Lentiviral Vector Mediated Gene Therapies Provide Stable Protection Against HIV Infection: The Use of Short-Hairpin RNA to CCR5 and Membrane Anchored C Peptide Entry Inhibitors
Scott Ledger, Annett Howe, John Murray, Geoff Symonds. Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia; School of Mathematics and Statistics, University of New South Wales, Sydney, New South Wales, Australia; Calimmune Pty Ltd, Sydney, New South Wales, Australia
Approximately 35 million individuals world-wide are currently infected with HIV, and whilst antiretroviral treatment has greatly improved viral load, T cell count, life expectancy and co-morbidity, it does not cure infection with obstacles being treatment adherence, side effects and emerging viral resistance. The report of a functional cure for an individual with AIDS/leukemia by transplanting hematopoietic stem cells with a CCR5delta32 mutation points to the potential for gene modified cells to impact on HIV. We examined the impact of gene therapy in vitro using a short-hairpin RNA to CCR5 (sh5), and a gene expressing a membrane-anchored C peptide (C46), both separately and in combination.
The therapeutic genes sh5 and C46 were cloned into eGFP tagged lentiviral constructs which were then transduced into Molt4 T cells previously modified to express CCR5. Replication incompetent HIV was made by pseudotyping pNL4-3-deltaE-mcherry plasmid with a variety of envelopes to produce 2 high CCR5 efficiency and 2 low CCR5 efficiency HIV. Single round infections were then performed and level of infectivity after 3 days was determined by measuring mcherry in cells via flow cytometry. Separate longitudinal infections over 3 weeks were performed by challenging cultures with R5-tropic Bal and observing viral load by p24 concentration and presence of gene-marked cells by flow cytometry.
The 3 constructs containing gene therapeutics provided varying degrees of protection from HIV infection. Control constructs showed no impact on cells or HIV infection. Cultures containing sh5, C46 and C46/sh5 gene therapeutics showed approximately 2-, 3- and 4-fold reductions in infectivity against high CCR5 and 6-, 2.5- and 8-fold-fold reductions in infectivity against low CCR5 efficiency HIV respectively.
Longitudinal challenges showed viral load reductions among all constructs, while therapeutic cells outgrew untransduced cells as a result of HIV-mediated selection over 3 weeks. The C46/sh5 construct showed the greatest selective advantage by increasing from 20 to 65% gene-marking.
These results demonstrate the potential for gene-containing lentiviral vectors to treat HIV infection; there is both stability of the insert and absence of negative effects. These results indicate that both of the examined therapeutic genes protect against HIV infection. They show, more importantly, that the combination of these genes in a single construct provides greater protection than either therapeutic alone, indicating an additive effect of the genes. This finding shows great promise for the use of combination gene therapies in the treatment of HIV infection.
Keywords: Lentivirus Vectors; Infectious Diseases; Cell Therapy - other
Session: Poster Session: Vaccines and Immunotherapy (5:15 PM-7:15 PM)
Date/Time: Friday, May 23, 2014 - 5:15 PM
Room: Hall A and B South