cell leukemia virus), HSV-1 (herpes simplex virus) and as an absolute preference, HIV. Under the name “HIV” are grouped together the different viral strains and isolates of the virus which is the etiological agent of AIDS.
“Trans-dominant mutant” is understood to mean a negative and dominant mutant, that is to say a mutant which is non-functional (incapable of exerting the function of the native protein from which it is derived) but capable of competitively and dominantly inhibiting the function of the latter. In practice, a trans-dominant variant can be obtained by deletion, substitution and/or addition of one or more amino acids of the native protein or of a functional fragment of the latter. A person skilled in the art knows the techniques which enable these modifications to be performed, as well as the regions which have to be modified according to the viral proteins under consideration.
However, and according to an advantageous embodiment, the first and the second trans-dominant variants employed in the context of the present invention are derived from viral proteins participating in an early stage of the infection, and in particular from proteins capable of interacting specifically with a particular nucleotide sequence (termed target sequence) present in the genome and/or the transcripts of the virus from which they originate.
It is preferable to employ a first trans-dominant variant derived from a protein exerting a trans-activating function, in particular from the VP16 protein of HSV-1 (Friedman et al., 1988, Nature, 335, 452-454), the p40 and p37 proteins encoded by the X region of the genome of the HTLV-I and -II viruses (Wachsman et al., 1987, Science, 235, 674-677) and, as an absolute preference, the TAT protein of HIV (see below). As a second trans-dominant variant, a variant derived from a viral protein participating in another step, preferably early, of the viral replication, for example at the level of transport of the viral mRNA to the cytoplasm of the host cell, will be employed.
A preferred composition according to the invention results from the combination of:
(a) a first trans-dominant variant derived from the TAT protein of the HIV virus; and
(b) a second trans-dominant variant derived from the REV protein of the HIV virus.
The choice of a trans-dominant variant of the TAT protein is very wide. It can fall on those described in the prior art, and in particular in Green et al. (1989, Cell, 58, 215-233) and Pearson et al. (1990, Proc. Natl. Acad. Sci. USA, 87, 5079-5083). But it can also fall on other trans-dominant variants, in particular a variant having the sequence as shown in sequence identifier NO: 1, beginning at amino acid +1 and ending at amino acid +86, in which:
(1) the phenylalanine residue at position +38 is replaced by an aspartic acid residue;
(2) the threonine residue at position +40 is replaced by an alanine residue;
(3) the lysine residue at position +41 is replaced by a glutamic acid residue;
(4) the isoleucine residue at position +45 is replaced by a serine residue; and/or
(5) the tyrosine residue at position +47 is replaced by an arginine residue.
Naturally, a trans-dominant TAT variant can combine several mutations.
As examples of trans-dominant variants of the REV protein, there may be quoted those mentioned in Malim et al. (1989, Cell, 58, 205-214) and Venkatesh and Chinnadurai (1990, Virology, 178, 327-330). However, it can b
Guss Tania
Mehtali Majid
Bui Phuong T.
Burns Doane Swecker & Mathis L.L.P.
Transgene S.A.
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