Institut Pasteur

Centre National De La Recherche Scientifique

Institut National De La Sante Et De La Recherche Medicale

Institut National De La Santéde La Recherche Médicale

Véronique Zennou

Pierre Charneau

Hüseyin Firat

Francoise Pflumio

Aride Sirven

Anne Dubart

Aude Sirven

Anne Dubart Kupperschmitt

Francois Clavel

Sylvie Paulous

The present invention provides nucleic acid, vectors, viruses, and recombinant cells comprising triple-stranded structures, such as those resulting from central initiation and termination of HIV-1 reverse transcription at the center of HIV-1 linear DNA genomes. These triplex structures can act as a cis-determinant of HIV-1 DNA nuclear import, allowing infection of non-dividing target cells. In one aspect, the presence of the DNA triplex sequence in an HIV vector strongly stimulates gene transfer in hematopoietic stem cells. The invention also provides methods of using these triplex structures for making recombinant cells, as well as methods of using the recombinant cells to express proteins of interest both in vitro and in vivo.

Lentiviral triplex DNA, and vectors and recombinant cells containing lentiviral triplex DNA

The invention relates to an immunogenic composition comprising a recombinant vector characterized in that it comprises a polynucleotide comprising the cis-acting central initiation region (cPPT) and the cis-acting termination region (CTS), these regions being of retroviral or retroviral-like origin, said vector comprising in addition a defined nucleotide sequence (transgene or sequence of interest) and regulatory signals of retrotranscription, expression and encapsidation of retroviral or retroviral-like origin, wherein the composition is capable of inducing or of stimulating a cell-mediated response for instance a CTL (Cytotoxic T Lymphocytes) response or a CD4 response, against one or several epitopes encoded by the transgene sequence present in the vector.

Lentiviral vectors for the preparation of immunotherapeutical compositions

The invention concerns a recombinant vector characterized in that it comprises a polynucleotide comprising a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), the regions being of retroviral or retroviral-like origin, and the vector further comprising a predetermined nucleotide sequence (transgene or nucleotide sequence of interest) and retrotranscription regulating, expressing, and packaging signals of retroviral or retroviral-like origin.

Use of triplex structure DNA in transferring nucleotide sequences

A method to induce an immune response in a host in need thereof, comprises administering to the host, recombinant lentiviral vector particles comprising:

The invention concerns a recombinant vector characterized in that it comprises a polynucleotide comprising a central initiation cis-active region (cPPT) and a termination cis-active region (CTS) of retroviral or retroviral-like origin, further comprising a predetermined nucleotide sequence (transgene or nucleotide sequence of interest) and retrotranscription regulating, expressing and packaging signals of retroviral or retroviral-like origin.

An in vitro, single cycle, recombinant virus assay (RVA) for determining inhibition of HIV replication by a protease inhibitor comprises transfecting a human epithelial cell line with amplified HIV protease sequences of an HIV virus; an HIV, envelope defective, molecular clone having a complete deletion of its protease coding sequence; and a plasmid containing HIV envelope coding sequence under the control of a promoter for phenotypic complementation of the envelope defective molecular clone. The transfected cells are cultured in the presence of a protease inhibitor to produce a testable stock of infectious particles that can be used to infect indicator cells containing an indicator gene without amplification of the infectious particles prior to infecting the indicator cells. Accumulation of indicator gene product is a measure of inhibition of HIV replication by the protease inhibitor.

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Paris, France

Véronique Zennou