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483. Phage C31 Integrase Induced Chromosomal Rearrangements in Normal Human Fibroblasts Involves DNA Damage Response
481. Structure-Based Prediction of InsertionSite Preferences into Chromosomes of Vectors Used for Gene Therapy Perry B. Hackeu,' Christopher S. Hackett.' Aron M. Geurts.'
'GCD and Beckman Center/or Transposon Research, Institute ofHuman Genetics, University ofMinnesota, Minneapolis, MN; ]Biomedical Sciences Graduate Program, University ofCalifornia, San Francisco, San Francisco, CA; "Human and Molecular Genetics Center; Medical College of Wisconsin, Milwaukee, WI.
Integration of therapeutic vectors is necessary for long term expression, Gene therapists hopc to avoid altering endogenous genc expression and the resulting risk in activation of oncogenes as a consequence of introducing genes meant to be expressed. The odds of either outcome depend on a vector's preference to integrate into genes and/or their transcriptional control regions. The variability in insertion preference between vectors varies considerably. We have examined the tendencies of several classes of retroviral and transposon vectors to target DNAsequcnccs, genes and genetic clements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. There are three principle benefits from elucidating factors that affect preferences in integration: I) in gene therapy, to assess the overall risks ofactivating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; 2) in genomic studies, to discern random from selected integration events, which is important for determining function; 3) in gene therapy as well as functional genomics, to design vectors and integrases that have greater targeting to specific sequences, which would be a significant advancement in the art of trans genesis. Although most vectors will integrate into a vast number of sites scattered throughout the genome, numerous studies have shown that these integrations arc not random with respect to several variables. Global preferences for vector integration can be governed by large-scale genomic context such as coding and regulatory regions ofgenes, and their transcriptional status, as compared to intragenic regions. The fine-tuning that determines specific sites of integration is governed by smaller scale, physical features, such as the specific sequences of'nucleotides surrounding insertion sites and DNA structural characteristics derived from these sequences. To this end, we have examined the following parameters ofintegration sites by various vectors proposed for gene therapy - Bendability (the curving ofa DNA sequence based on its sequence), A-philieity (the potential of a DNA sequence to shift from the B-form to A-form), Vstep (a single composite parameter ofprotein-induced DNA deformability), and Jaggedness (the degree to which Vstep values alternate from high to low). In particular, on average insertion sites for the Sleeping Beauty transposon system has a high trinucleotide bendability and high jaggedness values whereas those for piggyBac transposons have low trinucleotide bendability and low jaggedness sco res.
482. Construction and Testing of a LiverDirected Targeting Tether for Tissue-5pecific Delivery of Sleeping Beauty Transposons
Erik R. Olson,' Jeffrey M. Schreifels," Jason B. Bell,2 R. Scott C. Mclvor.t-' Perry B. Hack ett.'>
'Discovery Genomics, lnc., Minneapolis, AlN; ]Dept. ofGenetics, Cell Biology & Development, Beckman Center/or Transposon Research, University ofMinnesota, Minneapolis, MN. The Sleeping Beauty (SB) transposon system has is an effective non-viral vector for gene therapy in mice. Delivery of trans genes into livers ofmice can be achieved by rapid , high-volume (hydrodynamic) injection oftrans poson-containing plasm ids through the tail SI86
vein ofmice and recent studies suggest that localized hydrodynamic delivery can be achieved in large animals (Herweijer and Wolff (2007) Gene Ther. 14: 99-107). Nevertheless, alternative methods for tissue-specific delivery of transposon-containing plasmids will be useful for conditions that are not amenable to hydrodynamic methods. Our strategy is based on the findings that surface-tethered DNA complexes can enhance gene delivery (Segura & Shea (2002) Bioconjug. Chern . 13: 621-629). Hence we devised a strategy for tethering plasm ids to cells of animal organs and tissues while controlling the size of the molecular conjugate for more effective distribution (Icft figure). The vector is characterized by the blue , inverted arrowheads that designate the Inverted Terminal Repeats (ITRs) of the SB transposon and the gene of interest . The only two necessary functional motifs in the plasmid carrier are the origin of replication, ori, and a kan-selectable marker. Within the plasmid are cassettes with four tandem LexA-binding domains designated by the red diamonds to which the tether can bind. The structure ofthe tether is shown on the right. The LTT was expressed in E. coli, and is currently being tested for binding to the pKLAT2 vector. Results from injection of mice with the coupled system will be presented. a)
Steeping Beauty
Transposon
HBV
b)
LHBA
T8rgeUng
rethe r
87
Role: pKLAT2·b nding
13
27
Liver ligand
Liver-specific Tethering Polypeptid e (LIT)
Expression of Gene of Interest
483. Phage C31 Integrase Induced Chromosomal Rearrangements in Normal Human Fibroblasts Involves DNA Damage Response Jian Liu, Torben Gjetting, Thomas G. Jensen.
'Gene Therapy Laboratory; Kennedy Institute-National Eye Clinic, Glostrup, Denmark. Phage C31 integrase facilitate specific integration into human and mouse genomes and is considered as a potential non-viral gene therapy delivery system. However recent studies about Phage C31 integrase suggest that it induces various chromosomal abnormalities in primary human fibroblasts and other mammalian cell lines. (Liu el al. Gene Ther. 2006; 13: 1188-1190; Ehrhardt el al. Hum an Gene Ther. 2006; 17: 1077-1094). In this study we used normal human adult fibroblasts and generated stably transfected primary human fibroblasts expressing integrase confirming our previous findings using primary embryonic fibroblasts. All control cells were cytogenetically normal, but in cells expressing Phage C31 integrase, chromosomal abnormalities including various translocations were found. Since the chromosomal abnormalities were not unambiguously associated to pseudo-attB sites, the mechanisms need to be clarified. We used transient expression of'integrase followed by immuno-fluorescence to monitor H2AX phosphorylation - a primary event in the DNA damage response. H2AX phosphorylation was observed 3 days post-transfection, Thus our study suggests that Phage C31 integrase-induced chromosomal aberrations is linked to a DNA damage response.
Molecular Therapy Volume15.Supplement I•.\by:!007 Copyright © The Americ.. m Society { I t Gene Therapy
'GCD and Beckman Center/or Transposon Research, Institute ofHuman Genetics, University ofMinnesota, Minneapolis, MN; ]Biomedical Sciences Graduate Program, University ofCalifornia, San Francisco, San Francisco, CA; "Human and Molecular Genetics Center; Medical College of Wisconsin, Milwaukee, WI.
Integration of therapeutic vectors is necessary for long term expression, Gene therapists hopc to avoid altering endogenous genc expression and the resulting risk in activation of oncogenes as a consequence of introducing genes meant to be expressed. The odds of either outcome depend on a vector's preference to integrate into genes and/or their transcriptional control regions. The variability in insertion preference between vectors varies considerably. We have examined the tendencies of several classes of retroviral and transposon vectors to target DNAsequcnccs, genes and genetic clements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. There are three principle benefits from elucidating factors that affect preferences in integration: I) in gene therapy, to assess the overall risks ofactivating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; 2) in genomic studies, to discern random from selected integration events, which is important for determining function; 3) in gene therapy as well as functional genomics, to design vectors and integrases that have greater targeting to specific sequences, which would be a significant advancement in the art of trans genesis. Although most vectors will integrate into a vast number of sites scattered throughout the genome, numerous studies have shown that these integrations arc not random with respect to several variables. Global preferences for vector integration can be governed by large-scale genomic context such as coding and regulatory regions ofgenes, and their transcriptional status, as compared to intragenic regions. The fine-tuning that determines specific sites of integration is governed by smaller scale, physical features, such as the specific sequences of'nucleotides surrounding insertion sites and DNA structural characteristics derived from these sequences. To this end, we have examined the following parameters ofintegration sites by various vectors proposed for gene therapy - Bendability (the curving ofa DNA sequence based on its sequence), A-philieity (the potential of a DNA sequence to shift from the B-form to A-form), Vstep (a single composite parameter ofprotein-induced DNA deformability), and Jaggedness (the degree to which Vstep values alternate from high to low). In particular, on average insertion sites for the Sleeping Beauty transposon system has a high trinucleotide bendability and high jaggedness values whereas those for piggyBac transposons have low trinucleotide bendability and low jaggedness sco res.
482. Construction and Testing of a LiverDirected Targeting Tether for Tissue-5pecific Delivery of Sleeping Beauty Transposons
Erik R. Olson,' Jeffrey M. Schreifels," Jason B. Bell,2 R. Scott C. Mclvor.t-' Perry B. Hack ett.'>
'Discovery Genomics, lnc., Minneapolis, AlN; ]Dept. ofGenetics, Cell Biology & Development, Beckman Center/or Transposon Research, University ofMinnesota, Minneapolis, MN. The Sleeping Beauty (SB) transposon system has is an effective non-viral vector for gene therapy in mice. Delivery of trans genes into livers ofmice can be achieved by rapid , high-volume (hydrodynamic) injection oftrans poson-containing plasm ids through the tail SI86
vein ofmice and recent studies suggest that localized hydrodynamic delivery can be achieved in large animals (Herweijer and Wolff (2007) Gene Ther. 14: 99-107). Nevertheless, alternative methods for tissue-specific delivery of transposon-containing plasmids will be useful for conditions that are not amenable to hydrodynamic methods. Our strategy is based on the findings that surface-tethered DNA complexes can enhance gene delivery (Segura & Shea (2002) Bioconjug. Chern . 13: 621-629). Hence we devised a strategy for tethering plasm ids to cells of animal organs and tissues while controlling the size of the molecular conjugate for more effective distribution (Icft figure). The vector is characterized by the blue , inverted arrowheads that designate the Inverted Terminal Repeats (ITRs) of the SB transposon and the gene of interest . The only two necessary functional motifs in the plasmid carrier are the origin of replication, ori, and a kan-selectable marker. Within the plasmid are cassettes with four tandem LexA-binding domains designated by the red diamonds to which the tether can bind. The structure ofthe tether is shown on the right. The LTT was expressed in E. coli, and is currently being tested for binding to the pKLAT2 vector. Results from injection of mice with the coupled system will be presented. a)
Steeping Beauty
Transposon
HBV
b)
LHBA
T8rgeUng
rethe r
87
Role: pKLAT2·b nding
13
27
Liver ligand
Liver-specific Tethering Polypeptid e (LIT)
Expression of Gene of Interest
483. Phage C31 Integrase Induced Chromosomal Rearrangements in Normal Human Fibroblasts Involves DNA Damage Response Jian Liu, Torben Gjetting, Thomas G. Jensen.
'Gene Therapy Laboratory; Kennedy Institute-National Eye Clinic, Glostrup, Denmark. Phage C31 integrase facilitate specific integration into human and mouse genomes and is considered as a potential non-viral gene therapy delivery system. However recent studies about Phage C31 integrase suggest that it induces various chromosomal abnormalities in primary human fibroblasts and other mammalian cell lines. (Liu el al. Gene Ther. 2006; 13: 1188-1190; Ehrhardt el al. Hum an Gene Ther. 2006; 17: 1077-1094). In this study we used normal human adult fibroblasts and generated stably transfected primary human fibroblasts expressing integrase confirming our previous findings using primary embryonic fibroblasts. All control cells were cytogenetically normal, but in cells expressing Phage C31 integrase, chromosomal abnormalities including various translocations were found. Since the chromosomal abnormalities were not unambiguously associated to pseudo-attB sites, the mechanisms need to be clarified. We used transient expression of'integrase followed by immuno-fluorescence to monitor H2AX phosphorylation - a primary event in the DNA damage response. H2AX phosphorylation was observed 3 days post-transfection, Thus our study suggests that Phage C31 integrase-induced chromosomal aberrations is linked to a DNA damage response.
Molecular Therapy Volume15.Supplement I•.\by:!007 Copyright © The Americ.. m Society { I t Gene Therapy