1104 argonaute 2 is a key limiting factor for therapeutic RNAi in the liver

1104 Argonaute 2 Is a Key Limiting Factor for Therapeutic RNAi in the Liver triggers than our I" first generation trigger, HBVU6#2 Khovorova et al and Schwarz et al demonstrated that an siRNA''''s intern[.]

triggers than our I" first generation trigger, HBVU6#2 Khovorova

et al.and Schwarzet al demonstrated that an siRNA's

internalther-modynamic stability profile (ISP) determines whether the desired

antisense strandis efficiently incorporated into the RNA Induced

SilencingComplex (RISC) Weusedthe webtool,SFOLD to identify

HBV RNAi triggers most closelyconforming to the ISPdescribed by

Khovorovaet al.Wethen incorporated GU base pairs into the sense

strand that altered the thermodynamic profileto more closely match

the consensus.These triggers were designed to conform to reported

sequence preferences for efficient RNAi described by Reynoldset

at Wealso selected triggers that were predicted by SFOLD to target

regions in HBV RNAs that are accessible to hybridization RNAi

triggers embedded in endogenous miRNA scaffolds are more

effi-ciently processed into mature siRNAs than shRNAs Therefore,we

expressed our HBV RNAi triggers in the context ofthe endogenous

miRNA,miR30.This will also allow expression using liver-specific

and inducible promoters 14 out of 15 of the rationally designed

HBV RNAi triggers were more potent in cultured cells than our

I" generation construct, HBVU6#2 Northern blots indicated that

our rationally designed RNAi triggers favored incorporation of the

desired antisense guide strand into the RISC complex However,our

data also suggest that other downstream parameters

influencesilenc-ing efficiency These results demonstrate that rational approaches

can be used to reliably design more potent RNAi triggers Studies

with these optimized triggers in mice will be discussed Because

ofthe general nature of these approaches,they could be adapted to

the treatment of diverse infections and diseases

1102 Novel "Bifunctional" shRNA Knockdown

Therapeutics to Individualized Cancer Targets

John 1.Nemunaitis.P" Phillip B Maples.P Donald Rao,' Joseph

Kuhn,'Yuqiao Shen.! AlexW Tong.t-'Chris Jay",2Padmasini

Kumar,' NeilSenzer.I 2,3,~

[Cancer Research, Mary Crowley Cancer Research Centers ,

Dal-las, TX; 2Cancer Research, Murex Pharmaceuticals , lnc , Dallas,

TX; JCancer Research, Baylor Sammons Cancer Center; Dallas,

TX; 'Cancer Research, Texas Oncology;I~A , Dallas, TX

We have created a novel process of identifying cancer specific

and,potentially critical,molecular targets in individual patients and

have constructed corresponding RNA interference therapeutics to

test clinical impact Malignant and non malignant tissue from 81

cancer patients were harvested.Differentially overexpressed

pro-tein and correlated mRNA signals were determined (mRNAArray,

UTSWMC,Dallas, TX and 2D DIGE/MS;Applied Biomics,San

Francisco,CA) in 32 of these patients Gene expression pathway

modeling(GNS,Boston,MA) of prioritizedproteinswith pro-cancer

function overexpressed in cancer allowed for determination of intra

pathway proteinconnectivity in IS patients Novel"bifunctional"

shRNA constructs,which demonstrate both cleavage dependent

and cleavage independent RISC (RNA induced silencing complex)

mediated inhibition of the targeted homologous mRNA, were

con-structed and tested The "bifunctional" shRNA has produced up to

a 260 percent enhanced knockdown of the target protein (compared

to traditional siRNA knockdown to the same sequence at optimal

dose),at 48 hours after clonal cancer cell exposure Furthermore,

consequent functional modulation (e.g., apoptosis) was observed

Duration of target knockdown, measured by sequential Western

blot and alkaline phosphatase reporter assay, demonstrated a further

temporal advantage of bifunctional shRNA over siRNA (at optimal

doses) beyond 48 hours Subsequent treatment of malignant clonal

cells with known high expression ofthe target protein confirmedi

n-creased cell death over time followingexposure to the "bifunctional"

shRNA A clinical grade plasmid containing a unique promoter for

tumorspecificcontrolledshRNAexpression was engineered.Animal

efficacy and toxicology testing are underway using the target vector

MolecularTherapy Volume 15 ,SupplementI May2007

Copyrig ht © Th eAmericanSocietyo fG ene Therapy

packaged in a tumor antigen targeting immunoliposome nanopar-ticle for delivery.The clinicallND justifying patient treatment is in process Update of results will be presented

1103 Using Let-7 microRNA To Inhibit Pancreatic Cancer Cells Proliferation

Laurie Parmentier,'JeromeTorrisani, 'BarbaraBournet ,'Anny

Barthet,"N.Lesavre,' Philippe Ruszniewski,' DermotO'Toole,'

A.Aubert,'Pascal Hammel,' Philippe Levy,' Janick Selves," Lucien Pradayrol,I Louis Buseail,IPierre Cordclicr.'

IINSERM U858, 12MR, Toulouse , France; 2 Service de Gas -troenterologie , CflU Rangueil, Toulouse, France; 'Service de Gastroenterologie , Val d'Aurelle , Montpelliet; France; 'Service

de Gastroenterologie, CHU Nord et CIG, Marseille, France; ' Service de Gastroenterologie, Hopital Seal/jon, Clichy; France;

6 1NSERM U563, CHU Purpan, Toulouse, France.

Background; pancreatic ductal carcinoma is the fifth leading cause of cancer related deaths in Westerncountries, with an increas-ing incidence over the last 40 years So far, no effective therapies have been established to alleviate this devasting and often fatal end-stage condition Thus, there is an urgent need for the development

of new modalities to improve diagnosis and treatment of pancreatic

cancer MicroRNAs (miR) are small non-coding transcripts that regulate gene expression by inhibiting translation Emerging stud-ies suggest that many miRNAs may participate in human disease, including oncogenesis The aim of present work was to establish the pattern of expression of miR known to potentiate or to inhibit oncogenesis in PC-derived cell lines,tumors and fine needle aspi

-ration (FNA) Methods: Total RNA was purified from PC-derived cell lines, tumors and FNA RNA collected by aspiration was am-plified using Full Spectrum amplification kit As controls,we used RNA purified from normal,adjacent pancreas to pancreatic cancer miR levels were ascertained by Taqman RT-PCR Results: using

Taqman-based RT-PCR, we identified a global down regulation of anti-oncogenicmiR (miR-21,miR-22I,miR-222,miR-16,miR-15a, miR 17-5-p, Let-7a) in pancreatic cancer samples,when oncogenic miR were elevated (miR-211,mir-30I) Let-7 miR down regulation was further confirmed by Northern blotting andin situ RT-PCR

on pancreatic adenocarcinoma tissue micro array Eventually, we identified a global down expression of let-7 miR family members

in 30 FNA of pancreatic cancer tumors Because ki-RAS oncogene

is a validated target of Let-7 miR,we transfected pancreatic cancer

-derived cells with Let-? miR We observed a dramatic decrease in PC-derived cells proliferation,with a eoncomittant inhibition ofk

i-RAS protein expression Conclusion: a better understanding of the molecular mechanisms involved in pancreatic cancer oncogenesis could lead to the development of new molecular markers for either early diagnosis or targeted therapy Here we show that anti-onco-genic miR expression, such as let-7, is strongly down regulated in pancreatic cancer samples Taken together, this study describes the potential clinical andtherapeutic interest of delivering Let-7 for treating pancreatic cancer

1104 Argonaute-2 Is a Key Limiting Factor for Therapeutic RNAi in the Liver

Dirk Grimm, Lora Wang, Joyce S Lee,Theresa A Storm, Mark

A.Kay

I Pediatrics and Genetics, Stanford University, Stanford, CA.

We recently reported efficient and persistent suppression of Hepatitis B virus (HBV) in mice using double-stranded AAV-8 vectors expressing anti-HBV shRNAs A striking finding was that shRNA over-expression from a robust U6 promoter frequently caused hepatocellular toxicity, rapid organ failure and lethality

S421

Comprehensive analyses of various shRNAs and targets implied

es-pecially the sh/miRNA transporter Exportin-5 Here, we show

that compared to U6, shRNA expression from weaker HI or 7SK

promoters typically induces delayed but more persistent RNAi in

mice This substantiates the correlation of intra-cellular shRNA

levels and cytotoxicity We then tried to actively rescue mice from

it was only transient and the toxicity phenotype actually manifested

more rapidly This suggested that a second factor downstream in

the RNAi pathway had become limiting due to abundant Exportin-5

with robust shRNA constructs and plasm ids expressing key elements

competition with the active RISC complex in the cell Remarkably,

RISC which cleaves the target mRNA) enhanced RNAi activity

by at least 5-fold in vitro and in vivo, even with our most efficient

shRNAs The increase was greater than with Exportin-S, implying

that Argonaute-2 is the main limiting factor in the RNAi pathway

in mammalian cells and in murine liver To study the long-term

transgenic for human alpha-l-antitrypsin (hAAT), together with a

robust U6-anti-hAAT shRNA Control mice expressing only the

shRNA showed a transient knockdown that peaked at week 2 and

striking contrast, Argonaute-2 co-expressing mice continued to show

a stable 20-fold hAAT knockdown for over I month Mice expressing

Exportin-5 together with Argonaute-2 showed an even further 2-fold

the liver will be restricted by at least two endogenous factors

Be-cause the more limiting, Argonaute-2, is utilized by all conventional

a broad and general impact Critical to the success of therapeutic

liver RNAi will be to avoid Exportin/Argonaute saturation, either

by tightly controlling intra-cellular hairpin or siRNA concentrations

to co-express the limiting factors together with the shRNA Such

combinatorial strategies could broaden the therapeutic index of

1105 Prevention and Reversal of Autoimmune

Diabetes by Microsphere-Formulated Antisense

Oligonucleotiudes Targeting Costimulation

Gi-annoukakis.'

I Diabetes Institute University of Pittsburgh School ofMedicine.

Pittsburgh, PA ; 2 Epic Therapeutics Baxter Health Care

Corpora-tion Norwood, MA

Prevention and reversal ofautoimmune diabetes by

microsphere-formulated antisense oligonucleotiudes targeting costimulation

by a chronic autoimmune inflammation of the pancreatic islets of

Langerhans The inflammation compels migratory antigen

present-ing cells, dendritic cells most prominently, to acquire beta

cell-resident antigens derived from apoptotic and/or necrotic beta cells

The migratory dendritic cells then undergo an intrinsic "maturation"

program which renders them capable ofactivating T-cells (including

S422

the draining pancreatic lymph nodes We have previously shown that administration of dendritic cells with low-level expression of CD40, COSO and CDS6 costimulatory molecules into the geneti-cally-diabetic non-obese diabetic (NOD) mouse can considerably delay and prevent the onset of disease and can reverse new-onset disease Considerable logistics to generate these dendritic cell

in vivo Microparticle carriers can direct DC to the administration

unc-tional phenotype We have adapted PROMAXXTM microspheres

to formulate antisense oligonucleotides previously shown to render autologous dendritic cells diabetes-suppressive This formulation, when injected subcutaneously prevented type I diabetes in the non-obese diabetic (NOD) mouse strain and most importantly, exhibited

a capacity to reverse new-onset clinical hyperglycemia, suggesting reversal of new onset disease Mechanistically, we demonstrate that the formulation augments CD25+ Foxp3+ T regulatory cells which confer suppressive activity in vitro to NOD-derived pancreatic beta cell antigen without compromising global immune response to allo-antigen and nominal allo-antigen Furthermore, T-cells from diabetes-free recipients of the microspheres are capable of suppressing adoptive transfer of disease by diabetogenic splenocytes into secondary immunodeficient recipients We believe that these data form the foundation upon which a bona fide type I diabetes negative vaccine can be developed that is logistically-simple to produce and rapidly scalable to population levels

STEM CELLS - BASIC SCIENCE SESSION

1106 High Proteasome Activity Is a Common Feature among Stem Cells of Different Lineages and Restricts Lentiviral Gene Transfer

'Telethon Institute for Gene Therapy (TIGET), San Raffaele Sci-entific Institute, Milan, Italy; 21ljta-Salute University, San Raffaele Scientific Institute, Milan, Italy; 'Schoot of lIeterinmy Medicine, University ofTurin Turin, Italy; 'Stem Cell Research Institute, San Raffaele Scientific Institute, Milan, Italy; s Reproductive Technologies Laboratory; Istituto Sperlmentale L Spallanzani, Cremona, Italy.

An emerging field of studies has demonstrated that mammalian cells express factors that restrict retrovirus and retrovirus-derived vector infection Although Hematopoietic Stem Cells (HSC) can be

transduced by HIV-derived lentiviral vectors (LV) in short ex vivo

cytokine stimulation and high vector doses to reach high-frequency transduction We demonstrated that the use ofproteasome inhibitors

-ing reach-ing very high levels of vector integration in their progeny

in vivo, without apparent detrimental effect on their engraftment

and the molecular mechanism ofthis restriction Surprisingly, among

(ESC), murine mesenchimal stem cells (MSC) and murine neural stem cells (NSC) Remarkably, in cells differentiated from HSC

observed restriction seems to be a characteristic feature of stem

protea-Molecul ar Therapy Yofum e 15 Supplement I, \by 2007

Co pyright © '111C Am erican Socie tyo f G ene TI ICr.lpr