Title

Developing triplex forming oligonucleotides as a ‘block and lock’ strategy for an HIV cure

Presenter

Haoming Liu

Authors

H. Liu¹, C. Tumpach², J. Symons^2,3, S. Lewin^2,4,5, M. Roche²

Institutions

¹The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology, Melbourne, Australia, ²The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Department of Infectious Diseases, Melbourne, Australia, ³University Medical Center Utrecht, Translational Virology, Department of Medical Microbiology, Utrecht, Netherlands, ⁴Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Victorian Infectious Diseases Service, Melbourne, Australia, ⁵Alfred Health and Monash University, Department of Infectious Diseases, Melbourne, Australia

BACKGROUND: The main barrier to an HIV cure is the persistence of long lived and proliferating latently infected T cells. Virus can integrate in the host genome, stay transcriptionally silenced, and re-emerge upon reactivation of transcription. Triplex formation oligonucleotides(TFO) are single stranded DNA that can bind in the major groove of duplex DNA with high specificity and affinity. We hypothesized that TFO can bind to the integrated provirus and inhibit viral transcription leading to a ‘block and lock’ strategy for an HIV cure.
METHODS: We designed multiple TFOs targeting HIV gag(n=3), env(n=4) and to GFP(n=4). TFOs were delivered by lipofectamine or a novel nanoparticle to HEK 293T transfected with plasmids expressing gag, env, green-fluorescent-protein(GFP) and full length HIV NL-4.3. TFO impact on full length proviral transcription was assessed by incubating supernatant with the reporter TZM-bl cell line which contains an integrated HIV LTR, a portion of gag and the luciferase gene and quantification of cell associated unspliced HIV RNA by RT-qPCR. TFO activity on integrated provirus was assessed by transfection of TFOs and subsequent stimulation of TZM-bls.
RESULTS: In the plasmid transfection system, GFP-specific TFOs inhibited GFP expression (mean±SEM inhibition for 2uM GFP-TFO2 was 96.8±0.45%, p<0.001) and HIV-specific TFOs inhibited either HIV gag(reduction with 4 uM HIV-TFO7, 67.26±9.19%, p=0.03) or HIV env expression (reduction of env expression ranging from 75 to 88%). In HIV NL-4.3 transfected cells, 2uM HIV TFO4 potently reduced the infectivity of supernatant by 97±0.59%(p<0.001) and reduced unspliced HIV RNA 400±35.02 fold(p=0.03) consistent with the TFO inhibiting transcription initiation. Inhibition of HIV gene expression was achieved by both lipofectamine and nanoparticle delivery of TFO. In TZMBL cells, transfection of four different TFOs by lipofectamine had no effect on either basal or induced HIV transcription.
CONCLUSIONS: TFOs can potently inhibit HIV gene expression, however we were unable to demonstrate inhibition of transcription from an integrated provirus. This may be due to the lack of nuclear penetration by TFOs or target inaccessibity due to the chromatin environment. Further work to address these limitations is underway. TFOs present a novel HIV ‘block and lock’ option to limit HIV transcription.

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