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~taining) was consistent with the level of killing tions (intr~cellular 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-2 gene into T cells dramatically inhibited the efficicncy ofthc resultingstimulated CTL 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 unforeseen complexities in the mechanism ofaction orthese Th I-response cytokines in stimulating, promoting, robust CTL 833 Efficient Whole Body Muscle Transduction with Self-Complementary AAV 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 muscular 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 demonstrated that 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 expressing a secreted murine alkaline phosphatase (mSEAP) under the control of'the ubiquitousCMV promoter,allowing a global evaluation ofgene transfer efficiencyby measuring protein secretion into the circulation Locally,gene transfer efficiency into organs and muscles was evaluated using both qualitative histochemical detection and quantitative mSEAP assays in tissue Iysates In addition biodistribution was measured by vector genome copy quantification in tissues of interest by real time PCR analysis A single dose of x 10+ 11 vg 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 transduction 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 neuromuscular disorders 834 AAV8 Vectors Transduce Oligodendrocytes Efficiently Shin-ichi Muramatsu,' Hiroko Nishida,' Yuko Nara,' Naomi Takino,I Sayaka Asari,' Mika Kodera,I Wei-zhong Xiao.t-' Yasuo Sasaki.v' Satoru Kikuchi,':' Takashi Matushita,' Takashi Okada,' Minako Hoshi.v' Imaharu Nakano; Keiya Ozawa.' 'Neurology; Jichi Medical University, Shimotsuke, Japan; 2Genetic Therapeutics, Jichi Medical University, Shimotsuke, Japan; J Research Group for Alzheimer s Disease, Mitsubishi Kagaku Institute ofLife Sciences, Machida, Japan; "Btological Injonnation, 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 (AAV2) transduce neurons efficientlyand achieve long-termgene expression ~vith no apparent toxicity Several clinical trials of gene therapy us109 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 dcsirable for diseases that affect myelin, such as multiple sclerosis and leukodystrophy Among the various new AAV serotypes that have been cloned recently, serotype (AAV8) is of particular interest because they are highly efficient gene transfer vehicles for many organs and can transduce glial cells in the murine brain Weevaluated 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) promoter flankedwithAAV2 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 I O~ - 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, while 79A±9.l % of MAP2-immunoreactive cells were also positive for GfP fourweek-old male Wistar rats were injected with AAV8 vectors in the bilateral caudo-putaminal unit or collupus callosum (4 x 1010 vector genomelbrain) Although the majority of cells expressing GFP had neuronal morphology and were immunoreactive for the neuronal marker NeuN four weeks after injection of AAV8 vectors with CAG promoter, some GfP-positive celIs in AAV8 vector-transduced brains showed glial morphology and were positive for oligodendrocyte markers GfP-positive oligodendrocytes 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; J Research Institute for Biomacromolecules, University ofUlsan College ofMedicine, Seoul, Republic ofKorea ; "Molecuiar Biology, College ofNatural Sciences, Dankook University; Seoul, Republic ofKorea; sBiotechnology, 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 Molecular Therapy Volume 15 ~ Supplement I May2007 Copyright © T he American Society of G ene Th erapy S319 gene therapy In addition, recent studies have shown that various serotypes of self-complementary rAAV (scAAV) have advantages in effectivelytransducing various cell/tissue types for long duration Therefore, it would be important to examine the characteristics of transduction by differentscAAV scrotypes on various human cancer cells from differenttissue origins.Thus, we investigatedthe features of transductionby distinctscAAV serotypes in varioushuman cancer cells Then, we furtherexamined the long-termanti-tumoral effects To dissect the transduction properties, we infected a variety of human cancer cells (hepatocellular Sk-Hep l, cervical Hel,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 transduction efficiency of nearly complete transgene expressionat 1000MOl in most cancercells, regardlessoftissueorigins scAAV5could induce effective gene expression, even though gene transfer potency by scAAV5 was poorer than that by scAAV2 Substantial portion of transgene expression lasted 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 encoding HSV1-TK, transducedSKHepI cells, and examined the degree of cytotoxicity in the presence of ganciclovir; SK-Hep I expressing HSV1-TK sharply lost cell viability 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 and scAAV5 vectorscan be excellent gene transfer tools for cancer gene therapy, independently driving persistent transgene expression 836 Seropositivity Against AAV Serotypes 1, and in Cynomolgus Monkey Colonies Hiroaki Mizukami,' Akira Ishiwata.? Fumiko Ono,' Jun-ichiro Takano,' Koji Fujimoto,' Jun Mirnuro.! Masashi Urabe,' Akihiro Kume,' Keiji Terao,' Yoichi Sakata,' Keiya Ozawa: 'Div ofGenetic Therapeutics, Jichi Medical University; Shimotsuke, Tochigi, Japan; lDiv ofCell and Molecular Medicine Jichi Medical University, Shimotsuke, Tochigi, Japan; "Isukuba Primate Research Center; National Institute 0/Biomedical Innovation Tsukuba, Ibaraki, Japan AAV vectors, especially derived from serotypes 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 vectors successfully,selection ofseronegative 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 extensive immunosuppression,with the emphasis of excluding simian D retrovirus, simian cytomegalovirus, simian Ell virus and simian foamy virus None of the AAVs was considered as a target of this SPF project In this study, we compared the positivity of neutralizingantibody againstAAV serotypes 1,8 and 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 (6 to I and 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 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 also effective to reduce the prevalenceofthese serotypes in the colony,which adds the value of animals for preclinical experiments of gene therapy 837 Long-Term Effect and Biosafety of Recombinant AAV-Rat Cygb Expression Ruian XuY Xinyan LV Phillip Harriosn,' WeidongXiao,"Nagy Habib,' Farzin Farzaneh," 'Institute ofMolecular Medicine, Huaqiao University Fujian, China; lGRC, Hong Kong University Hong Kong, China; JDe_ partment 0/ Liver Studies and Transplantation, King s College London, United Kingdom; "Department 0/ Pediatrics University 0/Pennsylvania, Philadelphia, I'll; sDepartment ofTransplantation Imperial College, London United Kingdom; 6Department 0/ Haematological & Molecular Medicine, King s College, 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-rats were injected intraportallywith 3x10II I'AAVIrCygb and I'AAVleG FP respectively (n=5)after completingthe 8-wcckcourseofCCI injections Animals were subjected to another four weeks consectivc CCI4 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 CCI4 induced animals (Trivedi & Nowat 1983) all examined animals appeared normal in gross appearance and behaviour.All animals survived well except that CCI4-eGFP in which two rats were death during the experimental period No tumour or abnormal appearance was found in CCI4-rSTAPgroup There was not significant difference in body weight among three experimental groups, a substantialaccumulationoffat in abdominal cavity of both CCI.-eGFP and CC1 4-Cygb groups but not in normal group Previous investigators noted that side effect of CCI induction resulted in an increase in fat accumulation in induced animals To determine actual effect of induction of CCl4 and STAP expression on liver structure, Sections oflivertissues 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-eGFP had a characteristic appearance, i.e were enlarged, hard and nodular due to widespreadhepatic fibrosis after discontinuationoftreatment with CCI for 40 weeks Hydroxyproline content for normal group was 0.268 ±0.05mg/g liver 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 Cells In Vivo Chengwen Li,' Matt Hirsch, I Aravind Asokan,' Brian Zeithaml,' Hong Ma,' Tal Kafri,':' Richard Jude Samulski.'? 'Gene Therapy Center, UNC at Chapel Hill Chapel Hill, NC; ' Dep artment 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 applied in over 20 clinical trials Recent studies have demonstrated that AAV capsid expression and AAV vector transduction in vivo Molecular Therapy Volume15.Supplement t, \by 2007 Copyright © '111C American Society of Gene Therapy ... distinctscAAV serotypes in varioushuman cancer cells Then, we furtherexamined the long- termanti-tumoral effects To dissect the transduction properties, we infected a variety of human cancer cells (hepatocellular... groups but not in normal group Previous investigators noted that side effect of CCI induction resulted in an increase in fat accumulation in induced animals To determine actual effect of induction... Chapel Hill Chapel Hill, NC Adeno- associated virus (AAV) vector can initiate long- term transgene expression in pre-clinical experiments and has been applied in over 20 clinical trials Recent studies