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Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/16777

Title: RNA and DNA Inactivation Strategies to Prevent or Inhibit HIV-1 Replication via Gene Therapy
Authors: Nazari, Reza
Advisor: Joshi, Sadhna
Department: Laboratory Medicine and Pathobiology
Keywords: HIV-1 gene therapy
Interfering RNA
CCR5 co-receptor
Integrase
Hammerhead ribozyme
Group II intron
Issue Date: 20-Jan-2009
Abstract: AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.
URI: http://hdl.handle.net/1807/16777
Appears in Collections:Doctoral
Department of Laboratory Medicine and Pathobiology - Doctoral theses

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