Objective
The main goal of this project is the synthesis and study of new oligonucleotide-intercalator conjugates designed to inhibit specifically HIV DNA integration into the infected cell genomes. These oligonucleotides-intercalator conjugates will be designed to bind to the cognate sequences of HIV integrase, the viral enzyme that is responsible for the whole process of DNA integration. These sequences contain short alternated purine tracts susceptible to be targeted by appropriate triple helix-forming oligonucleotides (TFO) provided that the short triple helix be stabilized with a DNA intercalating agent. These oligonucleotides can form two contiguous triplexes switching from one strand to the opposite one. During the first part of the project, oligonucleotidic sequences will be systematically screened for their capability to form a stable switch triplex with the target DNA. Such oligonucleotides will fall into two classes: Oligonucleotide with a natural 5'-3' polarity and oligonucleotides with an alternated 5'-5' polarity. 5'-3' oligonucleotides will be synthesized by standard procedures whereas polymer supports containing thymidine residues modified at the 4-position of thymine will be prepared , in Moscow, Russia, and used for synthesis of 5'-5' oligonucleotides. Since most sequences should contain C-residues that are not able to form stable triplex at neutral pH, C-analogs such as 6-oxocytidine will be developped and synthesized in Frankfurt, Germany, in order to be incorporated in place of C residues. These oligonucleotides capable to form short triple-helices with HIV DNA extremity will be sequently functionnalized at both ends with intercalating agents. This functionnalization should i) stabilize dramatically the short triple-helical complex and ii) greatly improve the stability of the oligonucleotides against nucleases degradation. Several intercalators, including acridine, oxazolopyridocarbazole (OPC), daunomycine and anthraquinone will be tethered to both 5' and 3' ands of the oligonucleotides. Several approaches for oligonucleotide derivatization will be developped. In particular: i) New polymer support allowing 3'-tethering with anthraquinone will be prepared and used; ii) OPC-modified nucleoside will be synthesized and incorporated at the end of the oligonucleotidic sequence. Alternatively, OPC-oligonucleotides conjugates will be synthesized by treatment of activated oligonucleotide with amino-containing OPC. The ability of these conjugates to form stable triple-helical complexes with the HIV target will be determined in Chernogolovka, Russia, using quantitative footprinting, melting temperature measurements and non denaturing electrophoresis methods. Anti-integrase activity will be studied in Paris, France. In vitro assays involving recombinant integrase and oligonucleotidic strates will be performed. Finally, ex vivo activity against de novo infection of HeLa-CD4-ß-gal and CEM cells by HIV-1 will be undertaken with the best conjugate candidates. Should this approach prove successful, it could i) lead to a new class of HIV inhibitors and ii) lay the foundation for a new rationale of triple-helix forming oligonucleotides design. Moreover, during the course of this work, new methods for i) oligonucleotides-intercalators conjugates synthesis and ii) C-base analogs will be developped.
Programme(s)
Multi-annual funding programmes that define the EU’s priorities for research and innovation.
Multi-annual funding programmes that define the EU’s priorities for research and innovation.
Topic(s)
Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
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Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
Call for proposal
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Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.
Funding Scheme
Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
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Coordinator
94805 Villejuif
France
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.