753a 
Lentiviral Delivered VRX496 Long Antisense Sequences Exerts Molecular Pressure on HIV-1 in Human Subjects
Vladimir Lukashov*1, M Quinones-Mateu2, G McGarrity3, T Rebello3, and L Humeau3
1Academic Med Ctr, Amsterdam, The Netherlands; 2Diagnostic Hybrids Inc, Athens, OH, US; and 3VIRxSYS Corp, Gaithersburg, MD, US
Background: In a phase II open-label clinical trial,
40 HIV-infected subjects were infused with their own CD4 T cells previously
transduced ex vivo with VRX496, a lentiviral vector encoding for a 937
nt antisense against the env gene. VRX496 molecular pressures on the
diversity and the fitness of the HIV-1 quasispecies pre- and post-infusions
were investigated.
Methods: Plasma samples were collected at different time-points
during the course of the study. Samples from 9 subjects were randomly included
in this study. HIV-1 env sequences were amplified by real-time
polymerase chain reaction (RT-PCR), cloned into expression vectors, sequenced
for phylogenetic studies (5 of 9) and viral replicative fitness analyzed (6 of 9).
Results: Cloning and sequencing showed the presence
of wild type HIV-1 virions with deleted envelopes (3 of 5) in the area targeted
by the antisense. No deletion was found in any of the pre-dose samples.
Quasispecies diversities were dramatically reduced post-infusion, by as much as
70%, and nucleotide changes in the antisense targeted area were from G to A
(hypermutation) instead of the usual A to G in the non antisense targeted areas
of the envelope. Growth competition experiments showed an overall reduction in
viral replicative fitness in 5 of 6 subjects studied. Of interest, 1 subject
exhibited 2 distinct strains of HIV-1 at time of enrollment. At 1 month
post-infusion, no envelope containing HIV-1 virions were detected while
standard viral load assay detected gag sequences. At 2 month
post-infusion, viral load level were below limits of detection, and only 1 HIV-1
strain re-emerged, with significantly reduced diversity, major amounts of
envelope deletions, and impaired replicative fitness with decreased ability to infect
and propagate in cell culture.
Conclusions: This is the first in vivo
demonstration of anti-HIV antisense activity in cells delivered by a lentiviral
vector. Antisense pressures resulted in env deleted mutants, massive
quasispecies reductions and impaired replicative fitness. The parameters
studied indicate that VRX496 exerts high pressure on wild type HIV, which were not
reflected by the conventional read out methods to monitor HIV infection.
|
