CIBLE POTENTIELLE D'UNE IMMUNOTHERAPIE VISANT A PREVENIR LA REPLICATION DU VIH (VIRUS DU SIDA) CHEZ LES HUMAINS : ADN COMPLEMENTAIRE ET PROTEINE POUR UN REGULATEUR PUTATIF DE L'EXPRESSION D'UNE POLYPROTEINE DU GAG DU VIH, RECONSTITUEES A PARTIR DU BRIN ANTI-SENS DU GENE HUMAIN P53

POTENTIAL TARGET FOR IMMUNOTHERAPY TO PREVENT REPLICATION OF HIV (AIDS VIRUS) IN HUMANS: CDNA AND PROTEIN FOR A PUTATIVE REGULATOR OF HIV GAG POLYPROTEIN EXPRESSION RECONSTRUCTED FROM THE ANTISENSE STRAND OF HUMAN P53 GENE

Abrégé anglais

ABSTRACT
A cDNA reconstructed from the antisense strand of human p53 gene
encodes a 53 aa nuclear protein (Higaa) which has the potential
for interacting with a domain of very highly conserved sequences
in HIV gag polyprotein precursor. The protein which appears to
be an intracellular activator of gag may be important for the
successful replication of HIV in human cells. Since the protein
is constitutively repressed by the p53 gene in normal cells, it
is potentially a good target for a simple immunological approach
to prevent multiplication of HIV following integration of the
viral genome into the human genome.

Revendications

The Embodiments of the discovery in which an exclusive property
or privilege is claimed are defined as follows:
1. The cDNA and protein sequences described in the embodiment of
figure 1a and 1b.
2. Any diagnostic and or therapeutic reagents including
ribozymes produced using the cDNA described in the embodiment of
figure 1a or from any modified form of this cDNA.
3. Any diagnostic and or therapeutic reagents including
polyclonal antibodies, monoclonal antibodies, fusion proteins,
anti-peptide reagents etc., made from any part or all of amino
acid sequence described in the embodiment of figure 1b.
4. Any use of the cDNA and or protein described in the
embodiment of figure 1a and 1b or modifications of the cDNA or
protein to treat any type of cancer.

Description

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SPECIFICATION
HIV the AIDS causing virus will probably infect more than 100
million humans before the beginning of the 21 century, and
although there is a very slight chance that a vaccine may be
developed to prevent infection before the 21 century there i9 no
hope of curing an infected person. Certain drugs based on
nucleotide analogues, e.g., AZT, directed against HIV reverse
transcriptase gene, have had qualified success in slowing the
rate of intercellular spreading of the virus in small number of
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cases; however, the side effects of these drugs are almost
unacceptable in most cases, and the cost of treatment is
enormous. Replication of the virus, following integration into ;;
the human genome is a key step in the spread of infectivity. If
replication could be blocked or slowed down then the
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intercellular infection will be considerably curtailed. The HIV
virus `gag' and `gagpol' genes are involved in crucial steps of
viral replication; therefore, any interference with the
expression or processing of `gag' will have a negative effect
on viral replication. This invention relates to the discovery of
a protein HIV gag activator protein (Higaa) encoded by a gene,
antisense to a region of the human p53 gene, which may be
required for the correct expression or processing of HIV gag
polyprotein. p53 ls involved in protecting humans against
invading virus especially tumour causing viruses.

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(2)
Because `Higaa' mRNA is antisense to the sense strand of p53
mRNA it is probably not expressed in normal humans, but may be
expressed in humans suffering from certain virus, or other
infections, which could hinder, or stop, antisense repression by
p53 gene and hence allow `Higaa' to be expressed in sufficient
quantities to activate HIV gag.
A region of the antisense strand of p53 cDNA comprising
nucleotides 532 - 693 inclusive, is 100 % homologous to `Higaa'
cDNA. The protein deduced from the cDNA is made up of 53 amino
acids (53 aa) and by similarity appears to be a nuclear protein~
The structure of the cDNA is shown in the embodiment of figure
la and that of the deduced protein in the embodiment of figure
lb. The domain of the protein (16 aa) which is capable of
interacting with gag polyprotein is overlined.
`Higaa' cDNA is recon~tructed (and Hind III restriction enzyme
sites added) by oligonucleotide synthesis on an Applied
Biosystems DNA synthesizer using the column method according to
the recommendations of the manufacturer. The positive and
negative strands are synthesised separately, gel purified on
agarose, then mixed together in equimolar amounts and repurified
by agarose gel electrophoresis. The cDNA construct is ligated
into the pTrcHis Xpress vector and processed in the expresst
system (Invitrogen corporation pTrcHis Xpress Prokaryotic
Expression and Purification System) according to the
manufacturers instructions.

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(3)
The 16 aa domain, overlined in the embodiment of figure lb, is
probably all that is required to make antibodies, or very
specific anti-peptide reagents, for blocking reaction of `Higaa'
with HIV gag. Since the protein is not expressed in humans and
does not have homology to any known, normal human, protein,
blocking it should not produce undesirable side effects in AIDS
victims, and hence, a simple immunological approach to stop the
intercellular spread of HIV infection which leads to full blown
AIDS might be possible.
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