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Title: Studies on antisense RNA inhibition of HIV-1 replication
Author: Chadwick, D. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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Two vector systems were developed expressing antisense RNA (of two different sizes) complementary to three target regions in the 5' leader/LTR of HIV-1: the R (TAR) region, the primer binding site and the splice donor-packaging signal (ψ) region. After cloning these regions into an expression vector, pcDNA3, designed to express these sequences at high levels from the CMV IE promoter, stable constitutive expression in a T cell line was demonstrated by RT-PCR. After directly challenging these cell lines with HIV-1 at various doses cell lines expressing antisense RNA targeting the ψ-region showed significant inhibition of replication compared to cell lines expressing the same sequence in sense. A second, co-transfection assay using COS-1 cells was also developed to assess the antiviral capabilities of these vectors. Both sequences targeting the ψ-region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore analyses of relative levels of cellular and viral RNA from these assays suggested that each of these antisense molecules was exerting its effect at an early stage in the transcription-translation pathway. CXCR4, an important co-receptor for HIV was also targeted for down regulation by antisense RNA. Inducible expression of a sequence complementary to a region of this gene using the 'tet-on' system was accomplished leading to down-regulation of surface CXCR4 expression in T cells. Down-regulation appeared to result in reduced replication of HIV-1 in these cells. Genetic strategies for inhibiting HIV replication may provide an alternative and complementary approach to chemotherapy for infected individuals.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available