72 Retroviral Integration Sites in Mucopolysaccharidosis VII Dogs driven eGFP reporter gene followed by an IRES sequence and the puromycin resistance gene Several 1 IEK293 derived FRT target clones we[.]
driven eGFP reporter gene followed by an IRES sequence and the puromycin resistance gene Several 1-IEK293-derived FRT target clones were isolated for exploring selective site-specific integration from viral substrates We generated a lentiviral donor vector containing the FRT-hygromycin resistance gene without a start codon The vector was packaged in integration-defective viral particles containing defective integrase proteins By Southern Blotting of HIRT-extracted episomal DNA from transduced cells we could detect circular DNA molecules generated from the integration-defective lentiviral vector By co-expressing the Flp recombinase from transfected plasmid DNA in transduced cells we were able to facilitate integration of circular DNA site-specifically into the genomic FRT site Sequence analysis of hygromycin-resistent clones provided evidence that DNA circles containing one LTR or two LTRs were inserted by Flp recombination In addition, preliminary findings indicated that integration ofIentiviral circles could be facilitated by Flp recombinase expressed from a co-transduced integration-defective lentiviral vector We provide proof-of-principle that lentiviral DNA circles can act as substrates for Flp recomb inase-driven site-specific gene insertion based on virally transduced recombination substrates This technology allows Flp-mediated insertion ofcircular DNA that docs not contain a bacterial backbone, and may serve as a useful tool in genetic engineering as well as a platform for development of safer gene vehicles 70 Assessing Gammaretroviral Vector Integration Preferences for DNase Hypersensitive Sites Mingdong Liu,' Peter J Sabo,' Michael S Kuehn ,' John A Starnatoyannopoulos,' David W Emery.' I Department a/Medicine, University ofWashington, Seattle, Ifl;-'; 2Department ofGenome Sciences, University of Washington, Seattle, IVA Concerns surrounding the oncogenic potential of recombinant gammaretroviral vectors has spurred a great deal of interest into the integration site preferences ofsuch vectors Early studies ofmurine leukemia virus-transformed cell clones, and more recent studies with recombinant gammaretroviral vectors in unseleeted cell pools, indicated a correlation between viral integration sites and DNase hypersensitive sites, which mark open chromatin regions and are often associated with cis-regulatory elements such as enhancers and promoters Although compelling, these previous studies are difficult to interpret: the older studies involved the use ofwild-type virus and transformed clones , while the latter compared integration sites to DNase hypersensitive sites identified in a high throughput screen of an unrelated cell type In order to study this issue more directly, we looked for correlations between gammaretroviral vector integration sites identified in the absence of selection, and DNase hypersensitive sites mapped within the same cell line (the human fibrosarcoma HT1080) using robust conventional methods To date we have analyzed 12 independent integration sites located on different chromosomes Of these sites, a total of were found to be located directly within DNase hypersensitive sites, a frequency that is highly unlikely by chance The remaining sites were distributed over a distance of 521 bp to 979 bp from the border of the nearest DNase hypersensitive site, approximating a Gaussian distribution with a mean of473 bp and a median of574 bp for the overall dataset As a control, we used an extensive dataset ofvalidated DNase hypersensitive sites mapped in the ENCODE region using the DNase-array method (Sabo et al., Nature Methods 3:511,2006) Specifically, we calculated the distances between 753 DNase hypersensitive sites from extended segments of the same chromosomes used for the integration site analysis For this dataset, the distances between borders ofadjacent DNase hypersensitive sites ranged from 16 bp to over 200 kb, with an mean of9100 bp and a median of619 bp The Molecular Therapy Volume 15 ~ Supplement I May 2007 Copyright © Th e American Society of G ene Th erapy notable discrepancy between the mean and median values reflect a fat-tailed Cauchy-like distribution Although the median distances for both the experimental and control datasets were similar, comparison ofthese two datasets using the non-parametric, distribution free Kolmogorov-Smirnov test, showed a clear difference between the overall distributions (P=O.O16) Specifically, the integration site dataset lacked examples of integrations located at distances greater than I kb, whereas over 40% of the distances in the control dataset were greater than I kb Taken together, these results provide direct evidence that garnrnaretrovirus vectors have a preference for integration within , or at least near, DNase hypersensitive sites Current studies are focused on developing means ofassessing this preference on a genome-wide scale in clinically relevant cell types, as well as assessing the impact of vector provirus on the pattern of DNase hypersensitive sites surrounding sites of integration 71 Determination of the Profile and Site Preference of Lentiviral Vector Integration In Vivo and in Primary Astrocyte Cultures Caroline V Hacker,' Yohann D Moal,' Gareth S Ralph,' Susan M Kingsman ,' KyriacosA Mitrophanous,' James E Miskin ' 'Oxford BioMedica, Oxford BioMedica, Oxford, United Kingdom Oxford BioMediea has developed minimal lentiviral vectors based on Equine Infectious Anaemia Virus (EIAV), and these are currently being developed for use in the clinic One advantage of lentiviral vectors over other retroviral vector systems is that they can integrate in non-dividing cells such as neurons An EIAV-based vector for the treatment of Parkinson's disease ProSavin', has now developed ProSavin has demonstrated efficacy in industry standard models ofParkinson 's disease and it is hoped that ProSavin will enter clinical evaluation in the ncar future Integration ofretroviruses and retroviral vectors within the target cell genome has been shown to be non-random Human immunodeficiency virus-I (HIV-I) and HIV-I based vectors have a bias for integrating into genes , and more specifically genes that are active following infection with HIV-I Murine leukaemia virus (MLV) integration favours transcriptional start sites whilst Avian sarcoma-leukosis virus (ASLV) shows only a mild preference for transcriptional start sites Ligation mediated PCR (LM-PCR) was used to map 458 integration sites for ElAV based lentiviral vectors and 162 control sites for HIV-I based lentiviral vectors in vitro By comparison of these two data sets to a control data set that was generated in silica, we have shown that, like I-IIV-I, EIAV based vectors show a preference for active genes In contrast to published analysis ofMLV integration, EIAV and HIV-I not show a preference for the transcriptional start site or promoter region ofgenes The above data was obtained from transduction of transformed eclilines It would be of interest to know the integration profile of EIAV in primary cultured cells and also in non-dividing cells in vivo, such as neurons Therefore, an investigation of the integration profile ofEIAV-based vectors in primal)' astrocyte cultures has now been initiated In addition, an investigation into the in vivo integration profile of EIAV in cells within the rat striatum has commenced The results will be reported 72 Retroviral Integration Sites in Mucopolysaccharidosis VII Dogs Thomas M O'Malley,' Paula S Henthorn ,' Katherine P Ponder, Mark E Haskins 'School of Veterinary Medicine, University ofPennsylvania, Philadelphia, PA; 2SchoolofMedicine, Washington University St LOllis, 1110 Intravenous administration of an amphotropie gamma retroviral vector (RV) to neonatal dogs has been an effective therapy for Mueopolysaeeharidosis VII (MPS VII) Although this is primarily a S29 liver-targeted approach,bloodcells containaround 0.0I copiesofRV per cell at years after transduction This low-level hematopoietic marking and expression may prove to be an important component ofthe therapy However, hematopoietic cell transduction also presents an additional risk for insertional mutagenesis in a population of rapidly dividing cells As part of a long-term safety assessment, blood from RV-treated MPS VII dogs that received direct injections ofRV to 5.5 years previously was analyzed for RV insertion sites using ligation amplification-mediated (LAM) PCR There were relatively few insertionsites detected for each dog A total ofeleven non-artifactual genomic sequences were cloned from five dogs The results of a BLAT search of the UCSC May Z005 Dog Assembly (http://genome.ucsc.edu/cgi-bin/hgBlat)allowed assignmentofnine of these sequences to specific locations in the dog genome Of the genes closest to the nine identifiedsites, the mouse homolog of five of'these were presentin the Retroviral TaggedCancerGene Database (RTCGD(mm8) http://rtcgd.abcc.ncifcr[gov/) Assuming similar gene structures in dog as in human, the locations with respect to genes could be determined, Twowere located within kb upstream ofthe first known exon, three were within intron I, two were within other introns, one was 14 kb downstream of the nearest gene, and the last was at least 60 kb from the nearest annotated gene Three of these dogs had also been analyzed three years previously In that analysis, six unique sequences were identified Two of these corresponded to genes in the RTCGD Only one site, within the last exon of the HMGA2 gene, was detected in both analyses of one of the dogs 73 Identification of Genetic Insulator Elements for Safe Gene Therapy Viral Integrating Vectors Cecile Bauche,' Armelle Gaussin,' Julien De Royer,' Jean Francois Mouscadet,' Christian Auclair; Nicholas Merrnod,' Odile Y Cohcn-Hagucnaucr,'-' I Laboratoire de Biotechnologie et de Pharmacologie Genetique Appliquees, Ecole Normale Superieure, Cachan, France ; 21nstitute ofBiotechnology, University ofLausanne, Lausanne, Switzerland; JDep artment ofMedical Oncology, Hopital SaintLouis, Paris, France Insertional mutagenesis has been demonstrated following the integrationofgene transfervectors that includestrong enhancer/promoters Conversely, the insulator approach can also be investigated as a way to protectthe endogenousgenomic sequences/environment from the risk ofdysregulation resulting from integrating vectors In addition, recent reports have shown that genetic insulatorscurrently under usc, like the HS4 1,2 kp fragment or the 250 bp core clements cannot wholly insulate the genetic environment from potent viral regulatoryelements In this event, there will be no garantec that sideeffects will not reproduce followingclonal selection and expansion, In order to identify short genetic elements capable of insulating the strongest viral regulatory elements from the Fr-MuLV FB29 strain enhancer which are capable of both preventing insertional mutagenesis and the exctinction of transgene expression we have established a standard screening procedure This assay consists of a series of plasmids containing two reporter genes: one mimicking a therapeutic gene under thc control of strong viral enhancer/promoter, and the other one standing for an endogenous gene close to the chromosomalvector integration site, Potentialinsulatorelements interposed between the viral enhancer and the "chromosomal" gene promoter are expected to shield the latter gene from the influenceof the neighboringvector elements The setting up and assay validation make use of the chicken beta-globin 5'HS4 elements, whereas repeats of short genetic elements having more potent insulatoractivity are being screened and retained New insulators are challenged with both the full FOCHA-LrR and the Fr-MuLVenhancer alone SelfS30 inactivating gammaretrovirus-based vectors have been designed by other groups; the virus titers we have obtained from SIN Fr-MuLV FB29 derived, FOCHAvector, i.e., 106pfu/ml (Cohen-Haguenauer, unpublisheddata) are not standard.Aseries ofinsulated gamma- and lenti- SIN-retrovirus constructs have thus been engineered using two combinations of the shortest active stretches, respectively of 268 bp and 157 bp long in total, substituting the U3 LTRenhancer in full The lenti backbone is derived from Luigi Naldini's pSIN18 (with kind permission) Data form these vectors and related comparisons will be presented In adddition, we aim at combining the usc of insulated lentivectors and targeting their integration to heterochromatin Acknowledgement: EC FP6-NoE CLiNIGENE LSHB-CT-Z006-0 18933 (the European network of excellence for the advancement of clinical gene transfer and therapy) 74 Making Retroviral Gene Therapy Safer: Prevention of Retrovirus-Mediated Activation of Cellular Genes near the Integration Site by Engineering of the LTR Youngtae Hong, I Nam-Kyung Yoon, I Sujeong Kim, ' Sunyoung Kim.! Joong Gon Kim,' Jung Woo Rhim,' Hyoung Jin Kang,' Karim Lee,' Jiwon Jang.? I Department ofResearch and Development Viro1l4ed, Seoul, Republic ofKorea; 2Department ofBiological Sciences, Seoul National University, Seoul, Republic ofKorea; 'Depanment of Pediatrics Seoul National University Hospital, Seoul, Republic ofKorea The usc of retroviraI vectors has recently demonstrated its actual clinical benefit in a few inherited diseases However, the leukemia cases foundafter the x-scm gene therapy trial has raised the safety concern of the insertional mutagenesis inherent to the biology of the retrovirus.Although the retrovirus has long been known to integrate into the host chromosome, and thus have the potential to activate the nearby gene, there has been no convenient method of studying or assaying such a cis-activation phenomenon Here we report an in vitro assay system in which the effect ofretroviraI integration on the expression of the neighboring gene can be studied Using this assay, we found that the full-length LTR could indeed activate the neighboring gene expression from a distance and the magnitude of its activation was highly increased when this LTR was placed in the vicinity of the transcription start site of the gene, while the truncated LTR exerted little influence This system might provide a useful tool for selecting the appropriate vector structure as well as studying the molecular mechanism underlying the cis-activation by the viral LTR AAV PRODUCTION AND MODIFICATION 75 Targeting of Angiogenic Endothelial Cells Using a New AAV-2 Insertion Site Jorge Boucas,' Kerstin Lux; Anke Huber,' Sibille Hummc.P Michael Hallek.t-' Luca Perabo,' Hildegard Buning.l -' 'Clinic Ifor Internal Medicine University ofCologne Cologne Germany; 2Centerfor Molecular Medicine Cologne University of Cologne, Cologne , Germany The insertion of small peptides into the amino acid position 587 of the adcno-associatcd virus (AAV-2) capsid opened the field of AAV targeting Since then, recombinant AAV (rAAV) targeting vectors with 587 insertions have been proven to yield increased transduction efficiency in vitro and in vivo and to transduce their target cell via the new insertion receptor interaction Furthermore, combinatorial approaches using AAV display libraries and high throughput-screenings simplified the creation of targeting vectors with desired transduction abilities In this report we demonstrate Molecular Therapy Volume15.Supplement Iã \by 2IlQ7 Cop)Tight â The Amcricm Society o r Gene Therapy ... presented In adddition, we aim at combining the usc of insulated lentivectors and targeting their integration to heterochromatin Acknowledgement: EC FP6-NoE CLiNIGENE LSHB-CT-Z006-0 18933 (the European... the field of AAV targeting Since then, recombinant AAV (rAAV) targeting vectors with 587 insertions have been proven to yield increased transduction efficiency in vitro and in vivo and to transduce... dog as in human, the locations with respect to genes could be determined, Twowere located within kb upstream ofthe first known exon, three were within intron I, two were within other introns,