phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were d

phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were discovered using half-nested inverse PCR from cow fibroblasts. The genomic locations of these two pseudo attP sites were identified by direct sequencing and a BLAST search, and it was confirmed that they reside at positions 4q31 and 10q35 by fluorescence in situ hybridization analysis. Subsequently, the distinct integration frequencies of the four pseudo attP sites were examined. The BF4 site was identified as a hotspot where site-specific integration occurred in most of the cell clones examined, accounting for 74% (42/57) of the integration; much more than the integration frequency for BF10 (7%; 4/57), BpsF1 (7%; 4/57) and BpsM1 (0/57). Interestingly, similar to other hotspots identified in the human and mouse genomes, in which transgenes integrated at hotspots result in high expression, the GFP gene integrated at hotspot BF4 was expressed at high levels in cow fibroblasts, as confirmed by fluorescence microscopy and FACS analysis. Furthermore, ELISA showed that the expression level of the GFP gene integrated at the BF4 site averaged approximately 328 microg x mg(-1), which is more than twofold higher than that integrated at the BF10 site. This study suggests that somatic cells carrying a desired gene integrated at the BF4 site can be used as nuclear donors to generate valuable transgenic animals by nuclear transfer.

Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. M

Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. METHODS: First, we constructed a strong liver-specific expression plasmid for human factor IX (FIX). Next, we tested the vector by injecting two doses under hydrodynamic conditions into the tail veins of FIX knockout mice. RESULTS: A single injection resulted in an increase in FIX expression over 100% of normal plasma levels. The FIX resulted fully functional. Further, no anti-FIX antibodies were observed and expression levels were vector dose dependent. CONCLUSION: The high expression obtained in small animals give hope for further development of non-viral gene transfer for the treatment of hemophilia B in humans.

Mobile genetic elements with the ability to integrate genetic information into chromosomes can cause disease over short periods of time and shape genomes over eons. These elements can be used for functional genomics, gene transfer and human gene therapy. However, their integration-site preferences,

Mobile genetic elements with the ability to integrate genetic information into chromosomes can cause disease over short periods of time and shape genomes over eons. These elements can be used for functional genomics, gene transfer and human gene therapy. However, their integration-site preferences, which are critically important for these uses, are poorly understood. We analyzed the insertion sites of several transposons and retroviruses to detect patterns of integration that might be useful for prediction of preferred integration sites. Initially we found that a mathematical description of DNA-deformability, called V(step), could be used to distinguish preferential integration sites for Sleeping Beauty (SB) transposons into a particular 100 bp region of a plasmid [G. Liu, A. M. Geurts, K. Yae, A. R. Srinivassan, S. C. Fahrenkrug, D. A. Largaespada,J. Takeda, K. Horie, W. K. Olson and P. B. Hackett (2005) J. Mol. Biol., 346, 161-173 ]. Based on these findings, we extended our examination of integration of SB transposons into whole plasmids and chromosomal DNA. To accommodate sequences up to 3 Mb for these analyses, we developed an automated method, ProTIS, that can generate profiles of predicted integration events. However, a similar approach did not reveal any structural pattern of DNA that could be used to predict favored integration sites for other transposons as well as retroviruses and lentiviruses due to a limitation of available data sets. Nonetheless, ProTIS has the utility for predicting likely SB transposon integration sites in investigator-selected regions of genomes and our general strategy may be useful for other mobile elements once a sufficiently high density of sites in a single region are obtained. ProTIS analysis can be useful for functional genomic, gene transfer and human gene therapy applications using the SB system.

DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene

DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn--thus, answering the question "What genomic features affect integration?" requires carefully specifying the length scale of interest.

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.

When a retrovirus infects a cell, its RNA genome is reverse transcribed into a double-stranded DNA, which is then permanently integrated into the host chromosome. Integration is one of the essential steps in the retroviral life cycle. Many transposable elements also move around and integrate into th

When a retrovirus infects a cell, its RNA genome is reverse transcribed into a double-stranded DNA, which is then permanently integrated into the host chromosome. Integration is one of the essential steps in the retroviral life cycle. Many transposable elements also move around and integrate into the host genome as part of their life cycle, some through RNA intermediates and some through 'cut and paste' mechanisms. Integration of retroviruses and transposable elements into 'sensitive areas' of the genome can cause irreparable damage. On the other hand, because of their ability to integrate permanently, and the relatively efficient rates of transgenesis, retroviruses and transposable elements are widely used as gene delivery tools in basic research and gene therapy trials. Recent events in gene therapy treatments for X-linked severe combined immunity deficiencies (X-SCID) have highlighted both the promise and some of the risks involved with utilizing retroviruses. Nine of 11 children were successfully treated for X-SCID using a retrovirus carrying the gene mutated in this disease. However, later two of these children developed leukemias because of retroviral integrations in the putative oncogene LMO2 [1]. A third child has also been demonstrated to have an integration in LMO2, but is as of yet nonsymptomatic [2]. It is a bit difficult to explain the high frequency of integrations into the same gene using a random model of retroviral integration, and there has been evidence for decades that retroviral integrations may not be random. But the data were somewhat limited in their power to determine the precise nature of the integration biases. The completion of the human genome sequence coupled with sensitive polymerase chain reaction techniques and an ever-decreasing cost of sequencing has given a powerful new tool to the study of integration site selection. In this review, we describe the findings from several recent global surveys of target site selection by retroviruses and transposable elements, and discuss the possible ramifications of these findings to both mechanisms of action and to the use of these elements as gene therapy vectors.

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We m

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites ( /-5kb), near CpG islands ( /-1kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transpl

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

Reports on insertional "genotoxicity" in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese

Reports on insertional "genotoxicity" in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese diabetic/severe combined immunodeficient mice. A total of 186 retroviral vector integration sites were isolated by ligation-mediated PCR from chimeric mouse bone marrow of five PBPC donors, sequenced, and blasted against the human genome. Preferred integration near the transcription start regions, within CpG islands, and within Alu regions was observed. Detailed analysis of targeted RefSeq genes showed a favored integration within the first intron. Integrations were most common in genes coding for signaling proteins, transcription factors, and kinases. In all genes targeted independently multiple times the respective orientation of the provirus within the gene was identical, indicating integration hot spot regions and similar steric determinants for integration sites. Possible explanations for these findings could be nonrandom vector integration, clonal selection due to gene expression interference, or engraftment issues related to gene insertion in signaling and cell cycle genes. The low frequency of integrations in exons may be reassuring as to the safety of retroviral gene therapy with normal human PBPC.

OBJECTIVE: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in p

OBJECTIVE: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. METHODS: To allow large-scale and standardized comparison of insertion sites of viral vectors we developed two programs, IntegrationSeq and IntegrationMap. IntegrationSeq can trim sequences, and valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units, and further parameters like the distance from the transcription start site to the integration. RESULTS: We validated the method using 259 files originating from integration site analysis (LM-PCR). Sequences processed by IntegrationSeq led to an increased yield of valid integration sequence detection, which were shown to be more sensitive than conventional analysis and 15 times faster, while the specificities are equal. Output files generated by IntegrationMap were found to be 99.8% identical with results retrieved by much slower conventional mapping with the ENSEMBL alignment tool. CONCLUSION: Using IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the huma

To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the human immunodeficiency virus type 1 and visna virus integrases was found to be responsible for selecting non-viral target DNA sites when small alcohols provide the attacking nucleophilic OH group during non-specific alcoholysis assays. Testing deletion derivatives of the integrase protein in this assay, which has similarities to the DNA joining reaction that occurs during retroviral integration, defined a smaller central domain that is sufficient for activity. Thus, this core domain likely contains both the host DNA site and the nucleophile site. Surprisingly, the region of integrase responsible for selecting non-viral target DNA sites when the viral DNA end is the attacking nucleophile could not similarly be mapped with the standard oligonucleotide joining assay. We therefore tested the proteins in a more sensitive assay that displays preferred sites of viral DNA insertion in a plasmid DNA target. All 12 chimeras yielded novel patterns compared with the wild-type enzymes in this assay, although local insertion patterns indicated that the central domain plays an important role in target site selection. Together, these data suggest that other protein regions must be involved when the attacking nucleophilic group is provided by viral DNA. Because specific recognition of viral DNA ends was previously mapped to the central domain, two different regions of integrase must interact with retroviral DNA.

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.

The potential dangers of using viruses to deliver and integrate DNA into host cells in gene therapy have been poignantly highlighted in recent clinical trials. Safer, non-viral gene delivery approaches have been largely ignored in the past because of their inefficient delivery and the resulting tran

The potential dangers of using viruses to deliver and integrate DNA into host cells in gene therapy have been poignantly highlighted in recent clinical trials. Safer, non-viral gene delivery approaches have been largely ignored in the past because of their inefficient delivery and the resulting transient transgene expression. However, recent advances indicate that efficient, long-term gene expression can be achieved by non-viral means. In particular, integration of DNA can be targeted to specific genomic sites without deleterious consequences and it is possible to maintain transgenes as small episomal plasmids or artificial chromosomes. The application of these approaches to human gene therapy is gradually becoming a reality.

The administration of naked nucleic acids into animals is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models (Herweijer and Wolff, 2003; Hodges and Scheule, 2003). It i

The administration of naked nucleic acids into animals is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models (Herweijer and Wolff, 2003; Hodges and Scheule, 2003). It is also being used in several human clinical trials for genetic vaccines, Duchenne muscular dystrophy, peripheral limb ischemia, and cardiac ischemia (Davis et al., 1996; Romero et al., 2002; Tsurumi et al., 1997). Naked DNA is an attractive non-viral vector because of its inherent simplicity and because it can easily be produced in bacteria and manipulated using standard recombinant DNA techniques. It shows very little dissemination and transfection at distant sites following delivery and can be readministered multiple times into mammals (including primates) without inducing an antibody response against itself (i.e., no anti-DNA antibodies generated) (Jiao et al., 1992). Also, contrary to common belief, long-term foreign gene expression from naked plasmid DNA (pDNA) is possible even without chromosome integration if the target cell is postmitotic (as in muscle) or slowly mitotic (as in hepatocytes) and if an immune reaction against the foreign protein is not generated (Herweijer et al., 2001; Miao et al., 2000; Wolff et al., 1992; Zhang et al., 2004). With the advent of intravascular and electroporation techniques, its major restriction--poor expression levels--is no longer limiting and levels of foreign gene expression in vivo are approaching what can be achieved with viral vectors. Direct in vivo gene transfer with naked DNA was first demonstrated when efficient transfection of myofibers was observed following injection of mRNA or pDNA into skeletal muscle (Wolff et al., 1990). It was an unanticipated finding in that the use of naked nucleic acids was the control for experiments designed to assess the ability of cationic lipids to mediate expression in vivo. Subsequent studies also found foreign gene expression after direct injection in other tissues such as heart, thyroid, skin, and liver (Acsadi et al., 1991; Hengge et al., 1996; Kitsis and Leinwand, 1992; Li et al., 1997; Sikes and O'Malley 1994; Yang and Huang, 1996). However, the efficiency of gene transfer into skeletal muscle and these other tissues by direct injection is relatively low and variable, especially in larger animals such as nonhuman primates (Jiao et al., 1992). After our laboratory had developed novel transfection complexes of pDNA and amphipathic compounds and proteins, we sought to deliver them to hepatocytes in vivo via an intravascular route into the portal vein. Our control for these experiments was naked pDNA and we were once again surprised that this control group had the highest expression levels (Budker et al., 1996; Zhang et al., 1997). High levels of expression were achieved by the rapid injection of naked pDNA in relatively large volumes via the portal vein, the hepatic vein, and the bile duct in mice and rats. The procedure also proved effective in larger animals such as dogs and nonhuman primates (Eastman et al., 2002; Zhang et al., 1997). The next major advance was the demonstration that high levels of expression could also be achieved in hepatocytes in mice by the rapid injection of naked DNA in large volumes simply into the tail vein (Liu et al., 1999; Zhang et al., 1999). This hydrodynamic tail vein (HTV) procedure is proving to be a very useful research tool not only for gene expression studies, but also more recently for the delivery of small interfering RNA (siRNA) (Lewis et al., 2002; McCaffrey et al., 2002). The intravascular delivery of naked pDNA to muscle cells is also attractive particularly since many muscle groups would have to be targeted for intrinsic muscle disorders such as Duchenne muscular dystrophy. High levels of gene expression were first achieved by the rapid injection of naked DNA in large volumes via an artery route with both blood inflow and outflow blocked surgically (Budker et al., 1998; Zhang et al., 2001). Intravenous routes have also been shown to be effective (Hagstrom et al., 2004; Liang et al., 2004; Liu et al., 2001). For limb muscles, the ability to use a peripheral limb vein for injection and a proximal, external tourniquet to block blood flow renders the procedure to be clinically viable. This review concerns itself with the mechanism by which naked DNA is taken up by cells in vivo. A greater understanding of the mechanisms involved in the uptake and expression of naked DNA, and thus connections between postulated mechanisms and expression levels, is emphasized. Inquiries into the mechanism not only aid these practical efforts, but are also interesting on their own account with relevance to viral transduction and cellular processes. The delivery to hepatocytes is first discussed given the greater information available for this process, and then uptake by myofibers is discussed.

It is highly desirable to generate tissue-specific and persistently high-level transgene expression per genomic copy from gene therapy vectors. Such vectors can reduce the cost and preparation of the vectors and reduce possible host immune responses to the vector and potential toxicity. Many gene th

It is highly desirable to generate tissue-specific and persistently high-level transgene expression per genomic copy from gene therapy vectors. Such vectors can reduce the cost and preparation of the vectors and reduce possible host immune responses to the vector and potential toxicity. Many gene therapy vectors have failed to produce therapeutic levels of transgene because of inefficient promoters, loss of vector or gene expression from episomal vectors, or a silencing effect of integration sites on integrating vectors. Using in vivo screening of vectors incorporating many different combinations of gene regulatory sequences, liver-specific, high-expressing vectors to accommodate factor IX, factor VIII, and other genes for effective gene transfer have been established. Persistent and high levels of factor IX and factor VIII gene expression for treating hemophilia B and A, respectively, were achieved in mouse livers using hydrodynamics-based gene transfer of naked plasmid DNA incorporating these novel gene expression systems. Some other systems to prolong or stabilize the gene expression following gene transfer are also discussed.