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