mountain view, may 9 2005 · 2008. 3. 30. · progress to date. dr ... nef rt u6+1 pbs tat/rev rev...
TRANSCRIPT
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ASX/Media Release
Dr Rossi presents update on HIV lymphoma study at Keystone meeting
31 March 2008, Melbourne, Australia: Last Friday afternoon (US time), Benitec Limited (ASX:BLT) Scientific Advisory Board member Dr John Rossi presented an early update on the human pilot HIV lymphoma stem cell study at a Keystone meeting in Whistler, British Columbia. His talk was entitled “Applying RNAi for the treatment of HIV infection” and he was also one of the speakers in the workshop entitled “New Approaches to shRNA-Based Screening and Therapy” as part of the Keystone RNAi, MicroRNA and Non-Coding RNA conference. Dr Rossi is a key collaborator for the pilot human HIV study being undertaken at the City of Hope in Duarte, California. This study includes the use of Benitec’s technology as a clinical method to fight HIV-AIDS infection. In this ground-breaking pilot-clinical study, patients with AIDS-related lymphoma are being treated using vector expressed RNAi aimed at rendering the cells resistant to the HIV-1 virus infection. This study entitled “A pilot study of safety and feasibility of stem cell therapy for AIDS Lymphoma using stem cells treated with a Lentivirus vector encoding multiple anti-HIV RNA’s” commenced in Q3 2007. In his update, Dr Rossi outlined the specific aims, outline of the study design, eligibility and exclusion criteria and progress to date. Dr Rossi indicated that applying RNAi for the treatment of HIV infection is a powerful mechanism for targeted inhibition of gene expression. He also noted that several early reports have demonstrated the HIV was a highly susceptible target for RNAi and was amenable to the gene therapy approach. A copy of Dr Rossi’s slides are included with this release. CONTACT: BENITEC LTD Sue MacLeman Rudi Michelson Chief Executive Officer Monsoon Communications +61 437 211 200 +61 411 402 737
About Benitec Benitec is an Australian biotechnology company focused on licensing its extensive intellectual property portfolio and developing therapeutics to treat serious diseases using its proprietary ddRNAi technology. Its current therapeutic program is focused on Human Immunodeficiency Virus (HIV). For additional information, please visit www.benitec.com.
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Applying RNAi for the Applying RNAi for the treatment of HIV infectiontreatment of HIV infection
Powerful mechanism for targeted Powerful mechanism for targeted inhibition of gene expression.inhibition of gene expression.
Several early reports demonstrating Several early reports demonstrating that HIV was a highly susceptible that HIV was a highly susceptible target for RNAi.target for RNAi.
Amenable to a gene therapy Amenable to a gene therapy approach.approach.F
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Stem cell-based gene therapy
PluripotentStem Cell
CFU-Blast
NK-Pre
Lymphoidstem cell
CFU-GEMM
Pre-B Plasma Cell
T cell
NK cell
Pre-T
CFU-GM
BFU-Meg
BFU-E CFU-E Retic RBC
CFU-G
CFU-M
PMNL
Monocyte
Megakaryocyte
B cell
Transduction targets
Hematopoiesis
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•• CotransfectCotransfect target (pNL4target (pNL4--3) and siRNA PCR 3) and siRNA PCR construct into 293 cells construct into 293 cells
•• Collect supernatants at different times post Collect supernatants at different times post tranfectiontranfection
•• Assay p24 antigen as a marker of HIVAssay p24 antigen as a marker of HIV--1 1 expression expression
•• Compare Early tat/rev versus late Compare Early tat/rev versus late envenv targetstargets
PCR gene PCR gene ––HIV HIV cotransfectionscotransfections to identify most to identify most potent HIV targetspotent HIV targets
Si I Si II
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Cotransfection ShPCR
1
10
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day 1 day 2 day 3
days post transfection
p24
outp
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g/m
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YY4
YY6
NLSI
NLmSINLSII
nef
RT
U6+1
pBS
Tat/RevTat/Rev
RevRev
PolPol
Env4Env4Env6Env6
mTatmTat/Rev/RevNefNef
Vector Vector controlscontrols
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Targeting Early Transcripts encoding Tat and Rev has been most effective since these are
key proteins for subsequent steps in the viral life cycle. In particular the common exon
shared by Tat and Rev.
GagLTR LTRPol
Vif
Vpr VpuEnv Nef
RevTat
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Lentiviral vector construct stably Lentiviral vector construct stably expressing shRNAsexpressing shRNAs
CMV EGFPRRE/PPT
U6 S / U6 AS siRNA
LTRLTR
siRNAs
5’-------GCGGAGACAGCGACGAAGAGCUUUUCGCCUCUGUCGCUGCUUCUCG-------5’
S/AS(I) :
U6 promoter sense loop antisense ter
5’-GCGGAGACAGCGACGAAGAGCTTTGTGTAGGCTCTTCGTCGCTGTCTCCGCTTTTTT
5’-GCGGAGACAGCGACGAAGAGC3’-UUCGCCUCUGUCGCUGCUUCUCG
UU U GU
GUA G
siRNA
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GCGGAGACAGCGACGAAGAGC3’-UUCGCCUCUGUCGCUGCUUCU-5’
Viral escape mutants emerge
Mutant
Wildtype
CAP
AAA
A
Overlapping Overlapping Reading framesReading frames
Tat Tat ––GlnGln to Lysto Lys
RevRev--Asp to Asp to GluGlu
If mutant used to challenge cells expressing shRNA to tat/rev, mutation persists, but if grown on naive T-cells, wild type comes back as predominant speciesF
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Combinatorial RNA based Combinatorial RNA based therapy for HIVtherapy for HIV
•• Most efficacious drug therapy for HIV uses Most efficacious drug therapy for HIV uses combination of two or three drugs targeting combination of two or three drugs targeting HIV RT and protease. Combinations prolong HIV RT and protease. Combinations prolong and sometimes prevent resistant viral variants.and sometimes prevent resistant viral variants.
•• Can effective combinatorial gene therapy from Can effective combinatorial gene therapy from a single vector be accomplished?a single vector be accomplished?
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Each of these RNAs inhibits HIV-1 by a different mechanism Therefore it may be advantageous to combine these in a therapeutic setting
Combinatorial therapeutic RNAs
I. Nucleolar localizing TAR decoy(Michienzi et al. 2002, 2006)
II. Chimeric VA1CCR5 ribozyme(Cagnon et al, 1997; Li et al., 2003)
III. Anti –tat/rev shRNA
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pHIV-shI-TAR-GFPU6 TAR
pHIV-shI-CCR5RZ-GFP
U6 shI
CMV EGFP RU3 U5CMV RRER U5 ψ
pHIV-7-GFPWPREflap
VA1 RZU6 shI
VA1 RZU6 TARU6 shIpHIV-shI-TAR-CCR5RZ-GFP
pHIV-shI-GFPU6 shI
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Potent inhibition of HIV-1 replication
Triple combination provides better protection than individual shRNAs in transduced CD34+ derived monocytes/macrophages
Day
7D
ay 4
2D
ay 7
Day
42
Day
21
Day
28
Ladd
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GFP Triple
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Triple vector transduced CD34+ cells differentiate into thymocytes in vivo in SCID-hu mice. T-lymphocytes are resistant to viral challenge.
Purify T cells
HIV-1 challenge
60 days
1000
10000
100000
0 5 10 15Days PI
log
p24
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ml)
Non-transduced
GFP-alone
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-HIV-1 challenge of triple transgenic thymocytes
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1996 1996 –– established autologous BMT for AIDS lymphoma established autologous BMT for AIDS lymphoma
Surv Time (Months) Post-ASCT0 6 12 18 24 30 36 42 48 54 60 66 72
0
0.1
0.2
0.3
0.4
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0.6
0.7
0.8
0.9
1
% O
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Overall Survival95% Conf. Interval
From A. Krishnan et al. Blood 2005; 105:874-8
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A Pilot Study of Safety and Feasibility of Stem Cell Therapy for AIDS Lymphoma Using Stem Cells
Treated with a Lentivirus Vector Encoding Multiple anti-HIV RNAs
Specific Aims:The primary objective is to determine safety and feasibility of lentivirus-transduced hematopoieticstem cells in the setting of autologous HCT for thetreatment of AIDS lymphoma
The secondary objective is to determine the quantifyand duration of vector-marked peripheral blood cellsafter marrow engraftmentF
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Fill and FinishFill and FinishConcentration / Concentration /
Diafiltration Diafiltration 500K NWWCO500K NWWCO
Clarification ofClarification ofSupernatantSupernatant
ResuspendResuspend Pellet in Pellet in Final FormulationFinal Formulation
Harvest of PostHarvest of Post--Transfection SupernatantTransfection Supernatant
8.5L8.5L
50mL50mL
Transfection inTransfection in1010--layer cell factorieslayer cell factories
0.45 um0.45 um
High Speed High Speed CentrifugationCentrifugation
BenzonaseBenzonaseTreatmentTreatment
8.5L8.5L
EOP CellEOP CellCollectionCollection
Ongoing process developmentOngoing process development
In process sample collectionIn process sample collection
Batch release testingBatch release testing
Clinical grade triple vector production is now complete, with enough vector to treat 6 BMT and 6 Autologous T-cell patients
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Study #1: AIDS Lymphoma
CD34+ SelectionFraction A
Fraction B
Cryopreservation Untransduced
1 2 3 4 5 6 7 8...... HPC-A Mobilization (days)
G-CSF (10 ug/kg)
1 2 3 4 5 6 7 8...... HPC-A Mobilization (days)
G-CSF (10 ug/kg)
Aphereses
Lymphoma RxLymphoma Rx
ConditioningConditioningRegimenRegimen: BCNU BCNU BCNU VP16 Cytoxan
ConditioningConditioningRegimenRegimen: BCNU BCNU BCNU VP16 Cytoxan
-7 -6 -5 -4 -3 -2 0 +1Days Pre-and Post-transplant
Cryopreservation Transduction withHIV-shI-TAR-CCR5RZ
#1 #2 #3 #4
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Eligibility Criteria• Age 18-60 years• AIDS related lymphoma:
-Intermediate grade or high grade non-Hodgkin’s lymphoma (working formulations D-H and J), and >partial response or first relapse after remission with standard chemotherapy
-Hodgkin's lymphoma any subtype except nodular L&H lymphocyte predominant, and partial response or less, or relapse after standard chemotherapy
• HIV load
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Exclusion Criteria• History of grade III cystitis due to cyclophosphamide• CNS lymphoma• Prior other malignancy except treated basal cell ca, cervical
ca,or squamous cell ca • HIV-associated encephalopathy; dementia; seizures in past
year• No active bacterial, fungal, CMV infection; no OI in past year
except treatment-responsive MAI, candida, HSV, VZV, CMV• Other AIDS-related syndromes, infectious or otherwise,
perceived to cause excessive risk for morbidity post-HCT• Inability to undergo blood stem cell mobilization or any contra-
indication for undergoing HCT• CXCR4 tropism of HIVF
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AIDS Lymphoma Study Recruitment
Transplanted Mar 13, 2008Diffuse Large B cell
0305
Transplanted Feb 19, 2008Diffuse Large B cell
0304
Failed eligibility, low mobilization
Burkitt0303
Failed eligibility, infectionBurkitt0302
Cell Product failed releaseDiffuse Large B cell
0301
StatusDiagnosisUPN #
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First in man-Lentivirus-shRNA Stem Cell Transplant for HIV
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Outcome
• Safety as determined by AE reports• Vector marking in PBMC at months 1, 2, 3,
6, 12, 18, 24• HIV – vector recombination as measured by
RT-PCR of HIV positive plasma• Feasibility determinations
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Considerations for Future Developments
• Selection and expansion of transduced cells• Safe and efficient delivery to hematopoietic stem cells
• Direct delivery by injection• Targeted delivery to stem cells• Targeted expression in specific cell lineages
• Develop method for expansion of genetically protected cells• Development of methods for use early in HIV infection and
prior to antiviral chemotherapy
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Chimeric-gp120 aptamer –anti-tat/rev siRNA
Structural characterization of an anti-gp120 RNA aptamerthat neutralizes R5 strains of HIV-1ANTU K. DEY,1,2 CARLA GRIFFITHS,1 SUSAN M. LEA,2 and WILLIAM JAMES11Sir William Dunn School of Pathology and 2Laboratory of Molecular Biophysics, Department of Biochemistry,University of Oxford, Oxford, United Kingdom-RNA (2005), 11:873–884.
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Does aptamer allow internalization of aptamer-siRNA chimera?
• Test uptake in two cell lines: CHO-gp160 expresses HIV gp 120 envelope ectopically on cell surface:
• CHO EE does not express gp120• Compare wild type aptamer versus mutant
aptamer
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CHO-WT gp160 + chimera 1 CHO-WT gp160 + mutant-1CHO-WT gp160 control
CHO-EE + chimera 1 CHO-EE + mutant-1CHO-EE control
Uptake assay (confocal microscopy)Uptake assay (confocal microscopy)
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Linker
Chimeras L-1 (27 mer siRNA)
Chimeras L-2 (21 mer siRNA)Chimeras 1 (27 mer siRNA)
Chimeras 2 (21 mer siRNA)
Linker
Mutant-1
mutantLinker
Mutant-2mutant
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Infec
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EM +
Buffe
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27 m
ersiR
NA21
mer
siRNA
Aptam
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Ch L-
1Ch
1Ch
L-2
Ch 2
M-1
M-2
Infec
ted C
EMUn
infec
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EM +
Buffe
r2007-09-1927-sense probe and U6 probe
27 m
ersiR
NA21
mer
siRNA
Aptam
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Ch L-
1Ch
1Ch
L-2
Uninf
ected
CEM
Infected CEM Uninfected CEM
20 bp
30 bp
40 bp
50 bp60 bp70 bp
U6 probe
27-sense probe
HIV infected cells mixed with uninfected CEM Cells and three days later treated with Chimeric aptamers and controls. RNA extracted on day 7 and Northern gel analyses.
15% denaturing gel
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HIV-1 challenge of Infected CEM cells
0
1000000
2000000
3000000
4000000
5000000
6000000
Day 3 Day 5 Day 7 Day 9
P24
expr
essi
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g/m
L)
bufferCh L-1Ch L-2M-1M-2infected cellsuninfected cells
2007-09-22
HIVHIV--1 challenge of infected CEM cells1 challenge of infected CEM cells
After CEM cells were infected with NL4-3 for 10 days, 30% infected CEM cells and 70% uninfected CEM cells were incubated with different RNA. Supernatants were collected at different days (3, 5, 7, 9 days) for p24 assay.
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2008-01-20 (III-B)
HIV-1 challenge (aptamer)
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
D3 D5 D7 D9 D11
Days
P24
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L)
(1) Buffer(2) aptamer 1(3) A-1 RNA(4) B-68 RNA(5) A-28 RNA(6) 2nd-RNA pool(7) Infected CEM cells (8) uninfected CEM cells
A-1 B-68
KD (nM) 50.37 KD (nM) 148.6
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Future Directions• Use targeted aptamer-siRNAs and Tat inducible shRNA/micro RNA
expression systems to down regulate new cellular targets (Brass et al., Science 2008).
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Summary ConclusionsSummary Conclusions
•• First human clinical trial with First human clinical trial with expressed shRNA in combination with expressed shRNA in combination with ribozyme and TAR decoy is underway.ribozyme and TAR decoy is underway.
•• Alternative strategies for combinatorial Alternative strategies for combinatorial targeting of HIV include long hairpins, targeting of HIV include long hairpins, multicistronicmulticistronic microRNA mimics, microRNA mimics, shRNAshRNA--M10 fusion, and gp120 aptamerM10 fusion, and gp120 aptamer--siRNA chimeric molecules.siRNA chimeric molecules.
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Acknowledgments
• Mingjie Li• Nancy Lee• Alessandro Michienzi• Laurence Cagnon• Lisa Scherer• Daniela Castanotto• Haitang Li
• John Zaia• Amrita Krishnan• Larry Couture• David Hsu• Dave DiGiusto
• Ramesh Akkina-CSu
• Benitec Limited.• NIH NIAID
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Dr Rossi presents update on HIV lymphoma study at Keystone meeting