835 self complementary adeno associated virus serotypes can induce efficient long term therapeutic effects in human cancer cells

835 Self Complementary Adeno Associated Virus Serotypes Can Induce Efficient Long Term Therapeutic Effects in Human Cancer Cells higher killing efficiency than T cell delivery (42 9 to 23 6% killing)[.]

higher killing efficiency than T cell delivery (42.9 to 23.6% killing).

In all cases the IFNy:IL-4ratios within the stimulated T cell

popula-tions(intr~cellular ~taining) was consistent with the level of killing

Thus the.introduction of both IL-2 and IL-12 into DC (paracrine

gene delivery) resulted in CTL with superior killing abilities than

cytokinc gcnc delivery directly into T cells (autocrinc) In fact the

delivery ofthe IL-2gene into T cells dramatically inhibited the

ef-ficicncy ofthc resultingstimulatedCTL.These data strongly suggest

that AAV/IL-2 and AAV/IL-12 cytokine delivery into DC may have

utility in immunogene therapy protocols However, these data also

suggest unforeseencomplexities in the mechanism ofaction orthese

Th I-response cytokines in stimulating, promoting, robust CTL

833 Efficient Whole Body Muscle Transduction

with Self-Complementary AAV 9 Vectors

Following Systemic Delivery in Adult Mice

Christel Riviere; ValerieAllo,' Karine Poulard,' Patrice Deneflc,'

Anne M Douar.'

[R&D , Genethon, Evry; France

The challenge faced by viral vectors in gene therapy of

muscu-lar inherited diseases remains on targeting tissues throughout the

body with a single injection into the blood circulation leading to

widespread muscle gene transfer, Enhanced efficacyofAAVI based

vector followi.ng direct intramuscular administration was proved,

and a few studies demonstratedthat this serotype is able to transduce

skeletal and cardiac muscle following intravascular injection Based

on recent reports of the newly isolated serotype AAV9being able to

deliver gene to muscle, we intend to compare rAAVI and rAAV9

respectiveperformancein muscle followingsystemicadministration

In addition, we combined to capsid comparison the impact of self

complementary (sc) genome in these AAV vectors Normal adult

C57Bl/6 mice were injected with pseudotyped AAV vectors type I

and 9 expressing a secreted murine alkaline phosphatase (mSEAP)

under the control of'the ubiquitousCMV promoter,allowing a global

evaluation ofgene transferefficiencyby measuringprotein secretion

into the circulation Locally,gene transfer efficiency into organs and

muscles was evaluated using both qualitative histochemical

detec-tion and quantitative mSEAP assays in tissue Iysates In addidetec-tion

biodistribution was measured by vector genome copy quantification

in tissues of interest by real time PCR analysis A single dose of 3

x 10+11vg ofrAAVl and rAAV9, either single stranded (ss) or sc,

was injected into the tail vein.Equivalent circulating mSEAP levels

are achieved with ssAAVI and ssAAV9 vectors, whereas scAAV9

vectors yielded higher gene higher levels compared to scAAVI (4

folds) Tissue expression levels and patterns confirmed scAAV9

superiority with the observation of a widespread and robust

trans-duction pattern In addition to skeletal muscles,scAAV9 was also

able to deliver genes into multiple organs,especially the heart, but

also kidneys and lungs Vector DNA quantification is ongoing in

support ofthese analyses and will be presented.Altogether,scAAV9

vector appears as an improved and alternative gene transfer vector

especially in systemic approaches for the treatment of

neuromus-cular disorders

MolecularTherapyVolume 15 ~ Sup plement I May 2 007

Copyrig ht © T he American Socie ty o f G ene Th erapy

Oligodendrocytes Efficiently Shin-ichi Muramatsu,' Hiroko Nishida,'YukoNara,'Naomi Takino,ISayaka Asari,' Mika Kodera,IWei-zhongXiao.t-'Yasuo Sasaki.v'Satoru Kikuchi,':' Takashi Matushita,' Takashi Okada,' MinakoHoshi.v'Imaharu Nakano; KeiyaOzawa.'

'Neurology; Jichi Medical University, Shimotsuke, Japan; 2Ge-netic Therapeutics, Jichi Medical University, Shimotsuke, Japan;

JResearch Group for AlzheimersDisease, Mitsubishi Kagaku Institute ofLife Sciences, Machida, Japan; "Btological Injonna-tion, TOkyO Institute ofTeclmology, Nagatsuda, Japan

Adeno-associated virus (AAV) vectors have become one of the most popular vehicles for delivering therapeutic genes into the central nervous system, Vectors derived from serotype 2 (AAV2) transduce neuronsefficientlyand achieve long-termgene expression

~vith no apparent toxicity Several clinical trials of gene therapy

us-109 AAV2vectors are currently underway for neurological diseases, including Parkinson's disease and Batten disease, where neurons are the primary targets for gene delivery While AAV2 rarely transduces glial cells, transduction of oligodendrocytes is dcsir-able for diseases that affect myelin,such as multiple sclerosis and leukodystrophy.Among the various new AAV serotypes that have been cloned recently,serotype 8 (AAV8) is of particular interest because they are highly efficient gene transfer vehicles for many

organsand can transduce glial cells in the murine brain Weevalu-ated the transduction characteristics of AAV8 vectors in primary hippocampal cultures and in the adult rat brain AAV8 serotyped vectors encoded green fluorescent protein (GFP) under control ofa hybrid cytomegalovirus enhancer/chicken B-actin(CAG) promoter

or myelin basic protein (MBP) promoterflankedwithAAV2 inverted terminal repeats Mixed neuronal/glial cell cultures,prepared from the hippocampi of E17 Wistar Crj rats,were treated with AAV8 vectors with CAG promoter three days after plating at an estimated multiplicity of infection of lAx IO~ -GFP expression was evaluated three to ten days latcr Counterstaining with thc oligodendrocyte marker 0lig2 or neuronal marker MAP2 indicated that 39.1±2.6% ofOlig2-immunoreactive celIs were GFP-positive,while79A±9.l%

of MAP2-immunoreactive cells were also positive for GfP four-week-old male Wistar rats were injected with AAV8 vectors in the bilateral caudo-putaminal unit or collupus callosum (4x1010vector genomelbrain) Although the majority ofcells expressing GFP had neuronal morphology and were immunoreactive for the neuronal markerNeuN four weeks after injection ofAAV8 vectors with CAG promoter, some GfP-positive celIs in AAV8 vector-transduced brains showed glial morphology and were positive for oligoden-drocyte markers GfP-positive oligodenoligoden-drocytes were observed in the collupus callosum after injection of AAV8 vectors with MBP promoter AAV8vector may be useful for enhancing the efficacy of therapies for dysmylinating or demylinating diseases of the brain

835 Self-Complementary Adeno-Associated Virus Serotypes Can Induce Efficient Long-Term Therapeutic Effects in Human Cancer Cells Han Sacm Lee,' Oh Kyo Shin; Sung Jin Kim; Won II Lee,'Ji Yun Kim,'Sunjoo Jeong,'Kecrang Park,' Han Choc.s-'Heuiran Lee.':'

[Microbiology, University ofUlsan College ofMedicine , Seoul , Republic ofKorea ; "Phystology, University ofUlsan College of Medicine, Seoul, Republic ofKorea;JResearch Institute for Bio-macromolecules, University ofUlsan College ofMedicine, Seoul, Republic ofKorea ; "Molecuiar Biology, Colleg e ofNatural Sci-ences, Dankook University; Seoul, Republic ofKorea; sBiotech -nology, Juseong University Chung-Buk, Republic ofKorea.

The promising potential of recombinant adeno-associated virus (rAAV)as a gene delivery tool has been well documented in cancer

S319

gene therapy In addition,recent studies have shown that various

serotypes of self-complementary rAAV (scAAV) have advantages

in effectivelytransducingvariouscell/tissue types for long duration

Therefore, it would be important to examine the characteristicsof

transduction by differentscAAVscrotypes on various human cancer

cells from differenttissue origins.Thus,we investigatedthe features

of transductionby distinctscAAVserotypesin varioushuman cancer

cells.Then,we furtherexamined the long-termanti-tumoral effects

To dissect the transduction properties, we infected a variety of

hu-man cancer cells (hepatocellular Sk-Hep l, cervicalHel,a, colon

HCTI16, HT-29, lungA549, pancreatic Bx-PC3, and Pane-l, brain

glial U251) with scAAVI-6 or scAAV8 expressing GFP scAAV2

led to the best transductionefficiencyof nearly complete transgene

expressionat 1000MOl in mostcancercells,

regardlessoftissueori-gins scAAV5could induce effective gene expression, even though

gene transfer potency by scAAV5 was poorer than that by scAAV2

Substantial portion of transgene expressionlasted over a month

following gene delivery by both scAAV2 and scAAV5,indicating

that long-term gene expression can occur To validate anti-tumoral

effects,we constructedscAAV encodingHSV1-TK,

transducedSK-HepI cells, and examined the degree of cytotoxicity in the presence

of ganciclovir; SK-HepI expressing HSV1-TK sharply lost cell

vi-ability even on over 20 days post-infection, concomitantly with the

sustained expression of I-1SV 1-TK protein Moreover, co-infection

ofscAAV2 and seAAV5 could induce simultaneous expressions of

transgenes introduced via each vector.Therefore, the current study

providesrationalethat scAAV2 andscAAV5 vectorscan be excellent

gene transfer tools for cancer gene therapy, independently driving

persistent transgene expression

836 Seropositivity Against AAV Serotypes 1, 8

and 9 in Cynomolgus Monkey Colonies

Hiroaki Mizukami,'Akira Ishiwata.?Fumiko Ono,' Jun-ichiro

Takano,'Koji Fujimoto,'Jun Mirnuro.! Masashi Urabe,'Akihiro

Kume,'Keiji Terao,'YoichiSakata,' Keiya Ozawa:

'Div ofGenetic Therapeutics, Jichi Medical University ;

Shi-motsuke , Tochigi, Japan ; lDiv ofCell and Molecular Medicine.

Jichi Medical University, Shimotsuke, Tochigi , Japan; "Isukuba

Primate Research Center; National Institute0/Biomedical

In-novation Tsukuba, Ibaraki, Japan.

AAV vectors, especially derived from serotypes 8 and 9, hold

promise in clinical gene therapy In experiments with cynomolgus

macaques,we recognized high prevalence of neutralizing antibody

against capsid ofthese serotypes In order to carry out gene therapy

experiments using these vectorssuccessfully,selection

ofseronega-tive animals has a vital importance.Moreover, elimination ofthese

viruses from the colony would be ideal for preclinical studies In

Tsukuba Primate Research Center, specific pathogen free (SPF)

projects have been performed against various microorganisms

since 1978.As a result,a variety of pathogens has been eliminated

from the colonies,including simian B virus,simian varicella virus,

simian immunodeficiencyvirus and simian T Iymphotropievirus I

Currently,one colony is being developed for experiments using

ex-tensive immunosuppression,with the emphasis of excluding simian

D retrovirus,simian cytomegalovirus, simianEll virus and simian

foamy virus None of the AAVswas considered as a target of this

SPF project In this study,we compared the positivity of

neutral-izingantibody againstAAV serotypes 1,8 and 9 between the colony

(termed SPF) and a standard colony as a control By analyzing 10

animals from each group, significant decrease of seropositivity in

SPF group for both AAV8 and 9 (6 to I and 4 to I, respectively)

was observed Interestingly, positivity of AAVI was low in both

groups (I to I),and the animals seropositive for AAVI were also

positive for AAV8and 9.Analysis of the rest of the animals is now

underway and the results will be included Taken together,current

S320

SPF project concerningthe above virusesare alsoeffectiveto reduce the prevalenceofthese serotypes in the colony,which adds the value

of animals for preclinical experiments ofgene therapy

837 Long-Term Effect and Biosafety of Recombinant AAV-Rat Cygb Expression Ruian XuY XinyanLVPhillip Harriosn,' WeidongXiao,"Nagy Habib,' FarzinFarzaneh,"

'Institute ofMolecular Medicine , Huaqiao University Fujian, China; lGRC, Hong Kong University Hong Kong, China; JDe _

partment0/Liver Studies and Transplantation, KingsCollege London , United Kingdom; "Department0/Pediatrics University

0/Pennsylvania, Philadelphia, I'll; sDepartment ofTransplanta-tion Imperial College, London United Kingdom; 6Department0/

Haematological & Molecular Medicine, KingsCollege, London, United Kingdom.

To estimate potential of cytoglubin(Cygb) application in human liver fibrosis therapy, A new set of experiments to monitor the long-termeffect and safety was established CCl4-ratswere injected intraportallywith 3x10II I'AAVIrCygband I'AAVleG FP respectively (n=5)after completingthe 8-wcckcourseofCCI4injections Animals were subjected to another four weeks consectivcCCI4 induction, and then were kept under the normal condition for 40 weeks prior

to sacrifice A group of normal served as control We found that as previous report for CCI4induced animals (Trivedi & Nowat 1983) all examined animals appeared normal in gross appearance and be-haviour.All animals survived well except that CCI4-eGFPin which two rats were death during the experimental period No tumour or abnormal appearance was found in CCI4-rSTAPgroup There was not significantdifference in body weight among three experimental groups,a substantialaccumulationoffat in abdominalcavity of both CCI.-eGFP and CC14-Cygbgroups but not in normal group Previ-ous investigators noted that side effect of CCI induction resulted

in an increase in fataccumulation in induced animals.Todetermine actual effect of induction of CCl4and STAP expression on liver structure,Sectionsoflivertissues from differentgroup were subject histology and imrnuno-staining analysis,administration of rAAV

IrCygb significantly attenuated liver damage and fibrosis There

were still signs of fibrosis in rAAV/rCygbgroup, but accumulative collagen network can not be found in all sections of rAAV-Cygb group Furthermore, histological sections of livers revealed that all rAAV-Cygb had been healing,although complete resolution of fibrosis at the cnd is not clear In contrast,accumulative collagen network still can be found in the all scetions of CCI4-eGFPhad a characteristic appearance, i.e.were enlarged,hard and nodular due

to widespreadhepaticfibrosis after discontinuationoftreatment with CCI for 40 weeks Hydroxyproline content for normal group was

0.268 ±0.05mg/gliver tissue for normal, 0.309±0.05Img/g liver tissue for rAAV/STAP group and 0.387±0.06 mg/g liver tissue for CCI.-eGFP Taking all data together, Cygb might be a promising agent for liver fibrosis therapy

838 AAV2 Capsid Specific CTLs Do Not Eliminate AAV Vector Transduced CellsIn Vivo

Chengwen Li,' Matt Hirsch,IAravind Asokan,'Brian Zeithaml,' Hong Ma,' Tal Kafri,':' Richard Jude Samulski.'?

'Gene Therapy Center, UNC at Chapel Hill Chapel Hill, NC;

' Department ofMicrobiology and lmmunology; UNC at Chapel Hill Chapel Hill , NC; 'Department ofPharmacology; UNC at Chapel Hill Chapel Hill, NC.

Adeno-associated virus (AAV) vector can initiate long-term transgene expression in pre-clinical experiments and has been ap-plied in over 20 clinical trials Recent studies have demonstrated that AAV capsid expression and AAV vector transduction in vivo