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

ut p

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

(pg/

ml)

Non-transduced

GFP-alone

Triple

-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

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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

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

ted C

EM +

Buffe

r

27 m

ersiR

NA21

mer

siRNA

Aptam

er

Ch L-

1Ch

1Ch

L-2

Ch 2

M-1

M-2

Infec

ted C

EMUn

infec

ted C

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

on (p

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

expr

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(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