TUPDA0105 - Poster Discussion
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
Presented by Scott Ledger (Australia).
S. Ledger1, A. Howe1, J. Murray1, G. Symonds1,2
1University of New South Wales, Sydney, Australia, 2Calimmune, Sydney, Australia
Background: 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
Methods: 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.
Results: 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.
Conclusions: 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.
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