- Email: [email protected]
389. Directed Transposon Integration in Human Cells
DNA VECTOROLOGY: TRANSPOSONS AND CHROMATIN EFFECTS only by the transposase cDNA and inverted repeat (IR) elements. Mos1 required full internal sequences to be present for measurable transposition activity in human cells. The piggyBac transposable element could be reduced to a 311bp left IR and a 236bp right IR with retention of full activity. Evaluation of overall transposable activity revealed Mos1 << SB < piggyBac in cultured human cells. Hyperactive mutants of the Mos1 transposase were evaluated and showed little increase in transposition over native Mos1 in human cells. Interestingly, piggyBac showed more activity than native or even hyperactive SB (two-to-ten fold more active than hyperactive SB12 transposase combined with hyperactive pT3 transposon). Additionally, overproduction inhibition, a known major limitation of SB, was lacking with piggyBac. We evaluated piggyBac transposition following transient transfection of 50ng, 100ng, and 2µg transposon DNA while varying the transposase DNA amount. In all cases, piggyBac lacked overproduction inhibition while SB transposition could be driven to very low levels at higher amounts of transposase DNA. Such results revealed that piggyBac transposition was limited only by the amount of transposon DNA present. Finally, we evaluated the addition of a zinc finger DNA binding domain element to the N-terminus of the different transposase enzymes. Zinc finger DNA binding domain addition ablated Mos1 activity and decreased SB activity which was only reliably measured using a combination of hyperactive transposase and transposon mutants. However, chimeric-piggyBac transposase activity appeared unaffected by N-terminal zinc finger DNA binding domain addition when compared to the native system. These results demonstrate that the piggyBac transposon system has advantageous properties when compared to other transposon systems with activity in human cells thereby making it a viable alternative system for gene therapy.
analyzed, 7 (8.6%) integrated into chromosome 19 (19q13.31) and 9 (11%) into chromosome 12 (12q22). Most importantly, of all independent integration sites analyzed 13% were found to contain an integrated transgene which was flanked by DNA sequences originated from two different chromosomes. This observation strongly suggested that chromosomal rearrangements might have occurred. This assumption is further supported by the fact that if one uses the same experimental approach this phenomenon has not been reported in over 1000 Sleeping Beauty mediated integration events and in less than 0.25% (1 out of 400) of all sites of insertion after adeno-associated virus (AAV) mediated integration. To confirm chromosomal translocations, we determined the genotype of single cell clones by karyotype analysis in HCT116 cells, a cell line for which the chromosomal content is relatively stable and well characterized. In sharp contrast to wildtype HCT116 cells, all twenty-four metaphases examined in cell clone HCT-1A7 had the same chromosomal abnormality on chromosome 22 band q13, which confirmed our plasmid rescue data. For some clones, a mixture of transgenes flanked by DNA sequences originated from the same but also two different chromosomes was identified. Taken together, his feature will introduce novel ways to genetically modify mammalian cells and to study the fate of cells with chromosomal translocations but suggests that additional safety studies will be required before the use of this integrase in gene therapy approaches.
388. Chromosomal Rearrangements in Mammalian Cells Are Induced at High Frequency by the Bacteriophage Derived Integrase phiC31
The DNA transposon Sleeping Beauty (SB) can integrate efficiently into host cell genomes. Although this process makes these elements attractive vehicles for therapeutic gene delivery, the nonspecific nature of integration presents inherent hazards. Herein, we attempt to direct transposon integration into predetermined genomic sites by combining the integration capabilities of the SB recombinase with the site-specific DNA-binding activity of zincfinger proteins (ZFPs). We fused a hyperactive SB transposase mutant (HSB5) to the polydactyl zinc finger protein E2C, which binds a unique 18 bp sequence (e2c) on human chromosome 17. A total of 12 different chimeras of these two proteins were cloned under the control of a strong CMV promoter and evaluated for proper expression, target DNA-binding, and transpositional activity following plasmid transfection into human cells. Through systematic analyses and codon optimization, we ultimately identified a configuration and inter-domain linker that resulted in appreciable levels of E2C-SB expression and high-level transposon excision and integration activity in mammalian cells, with our best fusion protein showing ∼18% the level of unfused SB activity. We also characterized the DNA-binding activities for this fusion protein in vitro using an electrophoretic mobility shift assay and within human cells using a luciferase-based reporter system. In both instances, the E2C-SB protein bound specifically to E2C and SB recognition sites, but the affinity for the latter was partially impaired relative to unfused SB. Finally, we studied the potential for E2C-mediated targeting to the endogenous chromosome 17 site by mapping E2C-SB- and SBmediated (control) integrations in human cells via a ligation-mediated (LM)-PCR approach. Since this region of the chromosome is relatively GC-rich and has few adjoining TA target sites, it was understandable that we did not recover any integrations within the vicinity of the endogenous e2c site in either group. Nevertheless, we did observe a 3-fold higher frequency of chromosome 17 hits with E2C-SB protein (n=5/41; 12.2%) compared with SB (n=1/28; 3.6%). These data are consistent with E2C-mediated chromosomal tethering,
Anja Ehrhardt,1,4 Jeffrey A. Engler,2 Hui Xu,1 Athena Cherry,3 Mark A. Kay.1 1 Departments of Pediatrics and Genetics, Stanford University, Stanford, CA; 2Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL; 3Department of Pathology, Stanford University, Stanford, CA; 4 Department of Virology, Ludwig-Maximilians-University of Munich, Munich, Germany. Recent reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that the integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudo attP sites in the host chromosomes. However, there is strong evidence that cryptic lox sites for Cre mediated integration are present in the eukaryotic genome which result in chromosomal translocations. Thus, we investigated whether or not cryptic phiC31 recognition sites may result in chromosomal rearrangements. In this study we found that the integrase phiC31 induced chromosomal rearrangements in the mammalian host genome with a frequency of up to 13% as demonstrated by integration site analysis and limited karyotyping studies. Single cell clones from in human hepatoma cells (Huh-7), human embryonic kidney cells (293), and the human colon carcinoma cell line HCT116 were grown and sites of insertion were characterized based on an established plasmid rescue protocol which allows identification of both chromosomal DNA sequences flanking the integrated transgene. Out of 81 independent integration events S148
389. Cells
Directed Transposon Integration in Human
Stephen R. Yant,1 Yong Huang,1 Bassel Akache,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
DNA VECTOROLOGY: TRANSPOSONS AND CHROMATIN EFFECTS but suggest that in the absence of a suitable target site, the transposon/fusion protein complex will disassociate from its target site and then seek out a closely-linked locus in a manner analogous to when SB undergoes local chromosomal hopping. In a second approach, we characterized inter-plasmid transposition events using target plasmids containing five tandem e2c sites. Consistent with our chromosomal data, we found flanking target site duplications at all integration sites, suggesting that E2C-SB does indeed mediate integration via a cut-and-paste transposition mechanism. Additionally, when compared to unfused SB, there was an 8-fold integration bias with E2C-SB over a narrow targeting window, indicating that ZFP-mediated DNA-binding can significantly bias integration site selection in human cells. In summary, our results demonstrate the targeting potential of ZFP-SB fusion proteins and indicate that site-directed transposon integration in the human genome may be a suitable means to diminish the theoretical risks associated with SB gene transfer.
390. Phage c31 Integrase Induces Chromosomal Aberrations in Primary Human Fibroblasts Jian Liu,1 Iben Jeppesen,1 Kate Nielsen,1 Thomas G. Jensen.1 Gene Therapy Laboratory, The Kennedy Institute-National Eye Clinic, Glostrup, Denmark.
1
Phage c31 integrase is a recombinase with the potential to perform site-specific integration into a host chromosome. The enzyme mediates recombination between two different short sequences of DNA, attP and attB recognition sites. In mouse and human genomes, phage c31 integrase mediates the integration of plasmids bearing an attB site into sequences with partial sequence identity to attP, called pseudo attP sites. Based on the identification of phage c31 pseudo attP sites in the human genome it was estimated that the total number of human pseudo attP sites is between 102 and 103. Phage c31 integrase-mediated gene delivery is under investigation as an novel tool for human gene therapy. In order to investigate the safety of phage c31 integrase-mediated integration, we have studied potential effects of prolonged phage c31 integrase expression in primary human fibroblasts, derived from a spontanous abortion. Embryonic cells were used due to their high proliferative potential and genomic stability. Primary human fibroblasts were transfected with the plasmids pBabepuro or pBabepuroatt, containing the 285-base pair attB sequence, with or without the plasmid pCMVInt expressing the phage c31 integrase. The co-transfection experiments were performed with a 3:1 molar excess of pCMVInt compared to the pBabepuro/ pBabepuroatt plasmids to increase the likelihood that pCMVInt plasmids were integrated in puromycin resistant cells. After puromycin selection, besides the cytogenetic analysis, DNA and RNA were also isolated and analysed using primers specific for integrase gene and also for the most common used integration site (the aatP site A on chromosome 8). Cells transfected with pBabepuro or pBabepuroatt alone had normal karyotypes. In contrast highly abnormal karyotypes were found in cells transfected with the pCMVInt plasmids. No obvious pattern involving specific chromosomes was observed, and chromosomes containing the previously published pseudo attP sites were not involved more frequently than the other chromosomes in the aberrations. Only 1 of 14 identified breakpoints matched the localisation of a published human pseudo attP site. Analysis of
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
puromycin-selected clones expressing the integrase likewise showed abnormal karyotypes. The experiments were reproduced in primary human fibroblasts from another embryo, and again, abnormal karyotypes were exclusively found in cells transfected with plasmids containing the phage c31 integrase gene. Thus we have shown that the phage c31 integrase induces chromosome rearrangement in primary human cells raising serious safety concerns about possible clinical use. Another member of the family of site-specific recombinases, the Cre recombinase, has the ability to cause widespread DNA damage when expressed at high levels. Here pseudo-sites were suspected to act as target for the integrase and give rise to a mutagenic background. Since transgenic mice and Drosophila with phage c31 integrase expression have been generated with no adverse consequences, further work is required to understand the mechanisms behind phage c31 integrase mediated chromosome instability.
391. De Novo Human Artificial Chromosome Formation Is Predetermined by Initial Chromatin Assembly and Transcriptional Patterns after Transfection Hiroshi Nakashima,1 Megumi Nakano,2 Ryoko Ohnishi,2 Yasushi Hiraoka,4 Yasufumi Kaneda,3 Akio Sugino,3 Hiroshi Masumoto.2 1 Neurological Surgery, The Ohio State University Medical Center, Columbus, OH; 2Nagoya University, Nagoya, Aichi, Japan; 3 Osaka University, Suita, Osaka, Japan; 4National Institute of Information and Communications Technology, Kobe, Hyogo, Japan. De novo, human artificial chromosomes (HACs) with no functional difference from native chromosomes can be created from transfected naked DNA in human cells. Because HACs can be maintained stably without integration into host chromosomes, have a much greater transgene capacity than mega bases, and are expected to have lower immunogenicity than virus-based vectors, they have great potential as vectors for gene therapy. However, how inserted transcribing sequences and their chromatin structures affect HAC formation is unclear. To investigate this, we constructed BAC-based linear HAC vectors having a common selection marker gene on the right arms and containing βgeo coding sequences with or without a functional promoter on the left arms. Although HACs were successfully generated in HT1080 cells from the vectors lacking a functional promoter on the left arm, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, -B, and -C) assembled at the integration sites that correlated with a transcriptionally active state of both marker genes on the vector arms. With the stable HACs, on the other hand, chromatin immunoprecipitation analysis showed that HP1α and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA. This suggests that epigenetic assembly of heterochromatin is required to establish a stable HAC and that the presence of actively transcribed sequences around the alphoid sequences does not inhibit assembly of centromere chromatin, but does inhibit formation of necessary heterochromatin regions. These results will contribute to our understanding of de novo centromere formation and comprehensive design of functional HAC vectors for human gene therapy.
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analyzed, 7 (8.6%) integrated into chromosome 19 (19q13.31) and 9 (11%) into chromosome 12 (12q22). Most importantly, of all independent integration sites analyzed 13% were found to contain an integrated transgene which was flanked by DNA sequences originated from two different chromosomes. This observation strongly suggested that chromosomal rearrangements might have occurred. This assumption is further supported by the fact that if one uses the same experimental approach this phenomenon has not been reported in over 1000 Sleeping Beauty mediated integration events and in less than 0.25% (1 out of 400) of all sites of insertion after adeno-associated virus (AAV) mediated integration. To confirm chromosomal translocations, we determined the genotype of single cell clones by karyotype analysis in HCT116 cells, a cell line for which the chromosomal content is relatively stable and well characterized. In sharp contrast to wildtype HCT116 cells, all twenty-four metaphases examined in cell clone HCT-1A7 had the same chromosomal abnormality on chromosome 22 band q13, which confirmed our plasmid rescue data. For some clones, a mixture of transgenes flanked by DNA sequences originated from the same but also two different chromosomes was identified. Taken together, his feature will introduce novel ways to genetically modify mammalian cells and to study the fate of cells with chromosomal translocations but suggests that additional safety studies will be required before the use of this integrase in gene therapy approaches.
388. Chromosomal Rearrangements in Mammalian Cells Are Induced at High Frequency by the Bacteriophage Derived Integrase phiC31
The DNA transposon Sleeping Beauty (SB) can integrate efficiently into host cell genomes. Although this process makes these elements attractive vehicles for therapeutic gene delivery, the nonspecific nature of integration presents inherent hazards. Herein, we attempt to direct transposon integration into predetermined genomic sites by combining the integration capabilities of the SB recombinase with the site-specific DNA-binding activity of zincfinger proteins (ZFPs). We fused a hyperactive SB transposase mutant (HSB5) to the polydactyl zinc finger protein E2C, which binds a unique 18 bp sequence (e2c) on human chromosome 17. A total of 12 different chimeras of these two proteins were cloned under the control of a strong CMV promoter and evaluated for proper expression, target DNA-binding, and transpositional activity following plasmid transfection into human cells. Through systematic analyses and codon optimization, we ultimately identified a configuration and inter-domain linker that resulted in appreciable levels of E2C-SB expression and high-level transposon excision and integration activity in mammalian cells, with our best fusion protein showing ∼18% the level of unfused SB activity. We also characterized the DNA-binding activities for this fusion protein in vitro using an electrophoretic mobility shift assay and within human cells using a luciferase-based reporter system. In both instances, the E2C-SB protein bound specifically to E2C and SB recognition sites, but the affinity for the latter was partially impaired relative to unfused SB. Finally, we studied the potential for E2C-mediated targeting to the endogenous chromosome 17 site by mapping E2C-SB- and SBmediated (control) integrations in human cells via a ligation-mediated (LM)-PCR approach. Since this region of the chromosome is relatively GC-rich and has few adjoining TA target sites, it was understandable that we did not recover any integrations within the vicinity of the endogenous e2c site in either group. Nevertheless, we did observe a 3-fold higher frequency of chromosome 17 hits with E2C-SB protein (n=5/41; 12.2%) compared with SB (n=1/28; 3.6%). These data are consistent with E2C-mediated chromosomal tethering,
Anja Ehrhardt,1,4 Jeffrey A. Engler,2 Hui Xu,1 Athena Cherry,3 Mark A. Kay.1 1 Departments of Pediatrics and Genetics, Stanford University, Stanford, CA; 2Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL; 3Department of Pathology, Stanford University, Stanford, CA; 4 Department of Virology, Ludwig-Maximilians-University of Munich, Munich, Germany. Recent reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that the integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudo attP sites in the host chromosomes. However, there is strong evidence that cryptic lox sites for Cre mediated integration are present in the eukaryotic genome which result in chromosomal translocations. Thus, we investigated whether or not cryptic phiC31 recognition sites may result in chromosomal rearrangements. In this study we found that the integrase phiC31 induced chromosomal rearrangements in the mammalian host genome with a frequency of up to 13% as demonstrated by integration site analysis and limited karyotyping studies. Single cell clones from in human hepatoma cells (Huh-7), human embryonic kidney cells (293), and the human colon carcinoma cell line HCT116 were grown and sites of insertion were characterized based on an established plasmid rescue protocol which allows identification of both chromosomal DNA sequences flanking the integrated transgene. Out of 81 independent integration events S148
389. Cells
Directed Transposon Integration in Human
Stephen R. Yant,1 Yong Huang,1 Bassel Akache,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
DNA VECTOROLOGY: TRANSPOSONS AND CHROMATIN EFFECTS but suggest that in the absence of a suitable target site, the transposon/fusion protein complex will disassociate from its target site and then seek out a closely-linked locus in a manner analogous to when SB undergoes local chromosomal hopping. In a second approach, we characterized inter-plasmid transposition events using target plasmids containing five tandem e2c sites. Consistent with our chromosomal data, we found flanking target site duplications at all integration sites, suggesting that E2C-SB does indeed mediate integration via a cut-and-paste transposition mechanism. Additionally, when compared to unfused SB, there was an 8-fold integration bias with E2C-SB over a narrow targeting window, indicating that ZFP-mediated DNA-binding can significantly bias integration site selection in human cells. In summary, our results demonstrate the targeting potential of ZFP-SB fusion proteins and indicate that site-directed transposon integration in the human genome may be a suitable means to diminish the theoretical risks associated with SB gene transfer.
390. Phage c31 Integrase Induces Chromosomal Aberrations in Primary Human Fibroblasts Jian Liu,1 Iben Jeppesen,1 Kate Nielsen,1 Thomas G. Jensen.1 Gene Therapy Laboratory, The Kennedy Institute-National Eye Clinic, Glostrup, Denmark.
1
Phage c31 integrase is a recombinase with the potential to perform site-specific integration into a host chromosome. The enzyme mediates recombination between two different short sequences of DNA, attP and attB recognition sites. In mouse and human genomes, phage c31 integrase mediates the integration of plasmids bearing an attB site into sequences with partial sequence identity to attP, called pseudo attP sites. Based on the identification of phage c31 pseudo attP sites in the human genome it was estimated that the total number of human pseudo attP sites is between 102 and 103. Phage c31 integrase-mediated gene delivery is under investigation as an novel tool for human gene therapy. In order to investigate the safety of phage c31 integrase-mediated integration, we have studied potential effects of prolonged phage c31 integrase expression in primary human fibroblasts, derived from a spontanous abortion. Embryonic cells were used due to their high proliferative potential and genomic stability. Primary human fibroblasts were transfected with the plasmids pBabepuro or pBabepuroatt, containing the 285-base pair attB sequence, with or without the plasmid pCMVInt expressing the phage c31 integrase. The co-transfection experiments were performed with a 3:1 molar excess of pCMVInt compared to the pBabepuro/ pBabepuroatt plasmids to increase the likelihood that pCMVInt plasmids were integrated in puromycin resistant cells. After puromycin selection, besides the cytogenetic analysis, DNA and RNA were also isolated and analysed using primers specific for integrase gene and also for the most common used integration site (the aatP site A on chromosome 8). Cells transfected with pBabepuro or pBabepuroatt alone had normal karyotypes. In contrast highly abnormal karyotypes were found in cells transfected with the pCMVInt plasmids. No obvious pattern involving specific chromosomes was observed, and chromosomes containing the previously published pseudo attP sites were not involved more frequently than the other chromosomes in the aberrations. Only 1 of 14 identified breakpoints matched the localisation of a published human pseudo attP site. Analysis of
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
puromycin-selected clones expressing the integrase likewise showed abnormal karyotypes. The experiments were reproduced in primary human fibroblasts from another embryo, and again, abnormal karyotypes were exclusively found in cells transfected with plasmids containing the phage c31 integrase gene. Thus we have shown that the phage c31 integrase induces chromosome rearrangement in primary human cells raising serious safety concerns about possible clinical use. Another member of the family of site-specific recombinases, the Cre recombinase, has the ability to cause widespread DNA damage when expressed at high levels. Here pseudo-sites were suspected to act as target for the integrase and give rise to a mutagenic background. Since transgenic mice and Drosophila with phage c31 integrase expression have been generated with no adverse consequences, further work is required to understand the mechanisms behind phage c31 integrase mediated chromosome instability.
391. De Novo Human Artificial Chromosome Formation Is Predetermined by Initial Chromatin Assembly and Transcriptional Patterns after Transfection Hiroshi Nakashima,1 Megumi Nakano,2 Ryoko Ohnishi,2 Yasushi Hiraoka,4 Yasufumi Kaneda,3 Akio Sugino,3 Hiroshi Masumoto.2 1 Neurological Surgery, The Ohio State University Medical Center, Columbus, OH; 2Nagoya University, Nagoya, Aichi, Japan; 3 Osaka University, Suita, Osaka, Japan; 4National Institute of Information and Communications Technology, Kobe, Hyogo, Japan. De novo, human artificial chromosomes (HACs) with no functional difference from native chromosomes can be created from transfected naked DNA in human cells. Because HACs can be maintained stably without integration into host chromosomes, have a much greater transgene capacity than mega bases, and are expected to have lower immunogenicity than virus-based vectors, they have great potential as vectors for gene therapy. However, how inserted transcribing sequences and their chromatin structures affect HAC formation is unclear. To investigate this, we constructed BAC-based linear HAC vectors having a common selection marker gene on the right arms and containing βgeo coding sequences with or without a functional promoter on the left arms. Although HACs were successfully generated in HT1080 cells from the vectors lacking a functional promoter on the left arm, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, -B, and -C) assembled at the integration sites that correlated with a transcriptionally active state of both marker genes on the vector arms. With the stable HACs, on the other hand, chromatin immunoprecipitation analysis showed that HP1α and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA. This suggests that epigenetic assembly of heterochromatin is required to establish a stable HAC and that the presence of actively transcribed sequences around the alphoid sequences does not inhibit assembly of centromere chromatin, but does inhibit formation of necessary heterochromatin regions. These results will contribute to our understanding of de novo centromere formation and comprehensive design of functional HAC vectors for human gene therapy.
S149