AEGiS-11IAC: "Designer nucleases" with HIV-1 RNA specificity and their use in anti-HIV-1 gene therapy.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

"Designer nucleases" with HIV-1 RNA specificity and their use in anti-HIV-1 gene therapy.

Int Conf AIDS 1996 Jul 7-12; 11:24 (abstract no. LB.A.6011)
Joshi S, Melekhovets YF; Department of Microbiology. University of Toronto, ON, Canada. Fax: (416) 638-1459. E-mail: sadhna.joshi.sukhwal@utoronto.ca.

OBJECTIVE: An alternative and novel class of molecules that could be used for anti-HIV-1 gene therapy would include "designer nucleases" that would specifically recognize and cleave HIV-1 RNAs. Such nucleases could be engineered by conferring TAR RNA specificity to an RNase so that they will specifically recognize and cleave the TAR-containing HIV-1 RNAs. The feasibility of this approach was tested in vitro and is being assessed in vivo.

RESULTS: Tat is one of the key viral regulatory proteins. It binds to the TAR RNA element located within the 5' non-coding region of all HIV-1 RNAs. Thus the first 72 amino acids of Tat protein which include the TAR RNA binding domain were fused precisely to amino acids 433 to 560 of HIV-1 RT which include the RNase H domain. The resulting Tat-RNase H fusion protein was shown to specifically recognize the TAR RNA via Tat-RNase H-TAR RNA interaction and cleave the HIV-1 TAR RNA in vitro; cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA. Cleavage occurred at three specific locations within the TAR stem loop structure.

CONCLUSION: Design and engineering of other fusion proteins, containing a nucleic acid binding domain and a nucleolytic domain, is in progress to confer cleavage specificity to this and other nucleases against HIV-1 RNA. Usefulness of these "designer nucleases" in anti-HIV-1 gene therapy is being assessed in CD4+ cell lines and in human PBLs.

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LBA6011