Abstract Book Vol. 1 [International Conference on AIDS (16th: 2006: Toronto, Canada)]

Results: Of seven treated subjects, five were available beyond 12 months
post-infusion. Two subjects were lost to follow-up early in year one. Antisense
RNA was present in PBMC and/or CD4+ cells at the time of measurement in all
five. Four subjects were available for evaluation through month 60. At month
48, antisense RNA was present in both the PBMC and CD4+ fraction of one
subject, and in only CD4+ cells of two subjects. The fourth subject was negative
at month 48 and beyond. At month 60, antisense RNA was present in CD4+
cells of only one subject. Antisense RNA was found in CD34+ bone marrow cells
in all five subjects, the earliest at 6 months and the latest at 20 months. There
were no treatment-related adverse events, no evidence of clonal expansion
due to insertional mutagenesis, and no significant change in CD4+ cell count
or HIV-1 viral load.
Conclusions: These results are an example of the survival at low levels of
transduced CD34+ stem cells in nonablated subjects with sustainable transgene
activity as well as the long term presence of engineered cells in the'circulating
progeny, i.e., CD34+ and CD4+ cells.
MOPDA07
Inhibition of HIV-1 replication in human T cells by
RNA interference
Y. Meshcheryakoval, N. Gashnikoval, O. Plyasunoval, N. Pokrovskayal,
A. Pokrovsky', N. Petyovka2, N. Tchurikov2. 'State Research Center of Virology
and Biotechnology,'VECTOR', Research Institute of Molecular Biology,
Department of Retroviruses, Koltsovo, Novosibirsk region, Russian Federation,
2Enge/gardt Institute of Molecular Biology Russian Academy of Sciences,
Moscow, Russian Federation
Background: Silencing of gene expression by RNA interference (RNAi) offers
a new tool with potential therapeutic applications for the treatment of HIV-1
infection.
Methods: Two plasmid vectors specifically designed for expression of small
hairpin RNAs in mammalian cells were used. These are: 1) the pEGFP-N1
(Clontech); and 2) the siSTRIKET"M U6 Hairpin Cloning Systems (Promega), which
provide a cloning-based approach to allow fast, easy ligation and expression
of hairpin oligonucleotides. We targeted the Gag gene as degradation in this
region inhibits viral genomic RNA accumulation and production of p24 antigen,
the Pol region, which codes viral enzymes, and the gene for the regulatory
protein Tat. MT-4 cells were transfected with shRNA expression constructs
by electroporation with a Bio-Rad gene pulser on the third day of cultivation.
Transfection efficiency was determined by GFP fluorescence. The transfected
cells were infected 48 hours after transfection at a multiplicity of infection of
0,1-1,0 infectious units per cell. Four days later, cell-free supernatant was
collected, and viral production was quantified by measuring HIV-1 p24 antigen
levels by ELISA.
Results: The transfection efficiency under the most optimal conditions reached
60-65%. HIV-1 production in T-lymphocytes expressing shRNAs corresponding
to sequences in the Gag gene was reduced by more than five-fold compared to
untransfected cells (by 85%). The constructs containing tat and pol seguences
inhibited HIV-1 replication by less than two-fold (47 and 23%). There was also
correlation between the inhibition level of virus reproduction and the HIV-1
infecting dosage. Moreover, we confirmed the effectiveness of the shRNAs by
their dose dependence and the transfection efficiency.
Conclusions: Our experiments demonstrated significant inhibition of HIV-1
reproduction in cells containing expression vector constructions compared to
relevant controls. The obtained results showed the potential ability of using
these HIV-1 specific shRNA expression constructs for AIDS gene therapy.
MOPDA08
Inhibiting HIV-1 by augmenting cellular antiviral
pathways
H. Christensen', C. Ong', S. Chung', L. Frankel2, A. Daher2, S. Laine2,
S. Bannwarth2, A. Gatignol2, D. Purcell'. 'University of Melbourne, Microbiology
and Immunology, Parkville, Australia, 2Lady Davis Institute for Medical
Research, McGill AIDS Center / Molecular Oncology Group, Montreal, Canada
Background: High level expression of the cellular protein, TAR RNA Binding
Protein (TRBP), correlates with high level production of HIV-1. TRBP is a dsRNA
binding protein, which inhibits PKR activation. TRBP is also a component of the
RNAi system that directs siRNAs processed by RNase III family protein DICER
to the Ago-2 RNA silencing machinery. We investigated whether decreasing
high endogenous expression of TRBP in HIV-1 permissive cells resulted in
decreased HIV-1 production.
Methods: We identified siRNAs to TRBP that silenced an EGFP-TRBP reporter.
Next we confirmed that decreasing endogenous TRBP expression with TRBP
siRNAs reduced expression of the HIV-1 molecular clone, NL4.3, in HeLa cells.
Finally we assessed effects on TRBP and HIV-1 expression of shRNA and miRNA
expressing vectors that produce short stem-loop RNAs that are processed by
the RNA interference (RNAi) machinery into TRBP siRNAs.
Results: We validated the ability of the TRBP siRNAs to decrease HIV-1

expression. Vector delivery of functional siRNAs caused a dramatic 80% knockdown of EGFP-TRBP reporter expression, however, when co-transfected with
HIV-1NL4.3 no decrease in viral replication was observed. Our results suggest
that processing of vector delivered TRBP siRNAs is not impeded by the knockdown of TRBP, however at the same time the vectors appear to be ineffective at
decreasing HIV-1NL4.3 expression. We assessed whether the HIV-1 Tat protein
suppressed the biogenesis of RNAi from vector-derived dsRNA, an essential
step for the processing of vector delivered siRNAs.
Conclusions: The vector-derived TRBP dsRNA yielded insufficient RNAi and did

not reduce TRBP levels sufficiently to augment the PKR response to a degree
that reduced HIV-1 expression. However, jointly targeting HIV Tat and TRBP
with RNAi greatly enhanced the inhibition of HIV-1NL4.3 expression. Poor
silencing activity of vectors targeting TRBP in HIV-1 infected cells appears to be
linked to the silencing-suppressor activity of HIV-1 Tat.
Track B
MOPDB01
Risk of clinical progression over long term follow-up
in a wide cohort of patients by different patterns of
viro-immunological response assessed at month 18.
results of the Italian MASTER cohort
C. Torti1, G. Paraninfo', S. Casari', E. Quiros Roldan', F. Suter2, F. Maggiolo2,
T. Quirino3, G. Migliorino3, L. Minoli4, R. Maserati4, F. Ghinelli', L. Sighinolfi',
F. Mazzotta6, S. Lo Caputo6, A. Antinori', F. Antonucci', G. Pastore9, N. Ladisa9,
F. Castelnuovo1", C. Tinellill, A. De Silvestri", G. Carosil. 'University of
Brescia, Institute for Infectious and Tropical Diseases, Brescia, Italy, 2Ospedali
Riuniti di Bergamo, Department of Infectious Diseases, Bergamo, Italy,
3Ospedale di Circolo, Department of Infectious Diseases, Busto Arsizio, Italy,
4University of Pavia, Institute of Infectious Diseases, Pavia, Italy, 5S. Anna
Hospital, Department of Infectious Diseases, Ferrara, Italy, 6S. M. Annunziata
Hospital, Department of Infectious Diseases, Florence, Italy, 7IRCCS L.
Spallanzani ', National Institute for Infectious Diseases, Rome, Italy, 8IRRCS
L. Spallanzani, National Institute for Infectious Diseases, Rome, Italy,
9University of Bari, Institute of Infectious Diseases, Bari, Italy, "~Spedali
Civili of Brescia, Department of Infectious Diseases, Brescia, Italy, "IRCCS
Policlinico S. Matteo, Department of Biostatistics, Pavia, Italy
Background: HAART increases survival of HIV +ve patients. However, improved
knowledge of clinical predictors over long-term follow-up is required to optimize
HIV-disease management.
Methods: All HIV-positive patients in the observational longitudinal MASTER
cohort who started HAART between 1996-2002, with minimum follow-up
available at month 18 after HAART have been studied. New AIDS-definingevents (ADE) and/or AIDS-related-deaths have been considered as outcome
measure. Cross-check with Italian National AIDS Registry has been performed.
Three univariate and multivariable Cox proportional hazards regression models
were performed, respectively:
(i) baseline;
(ii) time-updated clinical variables, including initial viro-immunological
effectiveness ranked into three groups (V+/I-, i.e.: HIV-RNA<400 c/ml for 1
year after six month HAART and lack of CD4+ increase by at least 25% from
baseline; V+/I+ and; V-/I+) and;
(iii) both baseline & time-updated variables.
Results: Amongst 1,157 patients over a mean follow-up of 58 months (IQR:
39-77), 144 ADE's and 11 AIDS-related-deaths were recorded. When baseline
factors were tested, only clinical class C (CDC '93, modified) was independently
correlated with outcome (HR: 4.3; 95%C.I. 2.6-7.2; P<0.0001). In the timeupdated model, the following variables appeared to protect from the risk of
ADE/death: months on boosted-protease-inhibitor (PI)-based regimens (per
month HR: 0.98; 95%C.I. 0.96-0.997; P=0.025) and higher last CD4+ T cell
counts (>350 vs. <200/mm3 HR: 0.08; 95%C.I. 0.03-0.18; P<0.0001 and 200 -350 vs. <200/mm3 HR: 0.21; 95%C.I. 0.086-0.52; P: 0.001). In the baseline &
follow-up model, either baseline clinical stage and last available CD4+ resulted
independent predictors.
Conclusions: Diagnosis of HIV infection, treatment before ADE and
maintenance of high CD4+ T cell count induced by HAART remain important
priorities protecting from AIDS morbidity and mortality over a long-term followup even in this cohort of patients who followed a first line HAART for at least
18 months. Viro-immunological trends over this initial period did not appear to
influence subsequent clinical progression.
MOPDBO2
Causes of severe morbidity in HIV-infected patients.
Aquitaine cohort 2000-2004: the importance of
bacterial infections, cardio-vascular, digestive, and
psychiatric morbidity
F. Bonnet, G. Chine2, S. Lawson-Ayayi2, M. Dupon', J.-L. Pellegrin4,
R. Thibbaut2, P. Morlat', Groupe d'Epidemiologie Clinique du SIDA en
Aquitaine. 'Hopital Saint-Andrd, CHU de Bordeaux, Service de Mddecine
Interne et Maladies Infectieuses, Bordeaux, France, 'Universite Victor Segalen
Bordeaux 2, INSERM U593, Bordeaux, France, 'Hopital Pe//egrin, CHU de
Bordeaux, Service de Maladies infectieuses, Bordeaux, France, 'Hopital
Haut-Levbque, CHU de Bordeaux, Service de Medecine Interne et Maladies
Infectieuses, Pessac, France

Background: The increase of life expectancy of HIV-infected patients is
associated with a decreased incidence of AIDS events but little is known about
the evolution of other causes of severe morbidity because, in most of the
cohorts, only AIDS events are systematically recorded.
Methods: Among a large cohort of HIV-infected patients where all severe
events are systematically recorded and coded according to the International
Classification of Diseases 10th revision (ICD10), we aimed at studying the
evolution of the severe morbidities leading to hospitalisation and/or deaths
between 2000 and 2004.

Monday
14 August
Poster
Discussion

XVI INTERNATIONAL AIDS CONFERENCE * 13-18 AUGUST 2006 * TORONTO CANADA * ABSTRACT BOOK VOLUME 1