BSGT 2009 Poster Presentations

BSGT 2009 Poster Presentations

P 1

Studies on Expanded C-Termini Microdystrophin GeneTransfer Using rAAV9 Vectors in Skeletal Muscleof Dystrophin mdx Mice

Koo TY (presenting), Athanasopoulos P, Foster H, Graham I,Dickson G

Centre for Biomedical Sciences, School of Biological Sciences,Royal Holloway University of London, Egham, Surrey, UK

Duchenne Muscular Dystrophy (DMD) is a severe in-herited muscle-wasting disease and is caused by mutationsin members of a multi-component protein structure called thedystrophin protein complex (DPC). Previous studies haveattempted to transfer terminally truncated microdystrophins,however the role of C-terminal domain (CT) is yet to be de-lineated. CT domain of dystrophin recruits dystrophin asso-ciated protein complex (DPC) to form a transmembrane linkand perhaps act as mediator of signalling between extracel-lular matrix and cytoskeleton in muscle fiber. In this study,codon-optimised mouse micro-dystrophin cDNAs have beenexpanded at the C-terminal region (variants; JC= CC1, CC2,and CC3) allowing for specific=functional micro-dissectionof particular dystrophin regions (e.g., dystrophin regionalassociation=localisation studies of syntrophin, dystrobrevinand nNOS binding). These variants have been engineered inAAV plasmid vectors under SPc5=12 optimal muscle-specificpromoter, Kozak sequences and domain configurations. Wehave detected the localization of microdystrophin and part ofthe DPC at the sarcolemma of the skeletal muscle from mdxmice by western blotting and immunohistochemistry. Re-cently reported AAV 9 vector expressing C-terminal ex-tended microdystrophin variants are currently subjected tocomparative evaluation in mdx mice by intramuscular injec-tion. Complementation of dystrophin deficiency, transgenestability in muscle tissues and host immune modulation willbe monitored with the objective of developing safe and ef-fective muscle gene therapy protocols appropriate to moveforward towards clinical trials. The current study will presentthe role of C-terminal domain of dystrophin protein torestore=assist the function of dystrophin in DMD.

P 2

Long-Term Changes in Uterine Blood Flow andVascular Reactivity in Pregnant Sheep Following

Local Delivery of an Adenovirus Encoding VEGFto the Uterine Arteries

Mehta V (presenting)1, Abi-Nader K1, Torondel B2, WigleyV1, Tezcan B1, Fillipi E1, Petts G1, Boyd M3, Zachary I2,Martin J2, Peebles DM1, David AL1

1Institute for Women’s Health, University College London,London, United Kingdom, WC1E 6HX2Department of Cardiovascular Medicine, University CollegeLondon, London, United Kingdom3Biological Sciences Unit, Royal Veterinary College, London,United Kingdom, NW1 0TU

Impaired utero-placental perfusion leads to fetal growthrestriction (FGR), a challenging obstetric complication asso-ciated with high morbidity and mortality. Previously weshowed a significant increase in the uterine artery blood flow(UABF) and significant changes in the vascular reactivity ofpregnant sheep uterine arteries (UA) 4-7 days after local ad-enovirus vector mediated over-expression of VEGF. Thecurrent study investigated the long-term effects.

UABF was measured objectively using chronically im-planted flow probes placed around both the uterine arteries(n¼ 9) of mid-gestation pregnant sheep (90 days of gestation,term¼ 145 days). Maternal blood pressure was measuredusing a carotid artery pressure-sensitive catheter. BaselineUABF and haemodynamic values were recorded for oneweek. We then injected 5�1011 particles of adenovirus en-coding either the VEGF-A165 gene (Ad.VEGF-A165) or a con-trol non-vasoreactive b-galactosidase gene (Ad.LacZ) intoeach UA. UABF and maternal haemodynamics were mea-sured daily until term.

The percentage increase in blood flow from baseline inthe Ad.VEGF injected UAs was significantly greater than in theAd.LacZ injected UAs (39.57% vs. 16.01%, p< 0.05). In theAd.VEGF transduced vessels there was a diminished contrac-tile response to Phenylephrine (Emax 155.7� 13.65 vs. Emax

180.7� 10.39, p< 0.05) and significantly more vessels observedin the perivascular adventitia (n¼ 28, 11.64 vs. 8.75, p< 0.05).There were no significant changes in maternal haemodynamics.

Significant changes in UABF, vascular responses and re-modelling occur after local overexpression of VEGF longterm. We are investigating whether this effect could be usedto increase fetal size in growth restricted fetal guinea pigs,and therefore as a potential therapy for FGR.

P 3

A VAVgc Self-Inactivating Lentiviral Vector for the Gene

Therapy of X-Linked Severe Combined Immunodeficiency(SCID-X1)

Almarza E (presenting)1, Thornhill SI1, Howe S1, BlundellM1, Zhang F1, Bueren JA2, Thrasher AJ1

1Molecular Immunology Unit, Institute of Child Health, 30Guilford Street, WC1N 1EH, London, UK2Hematopoietic Gene Therapy Division,CIEMAT, Av.Complutense 22, 28040, Madrid, Spain

HUMAN GENE THERAPY 20:396–422 (April 2009)ª Mary Ann Liebert, Inc.DOI: 10.1089=hum.2009.1033

396

The efficacy of gene therapy for the treatment of SCID-X1has been demonstrated by the success obtained in clinicaltrials. However, it has been shown that there is a significantrisk of insertional mutagenesis due to transactivation ofnearby oncogenes through the strong promoter of the inte-grated vector. In the case of SCID-X1, modest expression ofthe transgene is likely to sustain the therapeutic effect, so theuse of promoters with a weaker activity and a reduced ca-pacity for gene trans-activation may be a safer alternative.The expression of the vav proto-oncogene is regulated by fiveDNAse hypersensitive sites (HS). The HS1 consists of theproximal promoter and mediates weak, homogeneous andstable expression of linked transgenes not only in cell linesin vitro but also in all hematopoietic lineages in vivo. It hasalso been shown that the vav promoter, although containinghigh number of CpG dinucleotides, is not susceptible tomethylation. We therefore generated self-inactivating lenti-viral vectors expressing the IL2RG under the control of thevav promoter. We observed that the vav promoter is suffi-cient to drive expression of IL2RG, enough to restore thecommon cytokine receptor gamma chain (gc)-mediatedsignaling in a SCID-X1 T-cell line. Moreover, the SINLVVAVIL2Rg is able to restore T and B cell populations andlymphocyte function in a SCID-X1 murine model followingex vivo gene therapy. With these results we suggest that thevav promoter may be a good alternative for the gene ther-apy of SCID-X1. The weak activity of this promoter woulddecrease the potential for dangerous insertional mutagenesiswhilst maintaining the therapeutic efficacy.

P 4

Gene Enhancement Magnetic Resonance Imaging

Holand T (presenting)1, Brewin J1, Kyrtatos P2, Price A2,Lythgoe M2, Qasim W1

1MIU, Institute of Child Health, London2Centre for Advanced Biomedical Imaging, University CollegeLondon

Magnetic resonance (MR) imaging is routinely enhancedthrough the administration of exogenous contrast agent, andsuperparamagnetic iron oxide (SPIO) particles have beenwidely used to label and track cells in vivo, both in animalsmodels and in humans. This approach is time-limited, as thecontrast agent is lost as cells divide. An alternative genetransfer approach could allow longer term tracking of cells.Previously Adenoviral vectors encoding heavy ferritin (FH)and light ferritin (FL) have been shown to enhance contrastfollowing intra-cerebral injection. For the purpose of modify-ing haematopoietic lineage cells, we have generated lentiviraldelivery systems, to compare FH, FL and an enhanced mutantof the ferritin light chain (FLmut). The vectors have a selfinactivating configuration and encode an internal SpleenFocus Forming Virus (SFFV) promoter. Expression of MRenhancement genes was linked to enhanced green fluorescentprotein, with gene expression being confirmed by flow cy-tometry and Western Blot analysis. The ability of these vec-tors to enhance MR signal was investigated in human:murinetumour engraftment models. Epstein Barr Virus transformedB-cell lines were transduced and expanded before subcuta-neous injection of 107 cells into the flanks of 6-week-old

NOD=SCID B2m� immunodeficient mice. Tumours werepalpable within 14 days. MRI measurements were performedon a 9.4 Tesla horizontal bore magnet. Contrast was found tobe enhanced in tumours formed by cells transduced with theHF gene. The technology will be useful for the serial imagingof tumour responses in a translational imaging system.

P 5

Long-Term Correction of Experimental Diabetes in Miceand Pigs After Nonviral Insulin Gene Therapy

Toporova OK (presenting), Gulko TP, Sukhorada OM,Ruban TP, Irodov DM, Kordium VA

Institute of Molecular Biology and Genetics of NAS of Ukraine,150, Zabolotnogo str., 03143, Kyiv, Ukraine

Background: Type 1 (insulin-dependent) diabetes resultsfrom the autoimmune destruction of pancreatic beta-cells inthe islets of Langerhans. Commonly employed therapy of Type1 diabetes requires lifelong insulin treatment , which does notprovide adequate glycemic control and cannot prevent dia-betes complications. Engineering of ectopic insulin synthesisin autologous non-beta cells is expected to overcome thisproblems and provide a steady supply of insulin.

Methods: The plasmid vector containing expression cas-sette for human preproinsulin gene has been constructed.The target gene is under the control of cytomegaloviruspromoter and flanked by inverted terminal repeats of humanadeno-associated virus 2. The hepatitis B viral enhancer 1 wassubcloned into the cassette to increase insulin expression.Diabetes was induced by administration of streptozotocinduring 5 days in mice (40 mg=kg ) and during 3 days in pigs(60 mg=kg). PEI=DNA polyplexes were used for intraliverdelivery of human preproinsulin gene.

Results: As a result of human insulin gene delivery intoliver cells of hyperglycemic animals, their serum glucose wasnormalized. All experimental animals exhibited human in-sulin and C-peptide immunoreactivity in the serum, thehepatic glycogen content did not differ statistically fromthat in intact animals. Constant normalization of glycemia for12 months in mice and for 18 months in pigs demonstratesfunctional activity of human insulin gene in liver cells ofmodel animals. The results of pathomorphological study andhistological analysis of animal tissues and organs after long-time experiments revealed that no detectable side effects ofgene therapy were seen.

Conclusion: The results have demonstrated the poten-tial for gene therapy approaches in the treatment of Type 1diabetes.

P 6

Expression Of O6-Methylguanine-DNA Methyltransferase

P140K via a SIN Retroviral Vector Encompassing a WeakInternal Promoter is Optimal forChemoprotection=Selection of Hematopoietic Stem Cells

Milsom MD (presenting)1, Jerabek-Willemsen M2, HarrisCE3, Schambach A4, Broun E5, Bailey J5, Jansen M5, WatsonA6, Margison GP6, Geiger H5, Moritz T4, Baum C4, ThomaleJ2, Williams DA1

BSGT 2009 POSTER PRESENTATIONS 397

1Children’s Hospital Boston=Harvard Medical School, Boston,MA, USA2University of Duisberg=Essen, Essen, Germany3Children’s Hospital Boston=Harvard Medical School, Boston,MA4Hannover Medical School, Hannover, Germany5Cincinnati Children’s Hospital, Cincinnati, OH, USA6Paterson Institute for Cancer Research, Manchester, UK

Retroviral-mediated delivery of the P140K mutant O6-methyguanine-DNA methyltransferase (MGMTP140K) intohematopoietic stem cells (HSC) has been proposed as a meansto protect against dose limiting myelosuppressive toxicityensuing from chemotherapy combining O6 alkylating agents(e.g. temozolomide (TMZ)) with pseudosubstrate inhibitorsof endogenous MGMT (e.g. O6-benzylguanine (6BG)). Sincedetoxification of O6-alkylguanine adducts by MGMT is stoi-chiometric, it has been suggested that higher levels of MGMTwill afford better protection to gene modified HSC. However,accomplishing this goal would potentially be in direct conflictwith current efforts in the gene therapy field, which aim toincorporate weaker enhancer elements to avoid insertionalmutagenesis. Using a panel of self inactivating gamma-retroviral vectors which express a range of MGMTP140K

activity, we demonstrate that MGMTP140K expression byweaker cellular promoter=enhancers is sufficient for in vivoprotection=selection following treatment with 6BG=TMZ.Conversely, the highest level of MGMTP140K activity did notpromote efficient in vivo protection despite mediating de-toxification of O6-alkylguanine adducts. Moreover, very highexpression of MGMTP140K was associated with a competitiverepopulation defect in HSC. Mechanistically, we demonstratea defect in cellular proliferation associated with elevated ex-pression of MGMTP140K, but not wild type MGMT. Thisproliferation defect correlated with increased localization ofMGMTP140K to the nucleus=chromatin. These data demon-strate that very high expression of MGMTP140K has a delete-rious effect upon cellular proliferation, engraftment andchemoprotection. These studies have direct translational rel-evance to ongoing clinical gene therapy studies usingMGMTP140K, while the novel mechanistic findings are rele-vant to the basic understanding of DNA repair by MGMT.

P 7

Adaptation of a Prosavin Producer Cell line to SuspensionCulture

Stevenson L (presenting), Stewart HJ, Mitrophanous KA,Radcliffe PA

Oxford BioMedica (UK) Limited, Medawar Centre, RobertRobinson Avenue, The Oxford Science Park, Oxford OX4 4GA

ProSavin� is Oxford BioMedica’s lentiviral vector-basedtherapy for Parkinson’s disease, and is based on a vector de-rived from Equine Infectious Anaemia Virus (EIAV) and en-gineered to express components of the dopamine synthesispathway. ProSavin has shown promising results in pre-clinicaltrials and is currently in a phase I=II clinical trial using vectormanufactured by transient transfection of HEK293T cells. Anadherent inducible ProSavin HEK293T based producer cellline (PCL) has been developed for the large scale manufacture

of ProSavin. This PCL has demonstrated controlled regulationof the packaging components, and vector yields were com-parable to transient production. Furthermore this cell lineproved to be stable, in the ‘OFF’ state, for up to 120 days inculture. However, manufacturing scale up can be restricted bythe adherent growth mode and therefore production wouldbenefit from the development of a suspension PCL.

Here we describe the adaptation of the adherently growinginducible ProSavin HEK293T based PCL to suspension cul-ture. When induced the cells in suspension demonstrated theproduction of functional vector, and furthermore displayedpopulation doubling times, reverse transcriptase (RT) activityand VSV-G protein expression levels similar to the adherentProSavin PCL.

P 8

Design, Assembly and Characterisation of NovelLiposomal Vectors for Systemic Delivery of siRNAto the Liver for the Treatment of Hepatic Diseases

Kolli S (presenting), Thanou M, Miller AD

Genetic Therapies Centre, Department of Chemistry, FlowersBuilding, Armstrong Road, Imperial College London, LondonSW7 2AZ

The ability to systemically administer siRNA and func-tionally modulate gene expression in disease sites is a criticalstep to opening up the therapeutic potential of RNAi tech-nology. Here we report on the synthesis and characterizationof novel siRNA-ABC nanoparticle systems designed for sys-temic delivery of functional siRNA to the liver. Initially, li-posomal nanoparticles were specifically formulated fromthe cationic cholesterol polyamine lipid N1-cholesterylox-ycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and theneutral co-lipid dioleoyl L-a-phosphatidylethanolamine(DOPE). These nanoparticles are then premodified with asmall molar percentage of a coupling aminoxy lipid choles-teryl-PEG350-aminoxy lipid (CPA) to provide aminoxygroups on their surface. The liposomes were then stabilizedby the post-coupling of polyethylene glycol2000-dialdehyde[PEG2000-(CHO)2], whereby rapid aminoxy-aldehyde cou-pling linked the liposomes to PEG via oxime bonds. The re-sulting nanoparticles have shown superior biological stabilityand have proven to be siRNA transfection-active in bothcancer and primary cell lines. The obtained siRNA-ABC na-noparticles appear to have pH-triggerable characteristics toogiven the fact that the resulting oxime chemical linkages weredemonstrated to be very stable at neutral pH but subject todynamic exchange under more acidic pH conditions andtherefore capable of breakdown in a crowded biological en-vironment at low pH such as in the endosome.

P 9

Ex Vivo Gene Therapy for the Inherited Skin Disease

Netherton Syndrome

Qasim W (presenting)1, Talbot G1, Thrasher AJ1, Larcher F2,Harper J3, Di W4

1Molecular Immunology Unit, UCL Institute of Child Health,London

398 BSGT 2009 POSTER PRESENTATIONS

2Cutaneous Disease Modelling Unit, Madrid3Dermatology, Great Ormond St Hospital, London4Immunobiology Unit, UCL Institute of Child Health, London

Netherton Syndrome (NS) is a severe inherited skin dis-order caused by mutations in the SPINK5 gene, which resultsin defective expression Lympho-epithelial Kazal-type-relatedinhibitor (LEKTI). Ex vivo gene therapy has been used tostably transduce keratinocyte stem cells form patients withother skin disorders, such as Epidermolysis Bullosa, andcorrected epidermal sheets generated can be used as viableskin grafts. This approach is highly attractive in NS, as genemodified autologous skin sheets could provide importantbarrier protection as well as serve as a source of systemicLEKTI protein delivery.

Self inactivating HIV-1 based lentiviral vectors were gen-erated to encode a codon optimised SPINK5 transgene linkedto the enhanced green fluorescent reporter protein. Forpre-clinical experimental purposes these vectors encodeSFFV-LTR promoter elements, as well as the HIV-1 centralpolypurine tract and Woodchuck post transcription regula-tory element. High levels of transduction were achievable inkeratinocyte cell lines (upto 100%) and in primary keratino-cytes (*30%) using both SPINK5=eGFP and eGFP controlvectors. Gene modified epidermal sheets were cultured on adermal support matrix to generate 3-D organotypic cultures.We demonstrate epidermal eGFP expression co-localisingwith reconstitution of LEKT1. Longer term ex vivo experi-ments in cell lines detected reductions in transgene expres-sion for SPINK5=eGFP vectors (but not for eGFP controlvectors). Differential promoter methylation, that was par-tially reversible by culture in Azacitidine, suggesting thatSPINK5 encoding vectors may be more prone to methylationmediated silencing over time, and this issue is being ad-dressed in further studies, which include the development ofhuman:murine chimeric models of skin grafting.

P 10

Targeted Skipping of Exon 53 of the Human DMD Gene:Recommendation of a Highly Efficient Antisense

Oligonucleotide for Clinical Trial

Popplewell LJ (presenting)1, Adkin C2, Arechavala-GomezaV2, de Winter C3, Aartsma-Rus A3, Wilton SD4, Morgan JE2,Dickson G1, Muntoni F2, Graham I1

1School of Biological Sciences, Royal Holloway-Universityof London, Egham, Surrey TW20 0EX, United Kingdom2Dubowitz Neuromuscular Centre, UCL Institute of Child Health,30 Guilford Street, London WC1N 1EH, United Kingdom3Center for Human and Clinical Genetics, Leiden UniversityMedical Center, 2300RC Leiden, The Netherlands4Centre for Neurological and Neuromuscular Disorders,Australian Neuromuscular Research Institute, Universityof Western Australia, Perth WA6009, Australia

Duchenne muscular dystrophy (DMD) is due to the lack ofdystrophin protein, as a result of various out-of-frame, or lesscommonly nonsense, mutations in the DMD gene. Modula-tion of pre-mRNA splicing with antisense oligonucleotides(AOs) to restore the reading frame, has been shown in vitro(in cultured cells and muscle explants) and in vivo (in animal

models and DMD patients) such that truncated, yet func-tional dystrophin is expressed. AO-induced skipping of exon51 of the DMD gene has now progressed to clinical trials. Thechoice of AOs for such trials was decided after the detailedcomparative study of AO sequences targeted to exon 51. Wedescribe here the methodical, cooperative comparison oftwenty-four AOs (of the phosphorodiamidate morpholinooligomer (PMO) chemistry) designed to target exon 53 of theDMD gene. PMO bioactivity was assessed systematically bytheir use in different preclinical models: in vitro in culturedhuman muscle cells (normal and DMD) and in vivo intransgenic mice expressing the human DMD gene. The col-laborative approach ensured independent validation of re-sults. This study strongly suggests the superiority of a PMOtargeting the sequence þ 30þ 59 of exon 53. This PMO wouldrepresent an ideal choice for future clinical trials for the tar-geted skipping of exon 53.

P 11

Indirect Tumour Cell Killing Following AAVP-Mediated

Suicide Gene Delivery to Vascular Endothelium

Trepel M1, Stoneham C (presenting)2, Mazarakis N2,Pasqualini R3, Arap W3, Hajitou A2

1University Medical Center Hamberg-Eppendorf, Departmentof Oncology and Hematology, Martinistrasse 52, D-20246Hamberg, Germany2Department of Gene Therapy, Division of Medicine, ImperialCollege London, Wright-Fleming Institute, St Mary’s Campus,Norfolk Place, London W2 1PG, UK3The University of Texas M.D. Anderson Center, 1515 HolcombeBoulevard, Houston, Texas 77030, U.S.A.

Suicide gene transfer is the most commonly used cyto-toxic approach in cancer gene therapy; however, a success-ful suicide gene therapy depends on the generation ofefficient targeted systemic gene delivery vectors. We recentlyreported that selective systemic delivery of suicide genes suchas the Herpes simplex virus thymidine kinase (HSVtk) totumour endothelial cells via a novel targeted AAVP vectorleads to suppression of tumour growth. This marked effecthas been postulated to result primarily from the death ofcancer cells by hypoxia following the targeted disruption oftumour blood vessels. Here we investigated whether an ad-ditional mechanism of action is involved. We show that thereis a heterotypic bystander effect between endothelial cellsexpressing the HSVtk suicide gene and tumour cells. Treat-ment of co-cultures of HSVtk-transduced endothelial cellsand non-HSVtk-transduced tumour cells with gancyclovirresults in the death of both endothelial and tumour cells.Blocking of this effect by 18a-glycyrrhetinic acid indicates thatgap junctions between endothelial and tumour cells are lar-gely responsible for this phenomenon. Moreover, the ob-served bystander killing is mediated by connexin (Cx)43 andCx26, which are expressed in both endothelial and tumourcell types. Finally, this heterotypic bystander effect is ac-companied by a suppression of tumour growth in vivo that isindependent of primary gene transfer into host-derived tu-mor vascular endothelium. These findings add an alternativenon-mutually exclusive cytotoxic mechanism to cancer genetherapy based on targeted AAVP, and further support the


promising role of non-malignant tumour stromal cells astherapeutic targets.

P 12

Study of Protein Biomarker for Early Diagnosis ofDiabetes Mellitus Type 2 and Role of Thiamine on Their

Level

Riaz S (presenting)1, Alam SS2, Rabbani N3, Thornalley P3,Akhtar MW4

1Department of Microbiology & Molecular Genetics, PunjabUniversity, New Campus (Quaid-e-Azam) Lahore, Pakistan2Dept of Pharmacology, Federal Post Graduate Medical Institue,Shaikh Zayed Hospital, Lahore, Pakistan3Experimental Systems Biology, Protein Damage and SystemsBiology Research Group, Clinical Sciences Research Institute,University Hospital, University of Warwick, Coventry CV2 2DX,UK4School of Biological Sciences, Punjab University, New Campus(Quaid-e-Azam) Lahore, Pakistan

This research project aims to characterize protein bio-markers which are specific to the various stages of diabetesmellitus type 2 and to assess their levels as a result of ad-ministration of high dose thiamine. Proteomic approachescan be used to identify proteins that can be exploited as po-tential diagnostic=prognostic biomarkers of acute and chronicdiabetes and pre-diabetes. 100 type 2 diabetic patients and thesame number of age- and sex-matched normal healthy con-trols were recruited from sheikh Zayed Hospital, Lahore.Clinical history and all baseline biochemical parameters havebeen assessed by different standard referred protocols.Thiamine (300 mg=day) and placebo was given for 3 monthsin a double-blinded design. The biochemical profiles of pa-tients was studied over a 6-month period and results sug-gested significant decreases in albumin excretion rate in 35%of thiamine treated patients as compared to placebo group.Other significant decreases such as Glycated HBA1c and tri-glycerides were also observed in the thiamine-treated group.Protein profile of the blood samples of type 2 diabetes pa-tients has been investigated and particularly the level of somemarker proteins like alpha-2 macroglobulin, CRP, RBP4, andApo-I were assessed. Identification and characterization ofprotein biomarker were done by a more recent proteinmapping technology using ProteomeLab PF 2D and massspectrometry MALDI-TOF and MASCOT software analysis.These studies have contributed to improved and more ef-fective treatment for type 2 diabetic patients with incipientnephropathy with expected decrease risk of kidney failure.

P 13

Towards the Generation of Induced Pluripotent Stem (iPS)Cells Using Non-Integrating Lentiviral Vectors

Mukherjee S (presenting), Almarza E, Kinnon C, Thrasher AJ

Molecular Immunology Unit, UCL Institute of Child Health

Direct reprogramming of mouse and human fibroblasts intopluripotent stem cell-like cells (referred to as induced plurip-otent stem cells or iPS cells) through retroviral or lentiviral

transduction of a combination of reprogramming factors hascreated a significant amount of interest in the field of cell-basedgene therapy. All the current protocols for generating iPS cellsemploy viral vectors which harbour the inherent risk of in-sertional mutagenesis due to random insertion of these virusesinto the genome of target cells. For the therapeutic applica-tion of iPS cells this constitutes a grave concern. To addressthis, we wished to employ non-integrating lentiviral vectorscarrying the reprogramming factors to generate iPS cells frommouse fibroblasts that have been derived from a mousemodel of X-linked chronic granulomatous disease (X-CGD).Non-integrated lentivectors which remain as episomal entitiespose reduced risk of causing insertional mutagenesis. It re-mains to be seen whether transient expression of the fourreprogramming factors (Sox2, Oct3=4, c-Myc and Klf4) fromnon-integrating lentivectors is sufficient to drive the adult fi-broblasts into pluripotency. It is quite evident that the trig-gering of the change for reprogramming must be silenced atsome point during the generation of iPS cells. Therefore it isreasonable to argue that expression of the transcription factorsfrom non-integrating lentivectors would be sufficient to pro-vide the impetus for the change in the epigenetic status of thecells. Here we present the preliminary results from our ex-periments to optimize the generation of iPS cells from mousefibroblasts using non-integrating lentivectors.

P 14

The Development of a Purpose Built Gene Medicines

Pharmacy Aseptic Unit

Stoner NS

Oxford Radcliffe Hospitals NHS Trust

Introduction: The Oxford Radcliffe Hospitals NHS Trust(ORH) new Cancer Centre opens in 2009 which has a purposebuilt gene medicines pharmacy aseptic unit, where the cancergene medicine clinical trials will be dispensed.

Aims: Gene medicines should be handled by pharmacystaff in purpose built pharmacy aseptic units to ensure theintegrity of the product and safety to patients and staff. Thegene medicine aseptic unit has been developed in line withnational and international guidelines on handling genemedicines and aseptic units.

Method: Work began on designing the new cancer centrein 2001. The Cancer Research UK Clinical Research Unit hasundertaken 12 gene medicine clinical trials, and has ensuredthat facilities are available in the new cancer centre for han-dling up to class 2 or 3 gene medicine clinical trials.

Results: A purpose built gene medicine pharmacy asepticunit was designed next to the research laboratory on the on-cology ward. There is an anteroom for changing in, whichleads to the aseptic room containing a pharmaceutical gradeD isolator, fridge, freezers, and workbenches. The room hasto be commissioned by pharmacy quality control and proce-dures are being written.

Discussion: A purpose built gene therapy aseptic unit isthe ideal facility in which to handle gene medicines. TheEuropean Association of Hospital Pharmacy guidanceon handling gene medicines is an essential tool for hospitalpharmacists handling gene medicine products.


P 15

NfsA Nitroreductase for Prodrug Activation Gene Therapy

Vass SO (presenting)1, Jarrom D2, Wilson WR3, Hyde EI2,Searle PF1

1CRUK Institute for Cancer Studies, University of Birmingham,Edgbaston, Birmingham B15 2TT, United Kingdom2School of Biosciences, University of Birmingham, Edgbaston,Birmingham B15 2TT, United Kingdom3Auckland Cancer Society Research Centre, Faculty of Medicaland Health Sciences, University of Auckland, Auckland, NewZealand

Prodrug activation gene therapy utilises the expression ofan enzyme to activate a systemically delivered, nontoxicprodrug within tumour cells. The cytotoxic metabolites gen-erated by this conversion can directly kill these cancer cellstogether with untransduced neighbouring cells as a conse-quence of the bystander effect. One such promising combi-nation that has entered clinical trials uses the E. coli enzymeNfsB, a NAD(P)H dependent nitroreductase that can activatethe prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide)to a potent bifunctional alkylating agent. The original isola-tion of NfsB was monitored with assays using menadione assubstrate and NADH as the cofactor so it was unknownwhether the major nitroreductase from E. coli, NfsA, aNADPH dependent enzyme, could preferentially activateCB1954 when compared to NfsB. Using purified enzymes wehave shown superior in vitro kinetics of CB1954 activation byNfsA, and demonstrated an increased potency for human cellsensitisation relative to NfsB. This also verified the preferencefor NADPH as cofactor over NADH. Adenoviral expressionof NfsA in human cancer cells was also shown to increasesensitivity to CB1954, relative to cells infected with an NfsB-expressing adenovirus. Whereas NfsB reduces CB1954equally at either the 4 or 2-NO2 position, we have shown thatNfsA reduces CB1954 almost exclusively at the 2-NO2 posi-tion. The 2-NO2-activation products were shown previouslyto have greater tissue-permeability, and in accordance withthis we have demonstrated an increased bystander effectusing NfsA rather than NfsB to activate CB1954. We have alsofound that NfsA is more effective than NfsB for cell sensiti-sation to a series of alternative dinitrobenzamide bromo-mustard prodrugs.

P 16

Oncolytic Adenovirus Vectors for Nitroreductase SuicideGene Therapy of Prostate Cancer

Herod M (presenting)1, Alemany R2, Mautner V1, Searle PF1

1CR UK Institute for Cancer Studies, University of Birmingham,Edgbaston, Birmingham B15 2TT, UK2Laboratori de Recerca Traslacional, Institut Catala d’Oncologia,Gran Via s=n km2,7 L’Hospitalet de Llobregat, 08907 Barcelona,Spain

Prostate cancer is the most common male cancer in the UKand USA, with a 1=13 chance of diagnosis and a 1=30 lifetimerisk of death from the disease. In a recent Phase I=II prostatecancer gene therapy trial we used a nonreplicating adenovi-

rus vector to express nitroreductase (NTR, encoded by E. coliNfsB), which converts the prodrug CB1954 into a cytotoxicDNA alkylating agent. Use of conditionally replicatingadenovirus vectors (CRAds) to deliver NTR should be ad-vantageous, combining the benefits of viral oncolysis withimproved prodrug susceptibility, due to amplification andspread of the virus within tumour tissue.

We have constructed a panel of CRAd vectors based on theICOVIR platform1; virus replication is controlled by an in-sulated E2F promoter driving expression of E1A-CR2-D24,and NTR expression is restricted to the late phase of infection,restricting replication and cell killing to tumour cells. To vi-sualise replication and spread, some constructs encode greenfluorescent protein in place of NTR.

Six gfp expressing CRAd vectors were compared in vitro,measuring virus replication, spread, transgene expression andcytotoxicity. The ICOVIR-based virus vMH6 demonstratedgreater therapeutic index than CRAds with just E1A-CR2-D24, or based on E1B-55K deletion, or wtAd5. A similarvector containing an RGD-retargeted fibre (vMH7) showedimproved transduction in cells lacking CAR (Coxsackie andAdenoviral Receptor). vMH7 was engineered to express NTRor catalytically improved variants from the adenovirus majorlate promoter. In combination with CB1954, these vectorsdemonstrated increased cytotoxicity to tumour cell lines com-pared to the parental vector.

P 17

Biological Role of DNp63alpha in Human PancreaticDuctal Adenocarcinoma, and Its Effect on Chemosensitivity

Choudhury N (presenting), Yuan M, Lemoine NR, Wang Y

Centre of Molecular Oncology and Imaging, Institute of Cancer,Queen Mary University of London, Barts and the London Schoolof Medicine and Dentistry, London, UK

Background: DNp63a is the predominant p63gene isoformthat is upregulated in many epithelial tumours. Its expres-sion is correlated to poorer outcomes and survival insome cancers.

Aims: To determine the biological role of DNp63a,and its effect on chemosensitivity, in human pancreatic can-cer.

Method and Results: Immunohistochemistry analysis from34 human pancreatic cancer tissue samples showed DNp63ais profoundly expressed in some invasive and poorly differ-entiated cancers. Pancreatic cancer patients not expressingDNp63a have a median survival advantage of seven months,compared to patients overexpressing DNp63a.

In order to assess the effect of DNp63a expression on cancercell biology, eight human pancreatic cancer cell lines werescreened for overexpression of DNp63a, using Western Blot.This confirmed two cell lines to show upregulation ofDNp63a.

RNA interference was used to conduct functional studies.Cell viability assays confirmed p63a expression promotescellular proliferation and survival. Wound healing assaysshowed decreased cell migration when p63a expression is

1Cascallo et al. (2007). Mol Ther 15, 1607–1615.


suppressed. In addition, soft agar assays have demonstratedthat this isoform promotes tumourigenicity and anchorageindependent growth. Most interestingly, we have demon-strated that silencing DNp63a significantly enhanced thesensitivity to gemcitabine and 5FU.

Conclusions: DNp63a is overexpressed in some pri-mary human pancreatic cancers and its expression correlateswith a worse survival. DNp63a expression enhances tumourcell proliferation, migration and tumourigenicity. SilencingDNp63a can induce a synergistic effect with chemotherapy ontumour cell killing. These findings suggest that DNp63a mayrepresent a promising therapeutic target in the treatment ofhuman pancreatic cancers.

P 18

Genetic Retargeting of Ad5 Using a Novel Class of Ligands

(Nanobodies) Belonging to the ImmunoglobulinSuperfamily and Having a Large Natural Diversity

Lindholm L1, Magnusson M1, Wising C1, Henning P1, BatyD2, Chames P2, Willemsen R (presenting)3

1Got-a-Gene AB, Ostra Kyviksvagen 18, 32930 Kullavik, Sweden;2University of Gothenburg, Gothenburg, Sweden2National Research Institute, CNRS LISM, 31 Chemin JosephAiguier, Marseille, France3ErasmusMC-Daniel den Hoed Cancer Center, Rotterdam, TheNetherlands

There is a great need to find ligands that can be used forgenetic retargeting of Ad. So far it has been difficult to findligands that both display a high affinity for their targets andare functional on the Ad capsid as well as belonging to afamily having a large enough diversity. This is due, amongother things to the constraints on S-S bond formation andstable folding that the reducing intracellular milieu of mam-malian cell imposes on ligands. We will present the successfulincorporation into the Ad5 fibre knob of a member of a classof ligands belonging to the camelid antibody (nanobody)family that, to our knowledge, has not been previously usedfor this purpose. The recombinant fibres bind their new targetand could be rescued into functional virions. The phenotypiccharacteristics of the virus are currently being assessed andwill be reported. The results may significantly impact thefield of genetic retargeting of Ad.

P 19

Gene Therapy Against Hepatocellular Carcinoma:Development of a Herpesvirus Saimiri-Based Vector

Turrell S J (presenting), Whitehouse A

University of Leeds

Herpesvirus saimiri (HVS) is a gamma-2 herpesvirus thathas potential as a cancer gene therapy vector. HVS-basedvectors have a large packaging capacity, can infect a varietyof human carcinoma cell lines, and can establish a latentpersistent infection in both in vivo and in vitro models.Moreover, our non-invasive optical imaging study using aluciferase reporter gene demonstrated that HVS-based vec-

tors have a tropism for liver tissue. This suggests potential foruse in gene therapy applications to treat liver diseases such ashepatocellular carcinoma (HCC). We show that HVS can ef-ficiently infect a variety of HCC cell lines and establish alatent infection. We have then assessed whether the cancer-specific cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has any efficacy for HCC tumourgrowth inhibition. Results suggest that recombinant TRAILcan induce apoptosis in HCC cell lines in combination withcertain enhancer molecules. To extend these promising find-ings we have engineered the TRAIL gene into an HVS-basedvector under the control of a tumour specific promoter. Re-sults demonstrate that the HVS-TRAIL vector can infect HCCcell lines efficiently and current experiments are ongoing todetermine the levels of apoptosis induction.

P 20

Herpesvirus Saimiri-Based Infectious Delivery of the APCGene Reduces Cell Proliferation of SW480 and SW620

Cells

Macnab S (presenting), Whitehouse A

University of Leeds

Bowel cancer is the second leading cause of cancer relateddeath in the United Kingdom. One form of bowel cancer isfamilial adenomatous polyposis (FAP). FAP is caused by agenetic mutation in the adenomatosis polyposis coli (APC) tu-mour suppressor gene, and leads to the growth of thousands ofpolyps in the colon. APC is a large gene, comprising of an 8.5 kbcDNA coding region, spliced from a 98 kb genomic locus. TheAPC protein is involved in the regulation of c-myc, an onco-protein associated with cellular proliferation, cell survival andapoptosis avoidance. Currently there is a bottleneck in the ef-ficient delivery of APC cDNA to target cells. In this study wehave constructed a number of wild-type and recombinantamplicon vectors containing the APC cDNA which are basedupon the g-2 Herpesvirus saimiri (HVS) bacterial artificialchromosome. HVS is a suitable vector for virally mediated APCgene therapy as it has been shown to be maintained as a non-integrated episome, allowing for long-term transgene expres-sion in both in vitro and in vivo studies. Our results show thatthe HVS-APC vectors are able to express the APC cDNAtransgene and reduce cellular proliferation of APC-mutantSW480 and SW620 cells in vitro. To our knowledge this is thefirst demonstration of virally mediated APC cDNA deliveryand expression.

P 21

Approaches to Increase the Level of Humoral ImmuneResponse to the Model DNA-Vaccine

Deryabina OG (presenting)1, Pokholenko IO2, Deriabin OM3,Gulko TP2, Kordium VA2

1Institute of Genetic and Regenerative Medicine AMSU, 67,Vyshgorodska, Kyiv, 04114, Ukraine2Institute of Molecular Biology and Genetics NASU, 150,Zabolotnogo str. Kyiv, 03143, Ukraine3Institute of Veterinary Medicine UAAS, 30, Donetskaya str.,Kyiv, 03151, Ukraine


The development of a new generation of vaccines has beenencouraged by the fact that genetic immunization is able toinduce protective immunity. Their efficacy depend on thenature of the individual antigen as well as on the vaccinationmode. The aim of the work was to develop the DNA-vaccinemodel against classical swine fever, study its antigenic prop-erties and find the ways for increase its efficacy in antibodyraising. The genetic construction was developed on the basis ofeukaryotic vector pTR-UF, which contained CMV promoter,AAV inverted terminal repeats and the CSFV E2 protein genefragment. The recombinant plasmids pmIL2-TR and pmI-L12el-TR carrying the cDNA of murine IL-2 and IL-12 re-spectively, were created. The female BALB=c mice wereimmunized using the different administration routes: intra-muscular, intrasplenic, subcutaneous with 10 to 100mg ofmodel DNA-vaccine. Several compositions with genetic ad-juvants were tested. Antibodies specific to model antigen inplasma were detected by ELISA. It has been shown that sub-cutaneous administration route failed to induce detectable IgGin plasma while intramuscular injection succeeded to induceIgG but not in all mice. The most successful and reproduciblewas intrasplenic administration. Usage of the recombinantplasmids with the murine interleukin genes together with themodel DNA—vaccine led to the enhancement of humoralimmune response The data obtained show the possible ap-proaches of increase the level of humoral immune response tothe model DNA—vaccine: correct choice of the immunizationroute and place, and use of genetic adjuvants.

P 22

Ocular Tolerance of a Lentiviral Vector-Based Angiostatic

Gene Therapy Product (RetinoStat�) in Rodent, Rabbit,and Nonhuman Primate Models

Kan O (presenting)1, Widdowson P1, Hamirally S1, BinleyK1, Nork M2, Miller P2, Bantseev V2, Christian B2, Iqball S1,Mitrophanous KA1, Naylor S1

1Oxford BioMedica (UK) Ltd, Oxford Science Park, Oxford, UK2Covance Laboratories Inc, Madison, WI, U.S.A.

Oxford BioMedica has developed a range of recombinantEquine Infectious Anemia Virus (EIAV) vectors expressingtherapeutic genes for the treatment of a variety of diseases.One such vector, ProSavin� is currently undergoing evalua-tion in a Phase I=II clinical trial for the treatment of Parkin-son’s disease. This is the first trial in which a lentiviral vectorhas been directly administered to patients.

EIAV-based vectors can transduce a broad range of di-viding and non-dividing human cell types, including ocularcells. Oxford BioMedica have also developed a vector, Re-tinoStat�, for the treatment of age-related macular de-generation (AMD) caused by choroidal neovascularisation.RetinoStat� vector expresses human endostatin and a-ngiostatin via the retinal pigment epithelial (RPE)-specificVMD2 promoter, thereby limiting transgene expression to theRPE layer following subretinal delivery. One key to success ofthese vector-based therapies is achieving delivery of vector totarget cells with acceptable ocular tolerance.

We have examined ocular tolerance in mice, rabbits andCynomolgus macaques following subretinal delivery of ei-

ther EIAV-VMD2 LacZ or RetinoStat� vector. The results oflacZ gene transfer showed that transgene expression wasconfined to RPE cells and, in mice, LacZ expression lasted forat least 1 year without signs of diminution. Ocular exami-nations in Cynomolgus macaques and rabbits revealedtransient dose-dependent ocular inflammation that resolvedduring subsequent weeks. No long-lasting pathophysiologi-cal changes on the retinal function or structure were associ-ated with subretinal injections. Results from these in vivoexpression and toleration studies will be presented.

These data support advancing to formal nonclinical studiesto support transition of RetinoStat� toward a first in Manstudy.

P 23

CEA Specific Engineered T cells: Absence of Auto-Toxicityin the CEA Transgenic Mouse

Gilham DE (presenting), Watson V, Sheard V, Robinson A,Cheadle EJ, Hawkins RE

Cell Therapy Group, Cancer Research UK Department of MedicalOncology, Paterson Institute for Cancer Research, University ofManchester

T-cells engineered to express tumour specific receptors arebeing explored as a cancer therapy. Mouse T cells harbouringa CEA-specific receptor can challenge the development of aCEAþ MC-32A tumour in a short-term syngeneic model.Critically, improved anti-tumour responses were observedwhen the T-cells were combined preconditioning chemo-therapy suggesting that engraftment of the T-cells was im-portant for optimal therapeutic effect.

In order to assess auto-toxicity, CEA transgenic (CEATg)mice, which express CEA in tissue locations similar to thatseen in humans, were conditioned with 10Gy TBI, given bonemarrow rescue 24 hours later and then CEA-specific mouse T-cells. Of the fourteen CEATg mice treated, four were culledsoon after conditioning with symptoms of radiation sicknessand three were culled at >300 days due to fighting. Noneshowed symptoms that could be associated with T-cell auto-immunity. The seven remaining animals survived greaterthan 400 days with five of these having detectable levels(>0.1%) of engineered T cells in spleens at the end of theexperiment. One mouse was found to have a liver tumourwhich did not contain detectable levels of engineered T-cellsand three further control mice also possessed similar livertumours suggesting that this was not a T-cell related effect.Although engineered T-cells were detected in the spleen andlung, these cells were rare in gastro-intestinal tissue sug-gesting that a lack of homing to sites of natural CEA ex-pression may be one reason to explain the absence of T-celldriven autoimmunity in the CEATg mouse.

P 24

Nonviral Gene Expression in the Lung Using

the Mini-CFTR Promoter

Connolly MM (presenting)1, Pringle IA1, Lawton AE1,Munkonge F2, Chan M2, Alton EWFW2, Boyd AC3,Doherty A3, Hyde SC1, Gill DR1


1Gene Medicine Research Group, Nuffield Department of ClinicalLaboratory Sciences, John Radcliffe Hospital, University ofOxford, Oxford, UK2Department of Gene Therapy, Imperial College, London, UK3Department of Medical Sciences (Medical Genetics), Universityof Edinburgh, Western General Hospital, Edinburgh, UK

Clinical studies are underway for the aerosol deliveryof Genzyme Lipid GL67A complexed with plasmid DNA(pDNA) to the lungs of patients with cystic fibrosis (CF). Theclinical plasmid utilises the ubiquitously expressing hCEFI(human Cytomegalovirus enhancer=elongation factor 1al-pha) promoter to express the CFTR protein. This plasmidand a similar luciferase (Lux) expressing version generatedpersistent high levels of gene expression (>8 weeks) fol-lowing aerosol delivery of GL67A=pDNA to the mouse lung.However, we are also investigating promoters that have thepotential to give tissue-specific CFTR gene expression, in-cluding a mini CFTR promoter encompassing the minimumsequence from the 50 region upstream of the CFTR gene ableto provide promoter activity and confer cell-type specificexpression in vitro. The promoter was cloned into our 4thgeneration CpG-free plasmid backbone in either a Native(promoter only) or Enhanced (plus human CMV enhancer)form. Versions of the Native and Enhanced plasmids withthe human CFTR transgene or the luciferase reporter genewere constructed and both expressed significant levels ofluciferase or CFTR mRNA in cell culture. CFTR protein ac-tivity was confirmed by iodide efflux. When gene expressionfrom the Native and Enhanced plasmids was compared withexpression from the synthetic hCEFI promoter followingdirect instillation into the mouse lung (80 mg pDNA=100ml,BALB=c, n¼ 6), Lux activity from the Enhanced plasmidgave 21% and 12% hCEFI levels at d1 and d14 respectively.These plasmids will also be assessed in human Air-LiquidInterface cultures to compare levels of expression in differ-entiated respiratory epithelia.

P 25

Optimisation of Viral Vector-Mediated Gene Delivery

to the CNS: Implications for Batten Disease

Ahmadi SM (presenting)1, Rahim AA2, Wong AMS1, BakerAH3, Cooper JD1, Waddington SN2

1Institute of Psychiatry, King’s College London2Royal Free and University College Medical School London3BHF Glasgow Cardiovascular Research Centre, Universityof Glasgow

The lysosomal storage disorder neuronal ceroid lipofusci-nosis (NCL) or Batten disease is a group of inherited neuro-degenerative disorders each caused by a mutation in adifferent gene, resulting in forms of NCL that progress atdifferent rates. Congenital, Infantile and Late Infantile NCLare caused by mutations in soluble lysosomal enzymes andare well suited to gene therapy, since therapeutically deliv-ered enzyme can diffuse to cross-correct deficient cells. Genetransfer in NCL mouse models is more effective if deliveredearly in disease progression, suggesting that prenatal deliv-ery may be required in some early onset forms. We haverecently demonstrated efficient gene delivery to the fetal CNS

using pseudotyped lentiviral vectors, displaying transduc-tion patterns that are distinct to those seen in the adult brain.To extend these studies and identify tools suited for prenataltherapy in NCL mouse models we have now taken a seriesof adenoviral, adenoassociated and lentiviral vectors andcompared their transduction capacity, stability and CNS ex-pression of reporter genes following intracranial administra-tion to mice in utero. Mice were injected on day E14 and thetransduction patterns of the viral vectors were analyzed at 21days after birth using the reporter genes luciferase and GFP.These vectors display pronounced regional and cell typespecific tropism with different degrees of efficiency and ex-tent of transduction within the CNS. We are now ideallyplaced to select vectors for the prenatal delivery of enzyme tothe Batten disease CNS and explore if this improves thera-peutic efficacy.

P 26

A Novel Chimeric Promoter Reconstitutes NADPHExpression and Function in X-CGD Cells

Santilli G (presenting)1, Almarza E1, Beilin C1, Blundell M1,Haria S1, Grez M2, Kinnon C1, Bueren JA3, Thrasher AJ1

1Molecular Immunology Unit, ICH, London2Division of Applied Virology and Gene Therapy,Georg-Speyer-Haus, Frankfurt, Germany3Hematopoiesis and Gene Therapy Division, CIEMAT andCIBER-ER, Madrid, Spain

X-linked Chronic Granulomatous Disease (X-CGD) is aprimary immunodeficiency caused by mutations in theCYBB gene encoding the phagocyte NADPH oxidase cata-lytic subunit gp91phox. A clinical trial for X-CGD using theretroviral vector SF71gp91phox has recently shown promis-ing results. However, the expression of gp91phox has de-clined over time, due to CpG methylation within the viralLTR. In this study we aimed to optimise gp91phox expres-sion in myeloid cells by using a novel chimeric promoter inthe context of lentiviral vectors. The chimeric promotercontains the c-Fes minimal promoter and the cathepsinGenhancer with binding sites for transcription factors highlyexpressed during granulocytic differentiation. The chimericpromoter was cloned in front of the eGFP gene in thepCCLsin lentiviral vector, which was used to transduce apanel of different cell lines. We found that the chimericpromoter induces higher GFP expression in PLB985, a my-eloid cell line, than in the other cell lines suggesting that ismyeloid specific. When transduced into primitive humanCD34 cells, the chimeric promoter drives a strong GFP ex-pression after granulocytic differentiation. We have en-gineered lentiviral vectors containing the chimeric or C-Fesinternal promoters to drive gp91phox expression. The vec-tors were assessed for their ability to reconstitute gp91phoxexpression and function in XCGD mice. We carried outtransplant experiments using Ly5.2XCGD bone marrow cellsas donors and X-CGD or Ly5.1Blck6 mice as recipients. Inboth systems the chimeric promoter could restore gp91phoxexpression and normal levels of NADPH oxidase function.These results suggest that the chimeric promoter could haveclinical value.


P 27

Characterisation of Nuclear Import Peptides as Adjuvants

for Liposome-Mediated Cancer Gene Therapy

Fox N (presenting)1, Lee TWR2, Blair GE1

1Institute of Molecular and Cellular Biology, University of Leeds,Leeds LS2 9JT, UK2Leeds Regional Paediatric Cystic Fibrosis Centre, St James’sUniversity Hospital, Leeds LS9 7TF, UK

Liposome-mediated gene therapy holds great promise asa therapy for epithelial cancers such as bronchogenic car-cinoma, particularly if detected at an early stage. However,current liposomes do not appear to be efficient in facilitat-ing nuclear import and expression of transgenes, particu-larly in non-dividing cells. We have identified two peptides(Mu and VII[25-54]) derived from adenovirus core proteinswhich complex with the viral genome during nuclearimport. These peptides significantly enhanced liposome-mediated plasmid DNA delivery to the bronchial epithelialcarcinoma cell line A549; between 2-3 fold increase in re-porter gene expression compared to liposome (DC-Chol=DOPE) and DNA alone. Previous work has beencarried out on subconfluent submerged cells, which isnot representative of the situation in vivo. We have thereforedeveloped a tumour cell culture model that is more repre-sentative of the respiratory tract by culturing primary hu-man polarised airway epithelial cells (AEC) derived fromnasal brushings. AEC were seeded with A549 cells to de-termine the levels of targeted expression of transgenes inco-cultures of normal and tumour cells. To distinguishthe A549 cells from AEC, A549 cells were pre-labelledwith a Celltracker Green dye (CFDA) prior to co-culturewhich were then transfected with a DS Red expressionplasmid complexed with peptide in DCChol=DOPE.Flow cytometry and fluorescence microscopy was used todistinguish cell populations and showed that unlabelledAEC could be distinguished from CFDA-labelled A549cells. We are also analysing the immune response againsteach peptide to determine whether an immune responsemight limit re-administration of the liposome:peptide:DNAcomplex.

P 28

Screening Gastrointestinal Cancers for AdenovirusReceptors: A Basis for Targeted Cancer Gene Therapy

Fox N (presenting)1, Verbeke C2, Blair GE1

1Institute of Molecular and Cellular Biology, University of Leeds,Leeds LS2 9JT, UK2Department of Histopathology, St James’s University Hospital,Leeds LS9 7TF, UK

Human adenoviruses (Ads) are potentially useful vectorsfor cancer gene therapy. The 51 human adenovirus serotypesare classified into six species (A to F). Most Ads utilise theCoxsackie B and adenovirus receptor (CAR) to interact withcells, however loss of CAR expression has been noted in anumber of tumour cell types, leading to a reduced ability of

these cells to be transduced by species C Ad5, the serotypemost frequently used in cancer gene therapy studies. Incontrast, CD46, CD80 and CD86 are proposed attachmentmolecules for species B Ads in cultured human cells.

We previously found that CAR expression was absent inmore than 50% of pancreatic ductal adenocarcinomas. Pan-creatic and colorectal cell lines and tissue samples weretherefore screened for expression of species B receptor mol-ecules. All cell lines studied expressed variable levels ofCD46, but did not express CD80 or CD86. The cell lines werethen infected with wild-type species B Ads (3, 11 and 35) todetermine their susceptibility to infection in comparisonwith Ad5, and results showed increased susceptibility toinfection by species B serotypes. Studies of pancreatic ade-nocarcinoma tissues by immunohistochemistry showedmembranous CD46 expression (88% moderate to high ex-pression) and heterogeneous CD86 staining (91% moderateto high expression), while the majority (72%) lacked CD80expression.

Overall, our results show that directing species B adeno-virus to tumours that express CD46 has potential promise forcolorectal and pancreatic cancer gene therapy.

P 29

Turning Monoclonal Antibodies into AdenoviralVectors—An Academic Perspective

Berrie EL (presenting)1, Bolam EJ1, Bird P1, Gallagher PA1,Andrews LA1, Oliveira CA1, Moyle SM1

1Clinical BioManufacturing Facility, University of Oxford, OldRoad, Headington, Oxford, OX3 7JT, UK

The CBF is an academic group, with over 13 years experi-ence producing biological Investigational Medicinal Products(IMPs) meeting EU GMP requirements. Our aim is to providethe link between novel academic research and clinical drugdevelopment; allowing collaborators to make rapid progressinto the clinic.

The first 11 years (when known as the Therapeutic Anti-body Centre) the CBF produced monoclonal antibodies and anumber of related biologics that have been used by cliniciansworldwide supporting more than 5,000 patients in clinicaltrials. Unlike commercial contract manufacturing organisa-tions the CBF specialises in iterative research with academicsand collaborators and is keen to support ‘proof of concept’trials. With the maturing of monoclonal antibody manu-facturing, a decision was made to switch direction to supportthe next generation of unmet needs in ‘proof of concept’clinical trials—the manufacture of novel vaccines and genetherapeutics using adenovirus vectors.

The CBF has a dedicated team of highly trained, moti-vated staff who learnt new skills and adapted to new chal-lenges associated with manufacturing different classes ofnovel products for early phase clinical trials. This presenta-tion describes our experiences in turning the manufacture ofmonoclonal antibodies into the manufacture of adenoviralvectors; three of which are currently in vaccine clinical trialsagainst malaria and Hepatitis C. Other vaccines and genetherapy products are currently in manufacturing and pro-cess development stages.


P 30

Inflammation-Free shRNA Expression Vectors for Cystic

Fibrosis Gene Therapy

Lawton AE (presenting), Smith SE, Gill DR, Hyde SC

Gene Medicine Group, NDCLS, University of Oxford, UK, OX39DU & UK Cystic Fibrosis Gene Therapy Consortium

We have recently developed CpG-free plasmid (pDNA)vectors that direct sustained, inflammation-free transgeneexpression when delivered to the lungs of animal models(Hyde et al, 2008 Nature Biotechnology 26:549). The deliveryof such vectors expressing the CFTR epithelial chloridechannel to the lungs of individuals with Cystic Fibrosis (CF) isbeing evaluated clinically. However CF lung pathology isalso linked to excessive sodium absorption via the epithelialsodium channel ENaC. Here we describe the development ofCpG-free pDNA vectors to express RNA interference (RNAi)molecules suitable for ENaC inhibition. Conventionally, suchvectors utilise polIII promoters to express short hairpin RNA(shRNA) sequences. However, the high expression levelsachieved by such vectors have proven toxic in some systems,limiting their duration of action (Grimm et al, 2006 Nature441:537). To facilitate benign, long-term RNAi, we utilised thehCEFI polII enhancer=promoter element and expressedshRNA sequences embedded in a miR30 miRNA backbone.As the native miR30 backbone contains 4 CpGs, we created aCpG-free form (miR30DCpG) containing additional com-pensatory nucelotide changes to conserve the RNA secondarystructure – essential for efficient miRNA=shRNA processingby Drosha=Dicer RNAi pathway. A version of this novelCpG-free RNAi expression vector fitted with an shRNA looptargeting luciferase, was co-transfected with a luciferasetransgene expressing plasmid into HEK293T cells. Encourag-ingly, efficient *80% knockdown of luciferase expressionwas observed (p¼ 0.042) in these cells, along with *60%knockdown in similar experiments using cell lines expressingluciferase constitutively (p¼ 0.025). Subsequent vector re-finements and in vivo studies will be discussed.

P 31

Nonclinical Studies for the Treatment of Parkinson’s

Disease for a First in Man Clinical Trial Using DirectAdministration of a Lentiviral Vector (ProSavin�)

Loader J (presenting), Day D, Ferrige G, Angell-Manning D,Esapa M, Mitrophanous KA, Miskin J

Oxford Biomedica UK Ltd, Medawar Centre, Oxford Science Park,Oxford, OX4 4GA

The EIAV-based Lentiviral vector, ProSavin�, is currentlybeing evaluated in a phase I=II first in man (FIM) clinical trialfor the treatment of Parkinson’s disease1 using direct in-trastriatal administration to convert cells into dopamine fac-tories. ProSavin corrects a variety of motor deficiencies in the

nonhuman primate (NHP) MPTP model of Parkinson’s dis-ease for up to 36 months.

Three nonclinical studies performed prior to gaining reg-ulatory approval for the ProSavin FIM study are brieflysummarised below.

Vector shedding and dissemination was investigated in therat at 1 hour, 24 hours and seven days (qPCR); approximately0.5% of the administered vector was detected in the CSF atone hour (this reduced *800-fold over 24 hours). Woundswab samples taken after surgery contained *0.1% of theadministered vector (this was reduced *10-fold with disin-fectant). Low, non-quantifiable levels of vector were detectedin some plasma and urine samples at 1 and 24 hours, butsequences were not detected by 7 days.

Biodistribution was investigated in the rat at 3 days, 30days and 6 months (qPCR). The vast majority of ProSavinsequences were detected in the brain.

Toxicology was assessed in rat and NHP; in-life moni-toring and toxicological endpoints revealed no ProSavin-related toxicities or adverse effects. Antibodies to VSV-Gwere detected in a subset of animals, but not to otherstructural components or the transgenes. Real-time PCRanalysis revealed insignificant ProSavin-derived circulatingvector RNA and DNA; vector RNA was not detected in theCSF.

P 32

Development of Persistently Expressing Vectors

for the Nonviral Gene Therapy of Choroideremia

Ostad-Saffari E (presenting), Wong SP, Tracey-White DC,Argyros O, Coutelle C, Harbottle RP, Tolmachova T,Seabra MC

Molecular Medicine, Imperial College London, London,SW7 2AZ, UK

Nonviral gene therapy is an attractive and promising al-ternative to a viral-based approach due to biosafety and lowimmunogenicity of plasmid-based vectors. Novel vectorsemploying scaffold=matrix attachment regions (S=MARs)provide stable episomal maintenance, resistance to epigeneticsilencing and are capable of sustained expression in murinetissues. Choroideremia (CHM) is an X-linked chorioretinaldegeneration, currently incurable, which is caused by the lossof function of the CHM=REP1 gene.

We generated S=MAR based vectors that contained eitherCHM=REP1 cDNA or EGFP driven by the EFS (humanelongation factor 1 short) promoter. pEOS-Rep1 plasmidprovides expression of hREP1 and similarly, EGFP expressionconferred by plasmid pEOS-EGFP was confirmed by FACSafter transfection of the B16-F10 cell line. We establishedpermanent AtT20 pEOS-EGFP cell lines. Stable maintenanceof pEOS vectors was monitored during culturing AtT20pEOS-EGFP cells over a period of time with and withoutselective pressure. Expression levels of the transgene did notdiminish after 6 weeks. Furthermore the cationic polymerpolyethylenimine (PEI) was used to condense vector DNA forin vivo experiments. DNA:PEI complexes were successfullydelivered to the eye by subretinal injections, were EGFP ex-pression was observed from pEOS-EGFP plasmid within theRPE cell layer of the eye.

1Prof. Stephane Palfi, Principle Investigator, Henri Mondor Hos-pital, France (EudraCT Number: 2007-001109-26).


We have developed an S=MAR vector expressing theCHM=REP1 protein and this vector has the potential tobe used as a therapeutic agent for the nonviral gene ther-apy treatment of CHM. Our experiments demonstrate thatS=MAR vectors are delivered to the RPE and thus could beused for the non-viral treatment of the RPE defects in oculardiseases.

P 33

Gene Therapy Studies in the Mouse Modelof Choroideremia

Tolmachova T (presenting), Tracey-White DC, Tolmachov O,Graham HJ, Seabra MC

Molecular Medicine, NHLI, Imperial College London

Choroideremia (CHM) is a progressive retinal degenera-tion caused by defects in the CHM=REP1 gene (Xq21.2). Pa-thological symptoms include night blindness and tunnelvision in teenage patients followed by slow demise of visionwithin next 2–3 decades and blindness. CHM=REP1 geneencodes Rab Escort Protein-1 (REP1), which participates inthe lipid modification of Rab GTPases. The resulting dysfunc-tion of Rabs is believed to be the trigger for retinal degener-ation in CHM. CHM is a perfect candidate for a gene therapysince patients can be diagnosed before major pathologicalchanges occur and slow progress of the disease provides atime window for the treatment. Recently we created a con-ditional knock-out mouse model of CHM that mimics humancondition. The aim of our current investigation is to performpreclinical gene therapy study in CHM mouse model.

Firstly we generated HIV-based lentiviral vectors expres-sing CHM=REP1 and=or GFP. We transduced canine cell lineD17 and primary fibroblasts derived from CHM patients withour lentiviral vectors and observed persistent expression ofthe transgenes. REP1 functionality was confirmed by in vitroprenylation assay using cell cytosolic extract as a source ofunprenylated Rabs. Rab prenylation defect in CHM fibro-blasts was rescued by Rep1 lentiviral vectors. Next we de-livered the vectors into subretinal space of the mouse eye andobserved long-term expression of CHM=REP1 and GFP in theRPE as long as 6 months post injection, while photoreceptorswere not transduced. In the RPE of injected CHM mouse eyeswe achieved correction of the prenylation defect. Similarstudy employing adeno-associated viral (AAV) vectors isunderway.

P 34

Towards Gene Therapy of the scid Mouse by Gene Repair

Abdul-Razak HH (presenting)1, Molina Estevez FJ1, GanCHV2, Roberts A3, Gregory PD4, Holmes MC4, Kinnon C2,Thrasher AJ2, Yanez-Munoz RJ1

1School of Biological Sciences, Royal Holloway-University ofLondon, Egham2Institute of Child Health, University College London3Department of Medical and Molecular Genetics, King’s CollegeLondon4Sangamo BioSciences, Inc., Richmond, California, USA

The ultimate goal for gene therapy of inherited diseases isto repair the defective gene at its chromosomal location. Thiscan be achieved by homologous recombination-mediatedgene targeting. Traditionally considered a low-efficiencyprocedure, gene targeting frequencies have recently been in-creased several orders of magnitude through two technicalimprovements: (i) efficient DNA delivery platforms, includingadeno-associated virus (AAV) vectors and integration-deficient lentiviral vectors (IDLVs); and (ii) designer nucle-ases, able to induce double-strand breaks at the target locus.However, no animal model of inherited disease has yet beencorrected by gene repair. We are developing an ex vivo sys-tem to correct the classical scid mouse model, which has apoint mutation in Prkdc, the gene encoding the catalyticsubunit of DNA-dependent protein kinase. We have pro-duced targeting constructs to correct the scid Prkdc mutationand incorporated them into IDLVs. We have also produced anumber of zinc-finger nucleases (ZFNs) directed against thetarget locus and included the ZFN genes into lentiviral vec-tors. Testing the plasmid targeting constructs on scid fibro-blasts we noticed extensive senescence in the clones initiallyrecovered. To overcome this problem and extend their life-span we are in the process of transducing the scid fibroblastswith a lentivector carrying TERT, the gene encoding the cat-alytic subunit of telomerase. We will first attempt to correctthe scid mutation in the immortalised fibroblasts. We willfollow up these experiments with attempts at ex vivo correc-tion of scid haematopoietic progenitors, transplantation ofthe corrected progenitors into scid mice and phenotypic an-alyses.

P 35

Correcting SMN2 Splicing with Tailed AntisenseOligoribonucleotides: A Potential Therapeutic Strategyfor Spinal Muscular Atrophy

Zhou H (presenting)1, Owen N2, Eperon I2, Muntoni F1

1Dubowitz Neuromuscular Unit, UCL Institute of Child Health,30 Guilford Street, London2Department of Biochemistry, University of Leicester, Leicester

In 95% of cases spinal muscular atrophy (SMA) is caused bythe homozygous deletion of the survival motor neuron gene,SMN1. The neighbouring SMN2 gene has nearly identical se-quence and can partly compensate the defect by expressinglimited amounts of functional, full-length SMN protein.However, a single-nucleotide C?T transition in SMN2 at po-sition 6 of exon7 causes predominant exon7 skipping and re-sults in an unstable truncated protein (SMND7). It is proposedthat an ESE recognized by the SR protein SF2=ASF has beendisrupted by C?T transition in SMN2 and then results inexon7 skipping. In order to increase the efficiency of the de-fective ESE in SMN2, we have developed bifunctional oligor-ibonucleotides that comprise an antisense sequence that canbase-pair with exon7 and a tail with motifs that can bindSF2=ASF. We named these targeted oligonucleotide enhancersof splicing (TOES), and demonstrate here that a series of themincrease the inclusion of exon7 both in vitro and in SMA patientderived fibroblasts. These TOES are capable of increasing theratio of full length SMN to SMND7 at RNA level detected by


quantitative real-time PCR and increase the level of SMNprotein by western blotting. One particular TOES appears to bevery efficient in inducing SMN2 exon 7 retention both in vitro,and in cellular models. The efficiency of this TOES is far su-perior to that demonstrated for other approaches such as his-tone deacetylase inhibitors, protein phosphatase inhibitors andantisense oligonucleotides targeting other SMN2 sequences,and will now be tested in vivo, using an animal model of SMNdeficiency.

P 36

Towards iPS Cell-Based In Vitro Models of SpinalMuscular Atrophy Using Integration-Deficient Lentiviral

Vectors

Wanisch K (presenting)1, Tilgner K2, Yang C2, Neganova I2,Ekonomou A3, Armstrong L2, Lako M2, Yanez-Munoz RJ1

1School of Biological Sciences, Royal Holloway-Universityof London, Egham2Institute of Human Genetics and NESCI, Newcastle University3Wolfson Centre for Age-Related Disease, King’s College London

In humans, the loss of function of SMN1 (survival motorneuron 1) in alpha motor neurons of the spinal cord causesSMA (spinal muscular atrophy). A variety of gene therapyapproaches are being tested for SMA treatment, includingdelivery of SMN1, trophic factors or constructs that modulatemRNA splicing. With the recent development of iPS (inducedpluripotent stem) cell technology, it is possible to derive ES(embryonic stem)-like cells from somatic cells like e.g. fibro-blasts. iPS cells are commonly generated using integratingretroviral or lentiviral vectors which introduce an establishedcombination of (mostly) 4 defined transcription factors.Subsequently, iPS cells can be induced to differentiation intomany cell types, including motor neurons. We are keen togenerate iPS cell models from a cohort of Spanish SMA pa-tients, with three major aims: (i) to improve our under-standing of SMA; (ii) to test potentially therapeutic vectors;and (iii) to produce clinically applicable patient-specific iPScells that could be used in a therapeutic setting. For the lattercase, it would be crucial to avoid stable integration of the iPS-inducing transgenes, which we hope to achieve with the useof integration-deficient lentiviral vectors (IDLVs). We havedemonstrated efficient transduction of human fibroblastswith IDLVs expressing eGFP. We have then compared ex-pression levels of iPS cell-inducing transgenes from IDLVs orstandard integrating lentivectors driven by the CMV or CAGpromoters. We are currently attempting to generate wild-type human iPS cells using IDLVs or integrating lentivectorsdriven by the more efficient CAG promoter.

P 37

Development of a Minimally Invasive and SelectiveTechnique for Hydrodynamic Gene Therapy of Liver

Segments in the Pig

Khorsandi SE (presenting)1, Zacharoulis D2, Jiao L1, LevicarN1, Jensen S1, Habib N1

1Hammersmith Hospital, Imperial College London2Larissa University School of Medicine, Larissa, Greece

Background: The clinical introduction of gene therapy hasbeen limited by technical issues. Hydrodynamic gene therapy(HGT) appears to be a promising solution, with rapid intra-venous injection of large volume plasmid DNA (pDNA)producing effective gene transfer in the liver. Moving fromthe small to large animal model has been problematic, withconcerns that the massive fluid shifts involved would pre-cipitate heart failure.

Methods: Twelve pigs were used. Under general anaes-thesia the internal jugular vein was exposed and a modifiedballoon catheter advanced under fluoroscopy into the lefthepatic vein. The balloon was inflated with 2–4ml of salineand hydrodynamic pDNA injection undertaken. Differentplasmids were constructed to express either human (n¼ 5) ormurine thrombopoietin (n¼ 1) or b-galactosidase (n¼ 1). Theamount of plasmid used per animal varied from 10–50 mgand volume of saline used for dilution varied from 200–300 ml. Blood samples were taken before and at various in-tervals afterward HGT. Immediately after HGT, ultrasoundassessment of the liver was performed. Animals were killedat intervals from Day 2 to 22 and liver biopsy taken.

Results: All animals tolerated the procedure. Liver ultra-sound did not demonstrate any gross changes. A transientrise in liver transaminases was observed on day 2 and 3,which returned to normal ( p> 0.05). b-galactosidase stainingwas low at 5%. A reproducible and persistent elevation inplatelet count was not observed, irrespective of alteration inplasmid design.

Conclusions: Minimally invasive selective HGT of liversegments appears to be technically safe but further work isrequired to optimise efficiency of gene transfer.

P 38

Efficient Transduction of the Spinal Cord with Integration-Deficient Lentiviral Vectors

Ahmed SGO (presenting)1, Peluffo H1, Foster E2, Lago N2,Moon L2, Hutson T2, Yip P2, Wanisch K1, McMahon SB2,Yanez-Munoz RJ1

1School of Biological Sciences, Royal Holloway-Universityof London, Egham2Wolfson Centre for Age-Related Disease, King’s College London

We are interested in the development of gene therapystrategies for the treatment of neurodegenerative diseases,including Spinal Muscular Atrophy (SMA). SMA is a neuro-muscular disorder characterised by degeneration of motorneurons, caused by homozygous loss or mutation of SMN1.We have recently produced integration-deficient lentiviralvectors (IDLVs), which fail to integrate as proviruses and areinstead converted into episomal circles. IDLVs are efficientgene expression vectors in vitro and in vivo, particularly inquiescent tissues. We have embarked in an extensive study tocompare the transduction efficiency of IDLVs and matchedintegrating lentivectors in spinal cord tissues in vitro andin vivo. Intra-spinal injections of IDLVs or integrating lenti-vectors expressing eGFP showed similar transduction levelsof cord motor neurons, reaching around 50% in the injectedarea. Much lower levels of transduction were observed inastrocytes. We have also transduced purified motor neurons,


astrocytes and microglia from the cord, and dorsal rootganglia (DRG) neurons. Transduction of purified motorneurons and DRG neurons reached similarly high levels re-gardless of the vector integration proficiency. Astrocyteswere very susceptible to transduction with both IDLVs andintegrating vectors, while microglia were clearly more effi-ciently transduced by the latter. In DRG neurons we have alsodemonstrated that IDLVs and integrating vectors are equallyefficient at mediating RNAi against TRPV1. Taken together,our results demonstrate that IDLVs are highly efficient fortransduction in the spinal cord. We are currently developingthem for delivery of potentially therapeutic transgenes toanimal models of SMA.

P 39

Gene Therapy Strategy for Oculopharyngeal MuscularDystrophy (OPMD)

Trollet C (presenting)1, Benstead J1, Bales O2, Foster H1,Foster K1, Malerba A1, Popplewell LJ1, Graham I1, Mouly V3,Antoniou M2, Butler-Browne G3, Dickson G1

1Royal Holloway University of London (RHUL), Biochemistry,School of Biological Sciences, Egham, United Kingdom2Department of Medical and Molecular Genetics, King’s CollegeLondon School of Medicine, Guy’s Hospital, London, UnitedKingdom3Institute of Myology, Pitie-Salpetriere University Hospital, Paris,France

A broad range of degenerative diseases is associated withintracellular inclusions formed by toxic, aggregation-pronemutant proteins. Intranuclear inclusions constitute a patho-logical hallmark of oculopharyngeal muscular dystrophy(OPMD), a rare inherited disease characterised by late-onseteyelid drooping (ptosis), swallowing difficulty (dysphagia)and proximal limb weakness. OPMD is caused by expansionof a trinucleotide (GCG) repeat in the gene that encodes fornuclear poly(A) binding protein (PABPN1). The mutationresults in an extended polyalanine stretch that has beenproposed to induce protein aggregation and formation ofintranuclear inclusions.

The aim of our study is to develop a gene therapy strategyfor OPMD by combining RNAi knockdown and gene re-placement approaches for PABPN1.

To test this strategy we are using various cellular and an-imal models that have been developed: one consists of clonesof conditionally immortalised primary myoblasts (H-2Kb)stably transfected to express wild-type or mutant humanPABPN1 under the control of the human desmin locus con-trol region (DES-LCR); another consists of a transgenic mouseexpressing wild-type or mutant bovine PABPN1 under thecontrol of the human skeletal actin promoter; We also haveaccess to myoblasts isolated from OPMD patients.

We have designed and validated optimal siRNA sequencestargeting PABPN1. We are currently testing shRNA adeno-associated viral vectors (AAV-H1-shRNA) for persistentRNAi-mediated knockdown of PABPN1 mRNA. We alsohave generated a codon-optimised sequence for wild-typehuman PABPN1 with base changes at siRNA target se-quences for a replacement strategy.

In parallel, we are using these genetic tools and models towork on a better characterisation of the disease, with thesearch for markers of OPMD.

P 40

Genetically Modified Adipose Tissue DerivedMesenchymal Stem Cells (AT-MSC) Overexpressing

CXCR4 Display Increased Motility, Invasiveness andHoming to Bone Marrow of NOD/SCID Mice

Bobis-Wozowicz SE (presenting)1, Zawisz A2, Majka MM1

1Department of Transplantation, Jagiellonian University, MedicalCollege, 265 Wielicka Street, Krakow, Poland2Private Clinic of Plastic Surgery, 3 Nowowiejska Street, Krakow,Poland

Bone marrow mesenchymal stem cells (BM-MSC) havealready been shown to be useful in cell-based therapies, dueto their ability to home and engraft into ischemic or damagedtissues and to support regeneration. However, the level ofengraftment is usually low and the therapeutic effect di-minishes in the long term. To overcome that obstacle MSCmight be engineered to overexpress a chemokine receptorCXCR4, which is essential for migration and homing. Thisstudy evaluates capability of genetically modified AT-MSCoverexpressing CXCR4 to migrate, invade, home and engraftto bone marrow of NOD=SCID mice.

AT-MSC were isolated from lipoaspirates of human heal-thy donors with liberases. Antigen profile was analyzed byFACS. Passage 3-4 AT-MSC were transduced with retroviralvector carrying cDNA for CXCR4 or GFP. Neo resistant col-onies were selected and used in experiments.

Genetically modified AT-MSC showed constant expressionof CXCR4 or GFP, approximately 85%. The cells maintainedthe ability to differentiate into osteocytes and expressed early-progenitor genes: Runx2, PPAR-g. CXCR4-ATMSC displayedenhanced migration toward increasing doses of SDF-1 withthe highest level of migration at 100 ng=mL. Up-regulation ofCXCR4 led to 188 times greater migration and 20 times higherinvasiveness through a matrigel comparing to GFP-AT-MSC.Transplantation of CXCR4-AT-MSC into NOD=SCID miceresulted in increased engraftment into bone marrow showingthe potential implications in cellular therapies of bone dis-orders.

Summarizing, we showed for the first time that AT-MSCover-expressing CXCR4 appeared to be a powerful tool forregenerative medicine and gene therapies, used as an alter-native to BM-MSC.

P 41

Towards the Generation of a Baculovirus Display Libraryfor Cell Targeting

Westenberg M (presenting)1, Soedling HM1, Nicholson LJ2,Mann DA3, Dolphin CT1

1King’s College London, Pharmaceutical Sciences Division,London SE1 9NH2King’s College London, The Rayne Institute, St Thomas’Hospital, London SE1 7EH


3Liver Research Group, Institute of Cellular Medicine, NewcastleUniversity, Newcastle upon Tyne NE2 4HH

The baculovirus (BV) AcMNPV is a promising gene de-livery vector—the virus can accept large DNA inserts, isunable to replicate in mammalian cells and is Class 1 status.However, its broad tropism, mediated by its envelope fusionprotein GP64, makes cell targeting difficult. At present, 43BVs have been sequenced of which the majority do not con-tain GP64. Instead, they express an unrelated envelope fusionprotein F with structural similarities to fusion proteins ofmammalian viruses, e.g., paramyxoviruses. Using counter-selection recombineering we have seamlessly replaced thegp64 ORF in an AcMNPV replicon with several exogenousF-protein genes. A gfp marker gene, driven by a dual (insect=mammalian) promoter, was also introduced to monitor in-fection and transduction. Transfection of these bacmids intoinsect cells led to progeny virions with reduced titres of ap-prox. 1 log. Transduction in Soas2, HeLa and HepG2 cellswith 100 pfu=cell was reduced from approx. 90% to 1–2%,probably because mammalian cells lack an F protein receptor.A cell-type-specific targeting peptide displayed on the virusmight rescue transduction via receptor-mediated endocyto-sis. Therefore a synthetic construct encoding the VSV-G stemdisplaying a c-Myc tag was introduced into the viral genomeand the presence of the c-Myc epitope on the virions con-firmed. The c-Myc epitope will be replaced, via recombiner-ing, by a random 7-mer sequence to generate a baculoviruspeptide display library. We aim to identify peptides confer-ring cell-type-specific targeting via in vitro and in vivo bio-panning protocols.

P 42

Minimised CD34 QBEND10 Epitope as a Clinical GradeMarker Gene

Philip B (presenting), Thomas S, Jathoul A, Brewin J, LinchD, Pule M

UCL Cancer Institute, Paul O’Gorman Building 72 HuntleyStreet London WC1E 6BT

Adoptive immunotherapy with engineered T-cellshas shown therapeutic promise. A marker-gene wouldbe useful for measuring transduction efficiency and sort-ing transduced cells. Previous marker-genes include the xe-nogeneic NeoR and human-derived dNGFR and dCD34genes; however these are limited by immunogenicity andsize.

An ideal marker-gene should be: non-immunogenic, havecell surface expression, be easily detectable, possess a com-pact coding sequence, enable clinical grade sorting whilstremaining biologically inert. We have minimized the CD34-epitope which binds the monoclonal antibody QBEND10used in Miltenyi CD34 CliniMACS systems. Cell-surfacedisplay was optimised by comparison of: flush expressionwith a transmembrane domain (TMD), versus attachmentto a CD8 stalk. The stalk-bound constructs demonstratedbinding affinity approaching full-length CD34. Epitopeminimisation involved sequential deletions producing anepitope of 16 residues. This complete marker-gene is 135

amino acids in length compared with 385 in native CD34and enables detection by FACS and clinical grade sortingvia CliniMACS reagents. Due to the minimal size andhuman origin, this protein should be non-immunogenicand biologically inert. The small coding sequence allows co-expression with additional genes using the FMD-2A peptide.CD34ep-8 should represent a useful marker-gene for T-celltherapy.

P 43

Novel Cleavable PEGylated Liposomes for Enzyme

Triggerable Release of Nucleic Acid in Tumours

Yingyuad P (presenting), Mevel M, Thanou M, Miller AD

Genetic Therapies Centre, Department of Chemistry, ImperialCollege London, London, UK. SW7 2AZ

A stealth surface modification of liposomes delivery sys-tem using polyethylene glycol (PEG) can increase liposomes’stability in blood circulation, however; it seems to blockintracellular uptake of the liposomes. Here we have devel-oped a dePEGylation approach based on specific proteolyticenzyme activities overexpressed on tumour cells. A shortpeptide, an enzyme substrate, was introduced between PEGand a fusogenic lipid via coupling reaction in order tosynthesize a cleavable PEGylated peptide lipid (PPL). ThePPL was then incorporated into liposomes. Luciferasetransfection of high charged liposomes on OVCAR cell linein 10% serum showed an increase in protein expression byliposomes containing PPL in the presence of the enzyme.The specificity of the peptide linker was confirmed by thedecrease in transfection efficiency of liposomes containingPEGylated lipid (PL), with the absence of the peptide linker.Low charge liposomes diminish non-specific interaction ofthe liposomes and cell membrane. Transfection studies werecarried out on MCF-7 cell lines and in various serum con-centrations. As the serum concentration was increased, thetransfection level of liposomes containing 4% PPL in thepresence of the enzyme was increased. The transfection ef-ficiency was significantly higher than that of the liposomesin the absence of the enzyme and the liposomes containingPL. The fluorescence microscopy correlated well with thetransfection data, suggesting that the peptide cleavage andhence dePEGylation has been catalyzed by the enzyme re-sulting in enhance cellular uptake.

P 44

Targeting Adenovirus via Endothelial Selectinsfor Cancer Therapy

Bachtarzi H (presenting), Roesen N, Briggs S, Carroll F,Fisher K, Seymour LW

Department of Clinical Pharmacology, Old Road CampusResearch Building, University of Oxford, Old Road Campus,Headington, Oxford, UK. OX3 7DQ

Tumour vasculature provides an accessible and vulnerabletarget for systemic gene therapy using targeted adenoviruses.This approach is particularly important for treatment of dis-seminated metastatic cancer.


We have explored the use of Streptococcus aureus protein G(SpG) as a versatile linker enabling antibody-mediated re-targeting of tropism-ablated polymer-coated adenovirus. Totarget viral infection via endothelial E-=P-selectins, a mouseanti-human E-selectin antibody (MHES mAb) and a chimericP-Selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion proteinwere linked (via their Fc regions) to SpG-modified polymer-coated virus encoding luciferase.

A concentration as low as 10mg=ml of MHES mAb couldspecifically target SpG-pcAdluc to TNF-a-activated E-selectin-expressing human endothelial cells resulting in a 4-foldincrease in transgene expression (relative to IgG2a control-modified virus) and a significantly increased transductionover unmodified virus. Competition with free MHES mAbresulted in 6.5 times reduction in transduction efficiency ofMHES-SpG-pcAdluc, while incubation with excess fiber in-hibited transduction by unmodified virus 5-fold, with no ef-fect on the retargeted virus.

Efficient delivery of transgene to P-selectin-expressing en-dothelium was demonstrated using PSGL-1-Fc on bEnd-3 cellline, resulting in improved transduction of the weakly per-missive mouse endothelium, with 27-times higher transgeneexpression compared with unmodified virus.

PSGL-1-Fc-SpG-pcAdluc showed better circulation kineticsthan Adluc following systemic administration in EL-4 tu-mour-bearing mice. Immunostaining of cryostatic tumoursections revealed more luciferase staining in tumours fromanimals treated with the retargeted virus than those treatedwith Adluc.

Optimal targeting is likely to be inhibited by susceptibilityof Fc-SpG interaction to competition from plasma IgGs. Fu-ture work will explore directly conjugated low molecularweight selectin-binding ligands.

P 45

Use of Macrophages to Target Therapeutic Adenovirus

to Hypoxic Areas of Human Prostate Tumors

Muthana M1, Giannoudis A2, Scott SD3, Morrow FJ1, CoffeltSB1, Murdoch C4, Cross N5, Burke B6, Mistry R7, Hamdy F8,Brown NJ9, Georgopoulos LJ (presenting)10, Hoskin P11,Lewis CE1, Maitland NJ10

1Academic Unit of Pathology, University of Sheffield, Sheffield, UK2Department of Haematology, University of Liverpool, Liverpool,UK3Medway School of Pharmacy, University of Kent, ChathamMaritime, Kent, UK4Dept. Oral & Maxillofacial Surgery, University of Sheffield,Sheffield, UK5Faculty of Health and Wellbeing, Sheffield Hallam University,Sheffield, UK6Department of Infection and Immunity & Inflammation,University of Leicester, Leicester, UK7Enterprise & Business Development Office, University ofLeicester, Leicester, UK8Academic Unit of Urology, University of Sheffield, Sheffield, UK9Academic Unit of Surgical Oncology, University of Sheffield,Sheffield, UK10YCR Cancer Research Unit, Department of Biology, Universityof York, York, UK11Mount Vernon Hospital, Northwood, Middlesex, UK

The systemic application of viral and nonviral vectors inanti-cancer gene therapies has met with little success, largelydue to poor delivery and penetration beyond the perivascularareas of tumors. In tumors, monocytes continuously extrav-asate from the bloodstream where they differentiate intomacrophages, and then accumulate in hypoxic=necrotic ar-eas. These areas are notoriously difficult to treat as they arepoorly vascularised and thus difficult to access using con-ventional, systemic therapies. We have devised a system touse macrophages to deliver therapeutic virus to abundanthypoxic centres in prostate cancers. Macrophages were co-transduced with a hypoxia-regulated E1A=B plasmid and anE1A=B-deficient adenovirus, containing a prostate-specificantigen (PSA)-GFP reporter cassette. When co-cultured withprostate tumor 3D spheroids, these ‘armed’ macrophagesmigrated into the hypoxic centres where E1A=B protein ex-pression was up-regulated, resulting in replication of the la-tent E1A=B-deficient adenovirus. Approximately 5000 virusparticles per macrophage were released and infected neigh-bouring prostate tumor cells, resulting in widespread GFPexpression. Systemic administration of co-transduced humanmacrophages into immunocompetent mice bearing prostate(PC3) xenograft tumours resulted in macrophage traffickinginto the hypoxic areas of these tumors, leading to viral rep-lication and infection of adjacent tumor cells. This novel,targeted gene therapy approach employs three distinct levelsof tumor-specificity; the homing of macrophages to tumors,the synthesis and release of therapeutic adenovirus only inhypoxia tumor areas, and the restriction of therapeutic geneexpression to prostate tumor cells.

P 46

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P 47

Naked DNA Encoding for Brain Derived Neurotrophic

Factor and TTC Fusion Protein Delays the DiseaseProgression and Prolongs Survival in SOD1G93A Mice

Moreno M, Calvo AC (presenting), Olivan S, Manzano R,Ciriza J, Munoz MJ, Zaragoza P, Osta R

Laboratorio de Genetica Bioquımica (LAGENBIO-I3A), Facultadde Veterinaria, Universidad de Zaragoza, C=Miguel Servet, 177,50013 Zaragoza, Spain

Amyotrophic lateral sclerosis (ALS) is one of the mostdevastating neurodegenerative diseases known. Neuro-trophic factors, such as BDNF, have been widely tested in anattempt to counteract neurodegenerative conditions. How-ever, unspecific neuronal access of neurotrophic factors mightcause that no effective therapy has been yet translated tohumans. The atoxic C-terminal fragment of tetanus toxinheavy chain (TTC) has not only been studied as a molecule-carrier to the CNS for its high neuron specificity, but also hasbeen described to possess neuroprotective properties. Re-combinant plasmid-vector genetically modified to expressBDNF-TTC fusion protein was administered by a single in-tramuscular injection in a mouse model of ALS. Our resultsindicate that the fusion-molecule or TTC treatment signifi-cantly delayed the onset of symptoms and functional defi-cits, and prolonged lifespan. However, BDNF treatmentalone did not give significant clinical improvement. Usingwestern blot analysis, we observed inhibition of proapoptoticpathways in the spinal cord of all treated SOD1G93A

mice groups, such as the activation of caspase-3 or down-regulation of Bax protein levels. Phosphorylation of Akt, anindicator of the activation of PI3K survival pathway, was alsodetected after treatments. Although BDNF-TTC treatmentshowed a trend of being the most effective treatment, nosignificant difference was found when compared to TTCtreated mice. These results suggest that nonviral gene therapyusing the TTC- or BDNF-TTC-encoding plasmids provides apotential therapy for ALS.

P 48

Chimeric Immune Receptor Function Depends onMultiple Activation Mechanisms and May Be PartiallyDependent on T-Cell Receptor Interactions

Bridgeman JS (presenting), Hawkins RE, Gilham DE

Cancer Research UK Department of Medical Oncology, PatersonInstitute for Cancer Research, University of Manchester,Manchester, UK

Chimeric Immune Receptors (CIR) are an attractive strat-egy for gene-modified T-cell therapy of multiple malignan-cies. In our lab we are investigating the function of CEAtargeted CIR consisting of CD3z fused to the CEA-specificscFv MFE23. As these receptors contain the CD3z transmem-brane domain required for endogenous CD3z-T-Cell Receptor(TCR) interactions, we sought to examine whether CIR wereincorporated into the TCR complex. We were able to dem-

onstrate that MFE23.CD3-z could reconstitute TCR surfaceexpression in a CD3z-deficient cell line and that this recon-stitution was dependent upon CIR dimerisation and a chargedaspartic acid residue in the transmembrane domain (D6).Using FRET we have demonstrated an interaction betweenMFE23.CD3z and the TCR in Jurkat T-cells. We have alsodeveloped a novel, bead-based FACS assay which supportsthe FRET data. To further clarify the signalling capacity ofCIRs we cloned tyrosine mutant receptors with Y-F aminoacid substitutions in the cytoplasmic domain. MFE23.CD3zY-F still retained much of its function, an observation attrib-uted to its capacity to heterodimerise with endogenous CD3z.However, a mutant CIR only capable of homodimerisationcould still upregulate CD69. This observation was not seenwhen repeated in TCR-negative Jurkat cells. We proposethat there are at least four independent mechanism underly-ing CIR activation of T-cells: 1) via the antigen ligatedchain; 2) via the antigen unligated chain of homodimers; 3)via the endogenous CD3z component of heterodimers; 4)via other components of the TCR complex. We are conduct-ing further experiments to elucidate these multiple activa-tion mechanism.

P 49

Optimising the Quality of RNA from Ovine Lung Tissuesfor Microarray Analysis

Yahaya B (presenting), McLachlan G, Baker A, Tennant P,Vrettou C, Collie DDS

The Roslin Institute & R(D)SVS, Easter Bush Veterinary Centre,University of Edinburgh, Roslin, Midlothian EH25 9RGEdinburgh Scotland

RNA quality and integrity are key factors that significantlyinfluence the utility of microarray analysis. In this study wesought to optimise RNA isolation techniques for ovine lungtissue that best preserved the quality and quantity of RNAavailable for subsequent microarray analysis. The primaryindices of quality were the OD 260=280 and 28S=18S ratiosand the RIN. Lung cell and tissue samples were harvestedeither by bronchoscopy under anaesthesia or at nec-ropsy examination and immediately processed to preservethe RNA. Thereafter subsequent processing of tissue samplesemployed a selection of protocols that facilitated an assess-ment of both the reproducibility of RNA extraction under setconditions and the selection of an optimised protocol forfuture studies. Samples were analysed and electrophero-grams generated by the Agilent microfluidic capillary elec-trophoresis BioAnalyzer 2100, allowing calculation of the28S=18S ratio and the RIN. Results were compared with vi-sual analysis of the electropherogram to determine the mostuseful RNA quality metric. We found that the 28S=18S andthe OD 260=280 ratios were poorly sensitive for detectingRNA quality, whereas the RIN was a sensitive index of RNAquality. Notably the quality of RNA available was depen-dent on the type of lung tissue (parenchyma vs cartilaginousairway vs bronchial brush biopsy samples) selected for ex-traction.


P 50

Development of Nonviral Vectors for Episomal

Maintenance and Mitotic Stability In Vivo

Wong SP (presenting), Argyros O, Coutelle C, Harbottle RP

Gene Therapy Group, Imperial College London, Exhibition Road,London SW7 2AZ

We recently developed the S=MAR vector system for in vivoapplication and demonstrated its utility to sustain transgeneexpression in vivo in the livers of mice. We reported that ourprototype plasmid vector, which contains an S=MAR domainand the luciferase reporter gene, showed transgene expres-sion for at least six months following a single administration.Subsequently, we have observed that transgene expression issustained for the lifetime of the animal. Following partialhepatectomy of the animals, however, we find no detectableS=MAR replication and subsequent propagation in vivo. Toovercome this, we developed a liver selection strategy to in-vestigate whether cells transduced with an S=MAR plasmidprovided them with a survival advantage over untransducedcells. This would confirm that these vectors are capable ofestablishing themselves as extrachromosomal entities andreplicate in the liver. We constructed a novel S=MAR plasmidexpressing a gene Bcl-2 that confers resistance to apoptotic-mediated challenges by a Fas-activating antibody Jo2. Fol-lowing hydrodynamic delivery into the livers of mice andfrequent Jo2 administrations, we show that the S=MARplasmid is capable of sustaining expression for over threemonths. Further analysis shows that the plasmid does repli-cate in vivo as shown by Southern blot. These results provideproof-of-principle that S=MAR vectors are capable of pre-venting transgene silencing, are resistant to integration andare able to confer mitotic stability in vivo.

P 51

Fetal Gene Therapy for Acute Neuronopathic Gaucher

Disease

Rahim AA (presenting)1, Wong AMS2, Hughes D1, BuckleySMK1, Karlsson S3, Cooper JD2, Thrasher AJ4, Mehta A1,Waddington SN1

1Department of Haematology, University College Medical School,London, UK2Department of Neuroscience, Institute of Psychiatry, KingsCollege London, London, UK3Molecular Medicine and Gene Therapy, Lund University, Lund,Sweden4Molecular Immunology Unit, Institute of Child Health,University College London, London, UK

Gaucher Disease (GD) is an autosomal recessive disordercaused by mutations in the glucocerebrosidase gene (GBA). Itis the most common lysosomal storage disorder and is di-vided into 3 main subgroups based on clinical phenotype andthe presence, and absence, of central nervous system (CNS)pathology. Although the visceral manifestation of the disease(hepatomegaly, splenomegaly, cytopenia and bone disease)

can be treated by enzyme replacement therapy, the neuro-degeneration in the CNS remains untreatable due to theblood brain barrier. As a result, patients with the infantileneuronopathic form of GD (Type II) usually do not survivepast 2 years of age. We have found that by administeringlentiviral vectors (both integrating and non-integrating) inutero to the brains of mice we are able to achieve morewidespread delivery compared to that observed in adultmouse brain. We constructed lentiviral vectors encoding theGBA gene driven by the spleen focus forming virus promoter(SFFV) and tested these in vitro. EBV transformed cells takenfrom Gaucher Type II and primary monocytes from GaucherType I patients were transduced and assayed for enzymeactivity. In all cases, glucocerebrosidase was expressed abovenormal physiological levels. These lentiviral vectors will beused to investigate the rapid neurodegeneration seen in thebrain of a mouse model of GD and whether they can slow-down its progress.

P 52

Development of Enhanced Lentiviral Vectors for Gene

Therapy of ADA-SCID

Montiel-Equihua CA (presenting)1, Monkeviciute A1,Antoniou M2, Thrasher AJ1, Gaspar HB1

1Molecular Immunology Unit, Institute of Child Health2Molecular Genetics, King’s College London

Deficiency of metabolic enzyme adenosine deaminase(ADA) leads to abnormal T, B and NK cell development,resulting in severe combined immunodeficiency (SCID).Promising results for correction of immunodeficiency havebeen obtained in phase I=II clinical trials in which a gam-maretroviral vector is used to transduce haematopoieticstem cells; however the risk of inadvertent gene activationobserved with this type of vector in other studies hasprompted the development of alternative vectors with im-proved safety profiles. Furthermore, LTR regulated vectorsmay be susceptible to gene silencing, and elements that en-sure high level long lasting expression may therefore bebeneficial. We are evaluating the inclusion of minimal ver-sions of the b-globin locus control region (LCR). The ratio-nale is to enhance expression of ADA in the erythrocytelineage to improve systemic detoxification, and at the sametime to achieve adequate expression in lymphoid precur-sors to ensure correction of immunodeficiency. To testthis system we have incorporated b-globin LCR hypersen-sitive sites HS4, HS3 and HS2 (LNT-432-EFS-GFP) upstreamof the housekeeping EF1alpha promoter. In murine ery-throleukaemia (MEL) cells, the inclusion of HS2, HS3 andHS4 in combination with the minimal EF1alpha promoter issufficient to enhance the expression of GFP upon differen-tiation into mature erythrocytes, with a *3-fold difference inthe mean fluorescence intensity (MFI) between the differ-entiated and the non-differentiated state. We have observedthat the presence of the minimal HS432 LCR does not alterthe expression of GFP in non-erythroid cell lines. These en-couraging results prompt further testing in relevant modelsystems of ADA-SCID.


P 53

Nonintegrating Lentiviral Vectors for Treatment

of Haemophilia B

Ward NJ (presenting)1, Apolonia L1, Cochrane M2,Buckley SMK3, Waddington SN3, Nathwani AC2,Philpott NJ1, Kinnon C1, Thrasher AJ1

1Molecular Immunology Unit, Institute of Child Health, UCL2Department of Haematology, UCL Cancer Institute3Department of Haematology, Royal Free and University CollegeMedical School

Integration deficient lentiviral vectors offer marked ad-vantages over currently used integrating vectors as side ef-fects caused by insertional mutagenesis are minimized.Previous work has shown that efficient and sustained trans-gene expression in nondividing cells, such as rodent ocular,brain and muscle tissue, using integration deficient vectorscan be achieved. Haemophilia B is an X-linked recessivedisorder caused by defects in the gene encoding coagulationfactor IX. Integrating lentiviral vectors have previously beenused to mediate long term expression of human factor IXin vivo. In this study we aim to investigate the use of non-integrating lentiviral vectors as treatment for Haemophilia Bwith liver cells, primarily nondividing hepatocytes, as aprincipal target.

Integrating and nonintegrating VSVg pseudotyped vectorsfor delivery of human factor IX (hFIX) cDNA were assessedfor duration of expression in vivo in adult C57=BL6 mice.After intravenous injection stable hFIX expression was de-tected from integrating vectors at 1500 ng=mL (30% normal),however no expression was identified from nonintegratingvectors. As assayed by quantitative PCR, mice injected withnonintegrating vector had significantly fewer viral genomesin liver compared with integrating vector. To further inves-tigate whether nonintegrating vectors could mediate expres-sion of a transgene in liver vectors expressing a luciferasetransgene were assessed in adult SCID mice. Overall, ex-pression from the nonintegrating vector was stable and sus-tained in liver but luciferase levels were significantly lowerthan seen with the integrating vector.

P 54

Lentivectors Expressing Chimeric Proteins Induce Specific

Tolerence to the Immunodominant Collagen PeptideAntigen in the Collagen-Induced Arthritis Mouse Modelof Disease

Ward EM (presenting)1, Eneljung T2, Gjertsson I2,Thrasher AJ1, Gustafsson K1

1Molecular Immunology Unit, UCL, Institute of Child Health2Department of Rheumatology and Inflammation Research,Goteborg University, Gothenburg, Sweden

Induction of specific tolerance to self-molecules is apromising potential future treatment in autoimmune dis-eases. However, it is not clear which method of delivery andpresentation of the offending peptide antigen would be mosteffective. We have used lentiviral vectors to express fusion

proteins designed to traffic the immunodominant collagen IIpeptide to MHCII-containing compartments of antigen pre-senting cells as a means of conferring specific tolerance tocollagen II. After demonstrating DC transduction, appropri-ate subcellular localization, and processing=presentation ofthe fusion protein by MHCII molecules to T-cells in vitro,initial in vivo studies in the mouse collagen-induced arthritismodel (CIA), have shown a protective effect. These resultsrepresent an encouraging indication that driving antigenpresentation in the context of MHCII could be an importantmethod of inducing specific tolerance to autoantigens.

P 55

Using the Sleeping Beauty Transposon Systemto Genetically Modify a Muscle Stem Cell Line

Muses S (presenting)1, Morgan JE2, Wells DJ1

1Gene Targeting Group, Department of Cellular and MolecularNeuroscience, Imperial College London, Hammersmith HospitalCampus, London, UK2Dubowitz Neuromuscular Unit, UCL, Institute of Child Health,30 Guilford Street, London, UK

Sleeping Beauty is a non viral gene transfer system thatconsists of a Class II DNA transposon (a transgene flanked byinverted terminal repeats) and a transposase. This system hasbeen previously used to stably integrate a variety of thera-peutic transgenes into the genomes of different of cell types.We have investigated Sleeping Beauty’s potential to stablyintegrate a detection marker (eGFP-IRES-neo) into the genomeof a new conditional immortal muscle stem cell line, H2K 2B4.The H2K 2B4 clone was generated from a single satellite cell.It proliferates and differentiates under selective conditionsand is able to differentiate in vitro, stably regenerate muscleand contribute to a functional satellite cell population in vivo.

To optimise the Sleeping Beauty system in the H2K 2B4 cellline transposition assays were set up comparing different ra-tios of Sleeping Beauty plasmids (Transposon:Transposase).H2K 2B4 cells which were co-transfected with the transpo-son and transposase had a greater number of neor colonies(up to an 8-fold increase) compared to the control, withouttransposase. This strongly suggested transposon mediatedintegration had occurred. Transposition sites are now be-ing sequenced to establish the distribution within thegenome. We are also using this system to stably integratemicro-dystrophin into a dystrophin deficient mouse cell line,H2K SF1, for subsequent transplantation into mdx mice.

P 56

Therapeutic Strategies for Rodent Models of Stroke

Rossetti T (presenting)1, Goncalves MB2, Yip P2, Doherty P2,Markus HS3, Hainsworth AH3, Yanez-Munoz RJ1

1School of Biological Sciences, Royal Holloway-Universityof London, Egham2Wolfson Centre for Age-Related Disease, King’s College London3Centre for Clinical Neuroscience, St George’s Universityof London


Stroke is a common cause of death and results in a majorburden to Health Services because of debilitating disabilitiesin survivors. Therapeutic options are limited and develop-ments leading to new treatments would be of significantimportance. The most common type of stroke is caused byocclusion of cerebral blood vessels resulting in widespreadcell death. Neurogenesis has been proposed as a possibletarget process for treatment, but endogenous adult neuro-genesis is very limited in humans. The goal of this project is tostudy and exploit ways of enhancing endogenous neuro-genesis, combining transgene expression mediated by safer,integration-deficient lentiviral vectors (IDLVs) and pharma-cological treatment. We will conduct these studies in a well-established rodent model of focal stroke, the middle cerebralartery occlusion (MCAo) model. To estimate the level of en-dogenous neurogenesis in rodents we have performed somepreliminary experiments injecting standard integrating len-tivectors or IDLVs encoding eGFP at the beginning of therostral migratory stream. This allows labelling of neurogenicprecursors in the subventricular zone (SVZ), one of the neu-rogenic areas of the rodent brain. Such injections have re-sulted in efficient labelling of neuroblasts, which we havedetected as differentiated neurons expressing eGFP in theolfactory bulb several weeks post-injection. The procedure isquantitative and we will next estimate levels of neurogenesisin the MCAo model with both IDLVs and integrating lenti-vectors. The use of efficient IDLVs would offer significantadvantages for potential therapeutic applications, becausethey have a much reduced risk of causing insertional muta-genesis compared with integrating lentivectors.

P 57

Secreted Gaussia Luciferase Is a Sensitive Reporterof Gene Transfer in the Sheep Lung

Griesenbach U (presenting)1, Vicente C1, Roberts M1,Green AM2, Cheng S3, Scheule R3, Pringle IA2, Gill DR2,Hyde SC2, McLachlan G4, Collie D4, Alton EWFW1

1Department of Gene Therapy, Imperial College and The UK CFGene Therapy Consortium2Gene Medicine Group, University of Oxford and The UK CFGene Therapy Consortium3Genzyme4Medical Genetics Section, College of Medicine and VeterinaryMedicine, University of Edinburgh and The UK CF Gene TherapyConsortium

Firefly luciferase (FLux) is a widely used reporter gene inmurine gene transfer studies. The cationic lipid GL67, is inour hands, the most efficient non-viral vector for lung genetransfer, leads to significant levels of FLux expression in themouse. However, we are unable to detect robust levels ofFLux in sheep after aerosolisation of GL67A=pCIK-FLux,despite significant expression of vector-specific mRNA. Re-cently a novel secreted luciferase reporter gene isolated fromGaussia princeps (GLux) has been described. We aerosolisedsheep with GL67A=pCMV-GLux complexes and quantifiedGLux in bronchoalveolar-lavage fluid (BALF), serum, bron-chial biopsies and brushings and in lung tissue pieces re-trieved at necropsy (n¼ 3 sheep). Compared with controls we

detected significant GLux expression in BALF (treated:6.9�104� 5.4�104 RLU=ml, control: 8.1�102� 1.4�102 RLU=ml) and lung tissue homogenate (treated: 3.2�104� 2.4�104 RLU=ml, control: 3.3�102� 1.6�102 RLU=ml). However,GLux expression was undetectable in biopsies and brushings,likely due to the very small amount of epithelial cells in thesesamples. Expression levels in the sheep lung were not highenough to lead to ‘‘spill-over’’ in serum, which was in contrastto mouse studies where GL67A=pCMV-GLux administrationto the lung by nasal sniffing led to significant levels of se-creted GLux in serum (treated: 2.9�102� 1.5�102 RLU=ml,control: 37.8� 14.8 RLU=ml). In conclusion, secreted Gaussialuciferase is a more sensitive reporter gene than firefly lucif-erase in the sheep lung. However, it is currently unclear, ifGLux is produced by alveolar or airway epithelial cells andsampling techniques have to be refined to address thisquestion.

P 58

Successful Transfection of Human Air Liquid InterfaceCultures with the Nonviral Gene Transfer Agent GL67A

Griesenbach U (presenting)1, Roberts M1, Vicente C1,Green AM2, Scheule R3, Cheng S3, Pringle IA2, Gill DR2,Hyde SC2, Xenariou S1, Alton EWFW1

1Department of Gene Therapy, Imperial College and The UK CFGene Therapy Consortium2Gene Medicine Group, Nuffield Department of ClinicalLaboratory Sciences, University of Oxford and The UK CF GeneTherapy Consortium3Genzyme

Human air liquid interface (ALI) are notoriously difficult totransfect and nonviral gene transfer has not been successfullydemonstrated in this model. We assessed GL67A-mediatedgene transfer in ALIs. We first complexed GL67A to a eu-karyotic expression plasmid carrying the commonly usedfirefly luciferase (pCIK-FLux). ALIs were transfected usingvarious amounts of DNA (2 and 10 mg), DNA:lipid ratios andtransfection times. 48 hr after transfection cell lysates wereprepared, but we were unable to detect lux expression in anyof the samples. Recently a novel secreted luciferase reportergene isolated from Gaussia princeps (GLux) has been de-scribed. We next transfected ALIs with GL67A complexed topCMV-GLux. ALIs were transfected using 10 mg pCMV-GLux=insert complexed to GL67A either at a 1:4 molar ratio(previously optimised for mouse nasal instillations) or at a 6:8molar ratio (previously optimised for clinical trials). This wasapplied to the apical surface of the ALIs. Significant levels ofGLux were secreted apically and basolaterally 48 hrs aftertransfection (1:4 ratio: 1.0�103� 8.8�102 RLU=ml, 6:8 ratio:1.3�104� 1.1�104 RLU=ml, control: 2.1�102� 41.5 RLU=ml,p< 0.05). Transfected ALIs were kept in culture for 14 daysand collection of apical and basolateral medium was repeatedto follow gene expression over time. Similar to in vivo modelsCMV-mediated expression fell to baseline levels within 1week after transfection. In conclusion: (1) secreted Gaussialuciferase is be a more sensitive reporter gene than fireflyluciferase, (2) GL67A transfects differentiated human ALIs,(3) gene expression can be followed over time by repeatedwashing of the apical cell surface.


P 59

Lentiviral Vector-Mediated Stem Cell Gene Therapy

for Mucopolysaccharidosis Type IIIA (MPS3A)

Langford-Smith AWW (presenting)1, Wilkinson FL1,Bennett W1, Sergijenko A1, Hopwood J2, Wraith E3,Wynn RF3, Bigger BW1

1MPS Stem Cell Research Group, The University of Manchester,UK2Lysosomal Diseases Research Unit, Women’s and Children’sHospital, Adelaide, South Australia3The Royal Manchester Children’s Hospital, Manchester, UK

MPS3A is a severe multisystem disorder caused by defi-ciency of the lysosomal enzyme, sulfamidase, which catabo-lises heparan sulphate. The disease affects children in the firstdecade of life, with progressive mental decline and death intheir mid teens. Enzyme replacement therapy and haemato-poietic stem cell transplant (HSCT) do not correct the severebrain degeneration. We propose that this is due to a doseeffect where enzyme is unable to cross the blood–brain-barrier and insufficient enzyme is being produced by donor-derived brain microglial cells. We are developing a stem celland gene therapy approach to overexpress sulfamidase(SGSH) and correct the disease in a MPS3A murine model.Lineage depleted bone marrow was transduced with anSGSH-lentiviral vector. 2.5�105 transduced or untransducednormal cells were administered to myeloablated MPS3Amice. Over 90% chimerism was achieved. Our preliminarydata shows 42% of WT SGSH activity in spleens and 44% inlivers of recipients of normal HSCT. Increases in SGSH ac-tivity are not detectable in the brains of MPS3A recipients ofnormal HSCT. By contrast, at 5 months post-transplant, re-cipients of SGSH-transduced HSCT showed 160% of WTSGSH activity in the spleen and 78% in the liver. We alsoshow 8% of normal WT activity in the brains of recipients ofSGSH-lentiviral transduced HSCT. This suggests that donor-derived microglia are present in the brain by 5 months andare secreting therapeutic levels of enzyme. This is a promisingstudy supporting the use of combined gene and cell therapyapproaches for the treatment of other lysosomal disorders.

P 60

Co-administration of AAV8 Myostatin Propeptideand Codon Optimised Microdystrophin in mdx Model

of Duchenne’s Muscular Dystrophy

Foster K (presenting)1, Graham I1, Foster H1, Trollet C1,Yaworsky P2, Walsh F2, Sharp P3, Wells D3, Dickson G1

1SWAN Institute of Biomedical and Life Sciences, RoyalHolloway, University of London2Clinical Discovery, Wyeth Research, Collegeville, PA, U.S.A.3Dept. Cellular and Molecular Neuroscience, Imperial CollegeLondon

Duchenne’s muscular dystrophy (DMD) is a severe musclewasting disorder affecting 1=3500 male births. DMD is causedby the lack of dystrophin in the skeletal muscle. Lack ofdystrophin within the muscle leads to muscle fibres which arehighly prone to exercise induced injury, which consequently

results in progressive rounds of degeneration and regenera-tion of the muscle, leading to respiratory or cardiac failure inthe third decade of life.

Use of myostatin propetide has been shown to improvepathology in mdx mice. Here we evaluate whether a genetherapeutic approach of myostatin inhibition leads to im-provement in the pathophysiology of the dystrophic pheno-type in the mdx female mouse. AAV8 myostatin propeptidewas administered by tail vein injection (4�1011 vg in 100ml)and AAV8 microdystrophin was administered by intramus-cular injection (5�1010 vg in 25ml).

There is no significant difference in the whole body massbetween control and treated groups, but codon optimisedmicrodystrophin normalised individual TA muscle mass toC57Bl10 controls and were significantly smaller than myo-statin propeptide treated mdx and mdx controls. Micro-dystrophin treatment, irrespective of myostatin propeptideadministration, resulted in a greater specific force comparedto mdx, although still significantly lower than C57Bl10 con-trols. Interestingly, irrespective of myostatin propeptideadministration, resistance to eccentric contractions was nor-malised to C57Bl10 controls following microdystrophin ad-ministration, however myostatin propeptide treatment alonewas significantly reduced compared to mdx controls.

P 61

Hey1 Gene Transfer Inhibits Late-Stage Prostate CancerGrowth and Promotes Sensitisation to Chemotherapy

Cheong SC (presenting), Sweeney K, Lemoine NR, Hallden G

Queen Mary University of London, Institute of Cancer,Charterhouse Square, London EC1M 6BQ

Androgen receptor (AR) signalling remains vital for themajority of prostate cancers to survive and proliferate,even when the disease has become hormone-refractory. Todate, there is no curative treatment for late-stage prostatecancer as the efficacy of standard treatments such as radio-therapy and chemotherapy is only limited. Novel treatmentsfor late-stage prostate cancer are therefore required. Hey1, amediator of the Notch signalling pathway, has recently beenidentified as a co-repressor of androgen receptor activity. De-creased expression of Hey1 has also been reported in prostatecancers suggesting an important role in prostate growth reg-ulation. We constructed the non-replicative adenoviral vectorAd5Hey1, expressing Hey1 from a CMV-promoter, in order toblock AR signalling. Efficient repression of AR activity byAd5Hey1 infection was confirmed by luciferase reporter as-says: virus doses as low as 10 particles per cell repressed ARactivity by 46.2� 0.9% in 22Rv1 cells. Cell viability assaysshowed that Ad5Hey1 could potently kill prostate cancercell lines LNCaP, 22Rv1 and DU145, up to 28-fold more effi-ciently than the corresponding GFP-expressing control-virus(Ad5GFP). Despite the lack of E1A, Ad5Hey1 could also in-duce sensitization to the chemotherapeutic drugs docetaxeland mitoxantrone. In cultured cells, up to 45% decrease of EC50

of chemodrugs could be induced by a low dose of Ad5Hey1.Chemosensitisation was also confirmed in vivo: a suboptimaldocetaxel treatment of DU145 xenografts could only signifi-cantly delay tumour growth when combined with Ad5Hey1.


Current work is focussed on elucidating the mechanisms un-derlying Hey1 cytotoxicity and chemosensitisation.

P 62

Myostatin Knockdown by Exon Skipping UsingAntisense Reagents

Kang JK (presenting), Dickson G, Graham I

SWAN Institute for Biomedical and Life Sciences, RoyalHolloway, University of London, Egham, Surrey, TW20 0EX

Myostatin is a negative regulator of muscle mass, andseveral strategies are being developed to knock down theexpression of the myostatin gene as a measure of bringingabout improvements in the muscle wasting conditions includ-ing Duchenne muscular dystrophy. Use of anti-myostatinantibodies and blockade strategies involving natural bindingpartners of myostatin have resulted in increases in musclemass in test animals. This approach has some flaws includingundesirable immune responses and low sustainability over alonger period of time.

Other strategies that involve the use of viral vectors such asretroviruses or lenti viruses to express short hairpin RNAsoffer a long term expression but are accompanied by risks ofuncontrolled insertion into the human genome. Here, wehave investigated a novel and controllable means of knockingdown myostatin production by the use of phosphorothiate-linked 20O-methyl RNA antisense oligonucleotides to induceskipping of exon 2 during pre-mRNA splicing. Also com-parative studies were done with naked morpholinos to in-duce exon skipping in vitro using nucleofection system.RT-PCR analysis of RNA from transfected muscle cell cul-tures demonstrated efficient exon skipping (almost 100% insome cases), which was also exemplified by increased cellproliferation in treated cultures, compared to controls( p¼ 0.002). Preliminary studies in vivo showed a small, butinsignificant, rise in the mass of treated muscles after a singletreatment.

P 63

Stress-Induced Duplex Destabilisation: An In Silico and

In Culture Analysis of Viral Vector Genomes IncorporatingS=MAR Elements

Kymalainen HE (presenting)1, Bode J2, Foster K1,Yanez-Munoz RJ1, Dickson G1

1School of Biological Sciences, Royal Holloway-Universityof London, Egham2Helmholtz Zentrum fur Infektionsforschung Inhoffenstr.,Braunschweig, Germany

Stress-induced DNA duplex destabilisation (SIDD) is theseparation of the two strands of the double helix that occurswhen negative superhelicity is imposed on the DNA mole-cule. In topologically constrained molecules the regions inwhich strand separation is likely to occur depend not only onlocal sequence but also on how the individual site competeswith all the others in the domain. The locations and extent ofSIDD occurring in linear or circular double-stranded DNAmolecules can be efficiently predicted with the web-based

tool WebSIDD [http:==orange.genomecenter.ucdavis.edu=benham=sidd=]. SIDD has been implicated in the mechanismsof activity of numerous biological processes, most notably theinitiation of transcription and replication. These functionshave also been attributed to the Scaffold=Matrix AttachmentRegions (S=MARs), which exhibit long regions of extensivedestabilisation. S=MARs have also been implicated in havinga positive effect on episomal transgene retention, and havebeen successfully incorporated into plasmid vectors, mostnotably the pEPI. We are interested in bestowing replica-tion and segregation capabilities to episomal vectors. Ourinitial approach is the inclusion of S=MAR elements into self-complementary adeno-associated viral vectors (scAAV) andintegration-deficient lentiviral vectors (IDLVs). We have usedWebSIDD to analyze the predicted forms that vector genomesincorporating or omitting the S=MAR element will takein vivo. We are now assessing whether WebSIDD results canbe used to predict the likelihood of persistent episomal es-tablishment in cell culture, and therefore whether they are avaluable tool in assessing prospective vectors prior to furtherin vitro and in vivo testing and analysis.

P 64

A Simple Single Affinity Chromatography Methodfor Purifying Multiple AAV Serotypes

McIntosh JH (presenting)1, Sotannde M1, Smith RH2, KotinRM2, Nathwani AC1

1UCL Cancer Institute, Paul O’Gorman Building, UniversityCollege London, 72 Huntley Street, London, WC1E 6BT2National Heart, Lung and Blood Institute, National Instituteof Health, Bethesda, MD, U.S.A.

Currently the only universal method for purification ofAAV is by density gradients, which are time consuming to setup and are only applicable for small scale purification. Ex-isting chromatography methods have been optimised foreach individual serotype of AAV, such as mucin for AAV5and heparin for AAV2.

AVB Sepharose High Performance consists of a recombi-nant 14KDa AAV Fab fragment bound to Sepharose by ahydrophilic spacer arm. We have used this media to purifyserotypes 2, 5, 7 and 8. A 5mL AVB sepharose column wasequilibrated in PBS for 5 column volumes, then the crudelysate was applied, unbound proteins were washed out with5 column volumes of PBS, AAV was then eluted with 5 col-umn volumes of 50 mM glycine pH2.7. 1 mL fractions werecollected into 30mL of 1 M Tris pH 8.8 to neutralise the pH.Pooled fractions were then dialysed against PBS overnight.Media was prepared by centrifugation at 18,000g to removesmall particulate matter followed by filtration through a0.8 mm filter. The media could then be added directly to thecolumn.

We have successfully used this method to purify AAV frommedia and crude cell lysate. Our data shows that 20% of thetotal rAAV produced is present in the media, which is usuallydiscarded, yields were between 1�1012 and 1�1013 per 40plate transfection depending on serotype. The vector was freeof cellular contaminants. Biological activity was equivalent torAAV purified by traditional column chromatography ordensity gradient.


P 65

The Sf9=Baculovirus Method Can Be Used to Successfully

Generate Self-Complementary AAV

McIntosh JH (presenting)1, Smith RH2, Kotin RM2,Nathwani AC1

1UCL Cancer Institute, Paul O’Gorman Building, UniversityCollege London, 72 Huntley Street, London, WC1E 6BT2National Heart, Lung and Blood Institute, National Instituteof Health, Bethesda, MD, U.S.A.

Large scale production of AAV is hampered by the use ofthe adherent cell line 293T in which to produce AAV. Urabeet al first reported the use of baculovirus and the insect cellline Sf9 to make AAV in 2002. In this system three baculo-viruses are created, one containing the AAV2 Rep, one con-taining the capsid, both under the control of an insectpromoter, and the third containing the transgene and ITRs.

Our existing self-complementary human Factor IX (hFIX)construct was cloned into the pFastBac plasmid. This wastransposed into bacmid DNA using the Bac-to-Bac kit fromInvitrogen. Baculovirus was made by transfection of adherentSf9 cells. To generate AAV, Sf9 cells in suspension weretransfected with three baculoviruses, rep, cap and transgene,the media and cell pellet was then harvested 72 hrs later andthe AAV purified.

A one litre production of AAV5 sc-LP1-hFIX gave 1.4�1013

virus particles, which is equivalent to 7�103 virus particles percell. The AAV was self-complementary when run on normaland denaturing agarose gels. A comparison of Baculovirusand 293T produced scAAV-LP1-hFIX showed comparablelevels in mice 189� 35 and 249� 98 ng=ml FIX respectively. Inconclusion, baculovirus produced AAV is equivalent to 293Tand is a feasible method to produce large quantities of AAV.

P 66

Novel Lentiviral Vectors for Haemophilia Gene Therapy

Kao VY (presenting)1, Waddington SN2, Thrasher AJ3,Antoniou M1

1Nuclear Biology Group, Department of Medical & MolecularGenetics, King’s College London School of Medicine, 7th Floor,Tower Wing, Guy’s Hospital, LONDON SE1 9RT2Department of Haemophilia Centre and Haemostasis Unit, RoyalFree and University College Medical School, Rowland Hill Street,London NW3 2PF3Institute of Child Health, 30 Guildford Street, London WC1N1EH

Novel lentiviral vectors (LV) have been designed for effi-cient expression in the haematopoietic system for haemo-philia gene therapy. The first LV consists of an expressioncassette based on the erythroid specific human b-globin genelocus control region (b-LCR) elements, promoter, secondintron=polyadenylation site) augmented with either (i) theGATA-1 gene HS2 insulator element (GATA-1=HS2) insertedwithin 30 LTR of the LV backbone, (ii) the ubiquitous chro-matin opening element from the human CBX-3=HNRPA2B1locus (A2UCOE) linked upstream of elements from the b-LCR. All LV were initially functionally analysed in erythroid

K562 and MEL and non-erythroid cell lines driving an eGFPreporter gene. Human Factor IX cDNA (hF.IX) will then beincorporated into what is found to be the most efficaciousexpression cassette for ex vivo haematopoietic stem cell and inutero delivery gene therapy in mouse model systems. MELcell-line tests shows that the A2UCOE is able to reduce in-sertion site position effects when combined with either HS2=3or HS3 elements from the b-LCR. The second and third LVsconsist of A2UCOE-hF.IX and SFFV-hF.IX that can provideexpression in all haematopoietic lineages. These two LVs arebeing assessed in an ex vivo bone marrow and in utero testdelivery systems in MF1 and 129sv mice.

P 67

Low-Dose Systemic Delivery of Morpholino AntisenseOligonucleotide Leads to Efficient Dystrophin Expressionin mdx Mouse

Malerba A (presenting), Dickson G, Graham I

School of Biological Sciences, Royal Holloway - Universityof London, Egham, Surrey, TW20 0EX

One of the most promising approaches for the treatment ofDuchenne Muscular Dystrophy (DMD), the most commoncongenital muscular disorder, is the administration of anti-sense oligonucleotides (AOs). The DMD is caused by muta-tions that create premature termination of dystrophintranslation; AO-induced exon skipping can restore the cor-rect reading frame in the dystrophin transcript, producing ashorter but functional protein. The phosphorodiamidatemorpholino oligomer (PMO) is one of the most promising AOchemistries thanks to the high affinity to the sequence target,resistance to endonucleases, and excellent safety profile.Future applications in clinical trials require a better knowl-edge of the pharmacokinetics, and the optimization of dose-regimens for the administration of this oligonucleotide. Sev-eral recent publications have demonstrated the efficacy ofpeptide-conjugated PMOs, but the clinical applicability ofsuch compounds is unclear at this stage. Here we report that,after systemic delivery, multiple injections of low amounts ofnaked morpholino show significantly more dystrophin ex-pression in a wide variety of muscle groups than a single highdose with the total amount. Some histological features, such asthe cross sectional area, centronucleation index and expressionof the dystrophin-associated protein complex, showed a sig-nificant improvement in mice treated by repeated injection.Importantly, four administrations of just 5 mg=kg induced asignificant amount of dystrophin expression. These data sug-gest that the repeated use of low doses of naked, unconjugatedPMO is likely to be clinically applicable for DMD patients.

P 68

Lentiviral Gene Therapy Strategies for Muscle Disease

Using Oculopharyngeal Muscular Dystrophy (OPMD)as a Model

Bales O (presenting)1, Trollet C2, van der Maarel S3,Dickson G2, Antoniou M1

1King’s College London, Department of Medical and MolecularGenetics, 8th Floor Tower Wing, Guy’s Hospital, London SE1 9RT


2School of Biological Sciences, Royal Holloway, Egham, SurreyTW20 0EX3Centre for Human and Clinical Genetics, Leiden UniversityMedical Centre, Leiden, The Netherlands

Lentiviral vectors (LVs) offer great potential to treat musclediseases by stable delivery of therapeutic molecules into bothmuscle precursor cells and differentiated muscle fibres. Weare testing several lentiviral gene therapy strategies in cellculture models for oculopharyngeal muscular dystrophy(OPMD), a late-onset autosomal dominant disorder withsymptoms including eyelid drooping and swallowing diffi-culties. OPMD is caused by small expansions of a polyalaninetract at the N-terminus of poly(A) binding protein nuclear 1(PABPN1), and is characterised by the appearance of nuclearinclusions containing this protein.

One potential therapeutic strategy is to use a PABPN1-specific intracellular antibody (‘intrabody’) to reducePABPN1 aggregation in muscle cells. We are expressing theintrabody 3F5, shown to prevent and reverse PABPN1aggregate formation in cell lines by transient transfectionassays, under the desmin promoter=enhancer. Another ap-proach we are pursuing is RNA interference using a smallhairpin RNA (shRNA) expressed under the H1 promoter toknockdown mutant PABPN1 mRNA levels.

Both LVs transduce human myoblasts at high efficiency,although transduction of a murine muscle cell model ofOPMD proved to be very poor. Therefore, we have generateda human muscle cell model of OPMD by expressing mutantPABPN1 in the immortalised myogenic cell line Hu5=E18.Initial tests on this line have shown that 3F5 intrabody ex-pression prevents aggregate formation and the shRNA re-duces PABPN1 protein levels.

Ultimately, we plan to test both LVs in cultured OPMDpatient myoblasts and an OPMD mouse model, providingfurther insight into the potential of gene therapy to treat de-generative muscle disease.

P 69

Investigating the Mechanism of Synergistically-Induced

Cell Death in Response to Combinations ofPhytochemicals and Adenovirus Type 5 (Ad5) in ProstateCancer Cells

Adam V (presenting), Lemoine NR, Hallden G

Centre for Molecular Oncology and Imaging, Institute of Cancerand the CR-UK Clinical Centre, Barts and The London, QueenMary’s School of Medicine and Dentistry, John Vane ScienceCentre, Charterhouse Square, London EC1M 6BQ

Replication-selective adenoviruses are a promising newtype of therapy for androgen-independent prostate cancer(PCa). Efficacy in clinical trials targeting PCa was reported tobe greatly enhanced by combining oncolytic mutants withconventional anti-cancer treatments, including chemotherapyand radiotherapy. We have identified a novel synergistic in-teraction between the phytochemicals equol and resveratrolwith replicating Ad5 mutants. Here we report on the role ofapoptosis in cell death induced by combination treatments inboth androgen receptor (AR)-expressing and non-expressingPCa cells.

Synergistic enhancement of cell death was detected in22Rv-1 and DU145 cells treated with combinations of equol(10–100 mM) or resveratrol (5–15 mM) and Ad5 (0.1–160 par-ticles per cell). Caspase-3 activation in DU145 cells was in-creased by equol (50 mM) and Ad5 (100 ppc) from 11.5% (Ad5alone) and 21% (phytochemical alone) to 30% cells (combi-nation) after 72 h treatments. In contrast, the highest levels ofcaspase-3 activation in 22Rv-1 cells were only 10% in cellstreated with resveratrol. In combination with Ad5, no furtherincrease was detected in 22Rv-1 cells. These differences be-tween cell lines paralleled similar changes in mitochondrialdepolarisation, determined by flow cytometry. While caspaseinhibition halved phytochemical- and combination-inducedcell death in DU145 cells, caspase inhibition only had a smallprotective effect in 22Rv-1 cells.

These results indicate that the role of apoptosis in syner-gistically-enhanced cell death induced by Ad5 and phyto-chemicals was cell line dependent. The involvement ofadditional modes of cell death is currently under investigation.

P 70

Functional Assessment of Recombinant DystrophinsArising from Exon-Skipping

Jones H (presenting), Sharp P, Wells DJ

Imperial College London

Recent clinical trials of antisense oligonucleotide inducedexon skipping have demonstrated that this therapeutic ap-proach can restore the expression of dystrophin in the musclesof patients with Duchenne muscular dystrophy. Exon skip-ping converts an out-of-frame mutation to an in-frame muta-tion. Although a number of patients with Becker musculardystrophy have been described with in-frame mutations and amilder clinical course, the clinical picture is not entirely clear asto how functional these recombinant dystrophins are. In ad-dition, many patients with duplications can also have thereading frame restored but it is not clear what the functionalconsequences of generating larger than normal dystrophinswill be. To assess the potential clinical benefit of different exon-skipping we are generating recombinant dystrophins with inframe duplications and are comparing these to full-lengthdystrophin and in-frame deleted dystrophins. Dystrophinconstructs can be delivered by electroporation into mdxmuscle and their ability to restore the dystrophin associatedprotein complex and exclude Evans Blue dye assessed. Withhigh transfection efficiencies physiological consequences canbe assessed using in-situ muscle physiology and a protocolexamining protection against the typical force drop in re-sponse to a series of eccentric exercise that is seen in mdx mice.The degree of protection is largely dependent on the number ofdystrophin positive fibres in the treated muscle.

P 71

Minicircle-DNA—A Safe and Efficient Vector System

Schmeer M (presenting), Blaesen M, Baier R, Schleef M

PlasmidFactory GmbH & Co. KG, Meisenstr. 96, D-33607Bielefeld


Conventional plasmid-DNA (pDNA) used in gene therapyand vaccination can be subdivided into a bacterial backboneand a transcription unit. Bacterial backbone sequences are(only) needed for pDNA production in bacteria. However, forgene transfer application these sequences are dispensable,reduce the overall efficiency of the DNA agent and, mostimportant, represent a biological safety risk. For instance thedissemination of antibiotic resistance genes, as well as theuncontrolled expression of backbone sequences in patientsmay have profound detrimental effects and unmethylatedCpG motifs have been shown to contribute to silencing ofepisomal transgene expression. Therefore, an important goalin vector development is to produce supercoiled DNA lack-ing bacterial backbone sequences.

PlasmidFactory’s minicircle production technology isbased on two processes: 1) An inducible, sequence specific,close to 100% efficient in vivo recombination process and 2) anovel affinity-based chromatographic purification technol-ogy for the isolation of the minicircle-DNA.

Quantitative real-time PCR analysis, capillary gel electro-phoresis and restriction analysis of the recombination prod-ucts and the minicircle-DNA revealed a recombinationefficiency greater than 99.5% and a purity of the isolatedminicircle-DNA of more than 98.5%.

Results demonstrate that the described technology facili-tates the large scale production of highly pure minicircle-DNA for applications in gene therapy and vaccination as wellas e.g. virus production.

Within this study, reporter genes for different types ofanalyses within various tissues, cells, animals and for testingthe mode of administration are used. Here, we show resultsdemonstrating a significant increase of gene expression ofminicircle-DNA in comparison with a standard plasmid.

P 72

Rapid Analysis of Adenoviruses with Prototype CIM� QAAnalytical Columns to Support the Development of High

Titre Manufacturing Platforms

Battom SE, Lindstrom KE, Whitfield RJ, Halsall JL(presenting)

Eden Biodesign Ltd, Estuary Banks, Estuary Commerce Park,Liverpool, L24 8RB

Adenoviruses are among the most commonly used vec-tors for the delivery of genetic material into human cells.Adenoviral vectors are currently being utilised in clinicalapplications such as suicide gene therapy, gene based immu-notherapy, gene replacement strategies and vaccine platforms.To support the development of a high titre purification pro-cess, various anion exchange methods have been evaluated.A method was designed and developed to quantify adeno-viral particles using a prototype CIM� QA analytical HPLCcolumn. This method was validated according to performancecriteria of repeatability, intermediate precision and linearity.We also report on conditions that efficiently regenerate thecolumn, extending its functional life. Our results demonstrateefficient separation of intact adenovirus type 5 particles fromcontaminating proteins and DNA. This method was used toanalyse samples taken throughout a downstream process.

These samples were detected at 260 nm and 280 nm using aphotodiode array detector (PDA).

P 73

Somatotransgenic Bioimaging: A Platform Technologyfor Disease Modelling and Drug Efficacy

McKay TR (presenting)1, Buckley SMK2, Rahim AA2,Howe SJ3, Thrasher AJ3, Waddington SN2

1Faculty of Medical & Human Sciences, University of ManchesterM13 9NT2Department of Haematology, Royal Free Campus, UniversityCollege London, NW3 2PF3Molecular Immunology Unit, Institute of Child Health,London,WC1N 1EH

Rodent models are an important platform for under-standing disease pathology and assessing drug efficacy. Pa-thology is analysed by peripheral or secreted=excreted bodyfluids and=or endpoint analyses. The former relies on anappropriate experimental variable, the latter on sacrificewhich increases animal numbers required. Recently, lightemitting luciferase transgenics have provided a platform forwhole body gene expression bioimaging in live animals.Transgenic luciferase mice, driven by a promoter activated bydisease induction or drug effect, are available but these areless useful when addressing biological processes in a singleorgan due to significant whole-body background noise.

Somatotransgenic Bioimaging is a versatile platform tech-nology for in vivo real-time investigations of disease and drugefficacy in small mammals. It utilises pseudotyped lentiviralvectors combined with neonatal administration to deliver a ge-netic biomarker to specific tissues providing continual, quan-tifiable light emission from a single animal over its lifetime.

We have transduced the bronchial epithelium of neonatalmice with a lentiviral vector expressing luciferase controlledby a NF-kB responsive promoter. We observe permanent, im-mune tolerated pro-inflammatory genetic effector restrictedto the conducting airways of the lung. These somatotrans-genic animals are predictably bioresponsive to pro-inflam-matory stimuli in the lung and as such represent a new modeof disease modelling whereby animal usage is significantlyreduced and data output substantially improved.

P 74

microRNA-Mediated Therapy: A New Conceptfor Treating Neurodegenerative Diseases

Abdelgany AS (presenting), Wood MJ

Department of Physiology, Anatomy and Genetics, Universityof Oxford, Oxford, OX1 3QX

Silencing of pathogenic-related genes using RNAi is anemerging field. However, it requires several investigations:identifying the correct genes, characterising their functionand pathogenesis, optimising their silencing, and investigat-ing the therapeutic effects of silencing them.

microRNAs are a class of non-coding small RNAs that alsowork via the RNAi pathway. They are key regulators of gene


expressions. microRNAs have been shown to be expressed ina tissue-specific manner.

Here, we hypothesis to recruit endogenous microRNAs todo the job for us by choosing the correct gene(s), target se-quences and more importantly, the correct physiologicalregulation pattern.

Neurodegenerative diseases share a common feature ofneuronal cell death such as the case in Parkinson’s diseasewhere patients loss 80% of dopamine neurons. Neurogenesisis a natural mechanism that produces new neurons in adultbrains. It is compromised by aging and also by neurodegen-erative disease. We hypothesise the use of brain-specific mi-croRNAs in inducing and promoting neurogenesis that cancompensate the neuronal loss. Our preliminary data usingboth human and mouse neuronal cell lines showed thatoverexpression of brain-specific microRNAs resulted in dif-ferentiating wild type cells into neuronal properties.

Our preliminary data demonstrate for the fist time a proof-of-principle that microRNAs has the potentials to be used astherapeutic agents in treating neurodegenerative diseases.This opens a new field of therapeutic approach practically inmodifying stem cells that can be used in therapies of severaldisease models.

P 75

Catheter-Mediated Naked DNA Delivery to Pigand Human Livers

Herrero MJ1, Lledo S2, Sabater L2, Noguera I3, Navarro V2,Sanchez M4, Alino SF (presenting)4

1Fundacion Hospital La Fe, Valencia, Spain2Servicio Cirugia, Hospital Clinico, Valencia, Spain3Servicio Central Investigacion, Universidad de Valencia4Dpto. Farmacologia, Fac. Medicina, Universidad Valencia

Hydrodynamic DNA injection in mice mediates highlyefficient liver gene transfer by a mechanism that involves si-nusoidal blood flow inversion and massive hepatocyte en-docytic process. Our aim is to translate the systemic largevolume i.v. injection performed in small animals into a ret-rovenous injection of selected organ in large animals byinterventionist catheterism (hydrocatheterism). Pigs wereanaesthetized and catheterized through the jugular veins toreach the hepatic vein, with a catheter having large flow ca-pacity and a tip able to be stuck in the desired caliber area,confirming the correct positioning by fluoroscopy. Then, aplasmid containing the human a-1-antitrypsin gene (hAAT)in saline solution (20mg=ml) was injected at 10–20 ml=s rate.Then, the catheter was removed and the animals awaken, tobe kept in the stall. Plasma samples were taken and kept at�208C for quantitative ELISA. After sacrifice, samples of livertissue were collected for morphological and molecular stud-ies. Liver transfection experiments show dose-dependenthAAT protein in porcine plasma, but the maximal effect islimited (*500 ng=ml). In ex vivo models, chirurgically re-sected human hepatic segments were catheterized, emulatingthe in vivo pig conditions, and injected with 20 mg=ml salinesolution of a plasmid containing eGFP gene. Preliminary re-sults show, in cryogenic slides of these segments, that the cellsin the perfused area were successfully transfected. However,

the conditions of catheter implantation and perfusion flowmust be adjusted in order to optimize gene transfer efficacy.Project financed by SAF-2007-64492 and GV-AP-071=08.

P 76

Application of RNA Interference to Target a Modelof Slow Channel Congenital Myasthenia Syndrome

McClorey G (presenting)1, Biba A2, Wood MJ1, Beeson D2

1Dept Physiology, Anatomy and Genetics, Le Gros Clark Building,University of Oxford, Oxford OX1 3QX2Weatherall Institute of Molecular Medicine, Universityof Oxford, Oxford OX3 9DS

Congenital myasthenic syndromes are disorders arisingfrom genetic defects in proteins of the neuromuscular junc-tion. In slow-channel congenital myasthenic syndrome(SCCMS), mutations in the nicotinic acetylcholine receptor(nAChR) cause prolonged opening of the nAChR channelresulting in abnormally slow decay of synaptic currents. Thiscauses calcium overload of the post-synaptic region andsubsequent loss of function at the neuromuscular junction.These slow-channel mutations are inherited in an autosomaldominant manner, have a toxic gain-of-function effect and areoften caused by single missense mutations. We therefore in-vestigated the potential of using allele-specific RNA inter-ference (RNAi) to target a slow-channel missense mutation inthe epsilon nAChR subunit (eL221F) to silence the mutant,but not the wild-type allele. This was performed using bothsiRNA and short hairpin microRNAs (sh-miRNA) systemstargeting a heterozygous cell expression system. We alsotested the feasibility of applying this approach in vivo bydelivery of siRNA and virally expressed sh-miRNA to aneL221F mouse model of SCCMS.

P 77

Adenovirus Serotype 11 Is Less Effectively NeutralisedThan Serotypes 3 and 5 at Low Concentrations ofMalignant Effusions from Cancer Patients

Thoma C (presenting)1, Seaton P1, Fisher K2, Davies HE3,Rahman NM3, Lee YCG3, Davies RJO3, Seymour LW2,Kehoe ST1, Morrison J1

1Nuffield Department of Obstetrics and Gynaecology, Universityof Oxford, UK2Department of Clinical Pharmacology, University of Oxford, UK3Department of Respiratory Medicine, Churchill Hospital, Oxford,UK

The use of oncolytic adenovirus is a powerful tool forcancer-treatment and its efficacy in eradicating tumours inanimal models has been proven. However, in humans, sys-temic delivery or regional delivery to the peritoneal or pleuralcavity encounter considerable obstacles. Apart from seques-tration onto erythrocytes, interaction with serum proteinsand especially pre-existing antibodies can severely impact onthe activity of a given dose.

Adenovirus serotype 5 (Ad5) is the most extensivelyinvestigated virus for oncolytic cancer therapy. However,


immunity to Ad5 is widespread and it has been proposed thatthe use of uncommon serotypes may provide a way toovercome neutralisation, at least for first application.

We investigated the capability of pleural effusions andperitoneal ascitic fluid from cancer patients to neutralise theoncolysis of tumour cells by adenovirus. To assess the pro-posed benefit of uncommon serotypes we used serotypes 3(Ad3) and 11 (Ad11) in addition to Ad5.

Individual screening of 25 samples revealed that the vastmajority effectively neutralise oncolysis by all serotypes atconcentrations lower than 5% vol=vol. Ad11 is significantlyless prone to neutralisation than Ad3 or Ad5 at these con-centrations. However, this difference may have only limitedrelevance in the clinical setting where the virus may en-counter a high concentration of pleural or ascitic fluid. Someclinical protocols permit reduction of the neutralising capa-bility by fluid drainage and subsequent washing of the peri-toneal or pleural cavity before application of the therapeuticvirus. These may provide the best setting for therapeutic as-sessment of these adenovirus serotypes.

P 78

Characterization of Transduction Efficiency of HSV-1

Amplicons in Mesenchymal Stem Cells

El-Sherbini Y (presenting)1, Stevenson M1, Seymour LW1,Wade-Martins R2

1Dept. Clinical Pharmacology, University of Oxford, OX3 7DQ2Department of Physiology, Anatomy and Genetics, Universityof Oxford, OX1 7QX

Bone marrow mesenchymal stem cells (MSCs) are useful asdelivery vehicles and for regenerative medicine. Here westudied MSCs genetically modified using large carrying ca-pacity (152 Kb) herpes simplex type 1 (HSV-1) amplicons.pHSVCMVGL is a plasmid containing the HSV-1 packagingsignal (pac) and origin of replication (oriS); it expresses EGFPunder control of the HSV-1 promoter (IE4=5) and luciferaseunder the CMV promoter. pHSVEF1GL, pHSVPUBGL andpHSVCA9GL are modified from pHSVCMVGL with theCMV promoter replaced by promoters for human elongationfactor (EF1), polyubiquitin and hypoxia-specific carbonicanhydrase (CA9), respectively. Amplicons successfully trans-duced MSCs at 6% efficiency (MOI 10); efficiency wasincreased up to 30% at the same MOI by applying centrifu-gation during transduction. The ability of the vector to delivera functional gene was shown by strong expression of EGFPand luciferase within cells post transduction. The levels ofluciferase expression driven by the four promoters varied,notably EF1, PUB and CA9 gave approximately one tenth theexpression from CMV. However expression from the EF1promoter was twice that from the PUB promoter. Under 5%hypoxia the CA9 promoter did not show upregulation com-pared to normoxia.

Transduction with amplicons altered neither the morphol-ogy nor surface markers of MSCs as shown by fluorescencemicroscopy and flow cytometry, respectively. Transductionalso did not affect plasticity of engineered cells as we wereable to differentiate them into adipocytes. This study dem-onstrates the possibility of using extrachromosomal vectorswith large carrying capacity to modify MSCs, thereby avoid-ing risks associated with integrating vectors.


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BSGT 2009 Poster Presentations