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Laboratory ofExperim ental VirologyLaboratory ofExperim ental Virology
Autologous T-cell therapy based on a lentiviral vector expressing long antisense RNA targeted against HIV-1 env
gene influences HIV replication and evolution in vivo
1Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands, and 2VIRxSYS Corporation, Gaithersburg, MD, USA
a.o.pasternak@amc.uva.nl
Abstract no. WEPDA0205
Alexander O. Pasternak1, Nik Korokhov2, Ben Berkhout1, Vladimir V. Lukashov1, and Laurent Humeau2
“Delivering the Promise of Genetic Medicine” TM
VRX496: a lentiviral vector encoding a 937-nt antisense RNA against a part of env gene (gp120)
This vector retains the full LTRs of HIV, and, therefore, expression of the antisense is conditional upon wild type HIV infection of the cell
Preclinical studies demonstrated complete inhibition of replication of heterologous HIV-1 strains in SupT1 cells and in primary CD4+ T lymphocytes from healthy donors, as well as in primary CD4+ T lymphocytes from HIV-1 infected patients
LTR psi cppt AS env RRE Gtag ppt LTR
SASD
Strategy: autologous CD4+ T lymphocytes from HIV-infected subjects are transduced ex vivo with the vector, expanded, and reinfused into patients
Phase I clinical trial (the first ever clinical trial with a lentiviral vector approved by FDA) demonstrated safety and tolerability of this approach (no evidence of insertional mutagenesis by deep sequencing) – Levine et al. (2006), PNAS 103(46):17372
LTR psi cppt AS env RRE Gtag ppt LTR
SASD
Step 1 –Leukapheresis: Extract WBCs from subject
Step 2 -Purification: Isolate CD4 T
CellsStep 3 -
Transduction: Modify CD4 T Cells
with VRX496TM
Step 4 -Expansion:
Expand modified CD4 cells from
~1 billion to ~80 billion
Step 5 –Harvest,
formulation, cryopresevationQCtestingsand
QA release
Step 6 –Re-Infusion: Infuse Lexgenleucel-T back
into subject
Phase II trial: 43 HIV-infected patients who failed >1 cART regimens
10-80 billion vector-modified cells were reinfused into patients
Longitudinal effects of the therapy on HIV-1 env evolution were analyzed in 17 subjects sampled both pre-infusion and monthly post-infusion for 6 to 12 months
Plasma-derived viral RNA from 144 samples was amplified, cloned, and the full-length gp120 coding region was sequenced in 8-10 clones for each sample
env
gp120 gp41
AS construct
c1 v1v2 c2 v3 c3 v4 c4 v5 c5
PCR I
PCR II
sequencing primers
10
100
1,000
10,000
100,000
1,000,000
0 50 100 150 200 250 300 350 400
09-07 (5.2) 09-05 (1.3) 07-11 (1.0) 07-18 (0.7)
09-02 (4.5) 09-04 (2.5) 09-14 (0.6)
Viral Loads: Bolus 1 DoseH
IV R
NA
per
mL
Days Post Infusion
Transient decrease in plasma viral load was observed in some patients
100
1,000
10,000
100,000
0 100 200 300 400
07-11 (1.0) 07-18 (0.7) 09-05 (1.3) 09-07 (5.2)
09-14 (0.6) 09-02 (4.5) 09-04 (2.5)
Persistence: Bolus 1 Dose
Days Post Infusion
VR
X496 P
rovir
al C
op
ies p
er
10
6 P
BM
Cs
Vector-modified cells were persisting for up to 1 year
Two AS-related factors:
(a) sequence SIMilarity of the AS RNA with the targeted HIV transcripts at baseline,
(b) PERsistence of the infused vector-modified cells during the follow-up period,
independently and cooperatively influenced HIV replication and evolution:
-4
-2
0
2
4
gen
eti
c d
ista
nce,
%
-2
-1
0
1
2
pla
sm
a R
NA
, lo
g1
0 P<0.001 P=0.003
PERlow SIMlow
PERhigh SIMlow
PERlow SIMhigh
PERhigh SIMhigh
PERlow SIMlow
PERhigh SIMlow
PERlow SIMhigh
PERhigh SIMhigh
Plasma viral load, change from baseline Degree of virus evolution from the pre-infusion to the post-infusion quasispecies
(relative MRCA distances)
The degree of virus evolution from the pre-infusion to the post-infusion quasispecies negatively correlated with virus replicative fitness, assessed ex vivo by growth competition assay
-1.0
-0.5
0.0
0.5
1.0G
D re
l M
RC
A i
nsid
e A
S,
%
Low replicative fitness
High replicative fitness
P=0.032
Conclusion:
Same AS-related factors were associated with enhanced viral evolution and with the relative decrease in plasma viral load,
suggesting that selective pressure exerted by the AS causes directional viral evolution, presumably towards escape, which is associated with the fitness loss
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