The first demonstration of anti-HIV activity by ribozymes took place over a decade ago. Since then, many strategies, ribozyme designs In addition, several gene therapy trials utilizing ribozymes in and demonstrations of efficacy both in vitro and in vivo have taken place. Ribozymes have the attributes of site-specific cleavage of the target RNA, the potential for turning over multiple substrates, and general lack of toxicity since they do not encode proteins In order to attain the goal of using anti-HIV ribozymes for treatment of HIV infection, we have been exploring several important areas of ribozyme function. These include identifying optimal RNA target sites for ribozyme interaction, co-localization of ribozyme and HIV RNAs and optimizing ribozyme expression from the backbones of lentiviral and retroviral vectors.
The specific aims of the proposed research are: 1) Mechanism of action studies, primary cell testing, and SCID-NOD mice toxicity studies of HIV-1 colocalized ribozyme constructs. A series of ribozyme-HIV-1 target co-localization strategies have been developed during the prior funding period. The genes encoding these constructs will be cloned in both lentiviral and retroviral vectors for transduction into cultured, HIV-1 susceptible cells as well as primary T-lymphocytes and hematotpoietic stem cells. Transduced cells will be challenged with HIV-1 (both lab and clinical isolates). The mechanisms of action for several of the HIV-1 target-colocalized ribozyme constructs will be assessed using various methods. The most efficacious constructs in the lentiviral vectors will be transduced into CD34+ hematopoietic progenitor cells and the transduced cells transplanted into SCID-NOD mice for analyses of engraftment potential. This latter work will be carried out collaboratively with Dr. Bruce Torbett at the Scripps Research Institute. 2) Functional analysis of nucleolar trafficking of HIV RNAs and regulatory proteins and in vivo selection for anti-HIV nucleolar localized ribozymes. A nucleolar localized anti-HIV-1 ribozyme as well as nucleolar localized TAR an RJ3E decoys have provided potent inhibition of HIV-1 infection in cultured cells. We will extend our analyses of these constructs to primary CD34+ cells and T-lymphocytes as part of specific aim 1.
In aim 2 further analyses of the functional role of HIV-1 RNA trafficking through the nucleolus will be explored. Specifically, we will investigate the possibility that 2'0-methyl covalent backbone modifications in the R region of HN-1 RNA are guided by a cellular small nucleolar RNA. We will engineer a small nucleolar RNA to misdirect 2'0 methylations to specific sites of protein interaction in the HIV TAR and Rl3E elements. Finally, an in vivo SELEX scheme will be developec to select for new targets, both in HIV-1 and cellular RNAs using a randomized binding arm library of nucleolar localized ribozymes. The results of the work proposed in this aim should extend our knowledge of the functional role of HIV RNA and protein trafficking through the nucleolus, and provide new ribozymes and other inhibitory RNAs for inhibition of HIV-1 infection. The overall objective of this work is tc enhance our understanding of HIV RNA localization via the use of ribozymes, and to identify the best ribozyme-HIV target strategies for future use in human gene therapy.
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