attB Site Research Articles

ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

Identification and sequence analysis of the region encoding the site-specific integration system from Leuconostoc œnos (Œnococcus œni) temperate bacteriophage φ10MC

Malolactic fermentation conducted by Leuconostoc oenos is an essential step in winemaking. L. oenos bacteriophages are thought to be responsible for fermentation failures, yet they have received little attention. The integration system of bacteriophage phi 10MC in the LOF111 L. oenos strain chromosome was studied and a 1,456 bp phage DNA fragment was cloned and sequenced. An open reading frame (int) showing homology with several temperate bacteriophage integrases was located upstream of the phage attachment site (attP). This organisation is comparable to other phage site-specific recombination systems. The same bacterial attachment site (attB) located in at tRNA(leu) gene was found in all 15 L. oenos strains studied and was involved in phi 10MC integration in the bacterial chromosome.

525. Mutants of phiC31 Integrase with Increased Efficiency and Specificity

The site-specific integrase from bacteriophage phiC31, catalyzes precise, unidirectional recombination between two |[sim]|30-40 bp sequences, a phage attachment site (attP) and a bacterial attachment site (attB). In mammalian cells, phiC31 integrase can recombine its own sites and can also integrate plasmids with the attB site into native sequences that bear partial identity to attP, referred to as pseudo attP sites. The enzyme recognizes a hierarchy of such sites and, in cell lines, appears to have several hundred potential integration sites, though most integration events occur at a subset of preferred sites. For the purpose of gene therapy, it appears desirable to obtain integration at only one or a small set of safe, well-expressed locations in the genome. In a previous study we demonstrated that by using a genetic screening assay in E. coli, we could create mutant forms of phiC31 integrase with enhanced preference for a pre-existing favored integration site in the human genome. However, this enzyme displayed a low overall integration efficiency. To create phiC31 integrase mutants with enhanced functionality, we devised a screen in mammalian cells to identify mutants with increased recombination efficiency at the attP site. We isolated a collection of such mutants, and combination of several of them led to the identification of an enzyme that mediated the majority of integration events at a single location, at a pre-integrated attP site. This enzyme may be useful for the rapid creation of cell lines with integration at a fixed location. The study also provided proof-of-principle for creation of integrases highly specific for endogenous sequences, a project is underway in the lab. Other mutants from the screen showed an elevated integration frequency at endogenous pseudo attP sites and are being tested in animals as improved gene therapy tools. We have also undertaken the structural characterization of phiC31 integrase. The location of the DNA-binding domain is being investigated by creating deletion mutants and examining them for att site binding with gel mobility shift assays. The increased efficiency and specificity attainable via mutagenesis and protein design will further enhance the utility of phiC31 integrase as a valuable tool in gene therapy.

863. Liver Gene Therapy for Hemophilia with phiC31 Integrase

The phiC31 integrase system effectively integrates plasmid DNA carrying an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, long-term expression of transgenes. The phiC31 system has been used to genetically engineer Drosophila and Xenopus and for in vivo and ex vivo gene therapy in several tissues in model organisms including mouse, rat, and rabbit. The integrase system appears to be well suited for long-term gene therapy in humans. In particular, the phiC31 system may allow long-term gene expression in hepatocytes in vivo. A simple and efficient delivery system for liver using naked DNA is available for mice by using high-pressure tail vein injection. With this delivery method and phiC31 integrase, we demonstrated lifelong, therapeutic levels of expression of human factor IX in mice. An adapted version of this DNA delivery method may be appropriate for use in patients. Our current study addressed the amount of liver perturbation and subsequent proliferation seen after the high-pressure tail vein procedure. Using a BrdU analog to label proliferating hepatocytes, we showed that a sizable fraction of the hepatocytes entered S phase within two days after injection. By one week post-injection, however, hepatocytes had stopped proliferating and the livers appeared normal by histological analysis. We also determined the immunogenicity and ability to re-deliver phiC31 integrase by measurement of expression levels of a reporter gene after repeated doses. The level and duration of gene expression achievable with the phiC31 integrase system in liver makes it an attractive strategy for clinical development of gene therapies for diseases such as hemophilia.

390. Phage c31 Integrase Induces Chromosomal Aberrations in Primary Human Fibroblasts

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.

Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase

The integrase from the Streptomyces bacteriophage phiC31 carries out efficient recombination between an attP site in the phage genome and an attB site in the host chromosome. In the present study, we have used the phiC31 integrase system to mediate site-specific recombination in the cultured silkworm cell line BmN4. A plasmid containing a cDNA encoding DsRed flanked by two phiC31 attP sites was co-transfected together with a helper plasmid encoding the phiC31 integrase into a cell line in which phiC31 attB sites inserted between a baculovirus IE2 promoter, and a polyadenylation signal are present in one chromosome. Seven days after transfection, expression of DsRed was observed in transformed cells. Nucleotide sequence analysis demonstrated that the expected recombination between the attB and attP sites had been precisely carried out by the phiC31 integrase. These results indicate that the phiC31 site-specific recombination system should be widely applicable for efficient site-specific gene integration into silkworm chromosomes.

Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis.

So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus Bifidobacterium. In this report we analyzed three prophage-like elements that are present in the genomes of Bifidobacterium breve UCC 2003, Bifidobacterium longum NCC 2705, and Bifidobacterium longum DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNA(Met) gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the attB sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a B. longum DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among B. breve strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.

GroEL1: A Dedicated Chaperone Involved in Mycolic Acid Biosynthesis during Biofilm Formation in Mycobacteria

Mycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential--presumably providing the housekeeping chaperone functions--while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates--long-chain fatty acid components of the mycobacterial cell wall--specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56-C68) fatty acids, and strains with altered mycolate profiles--including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA--are defective in biofilm formation.

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the phiC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.

Rearranging the centromere of the human Y chromosome with C31 integrase

We have investigated the ability of the integrase from the Streptomyces φC31 ‘phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a φC31 integrase attB site and the chicken genome.

Combinatorial biosynthesis of lipopeptide antibiotics in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. Cubicin (daptomycin-for-injection) was approved in 2003 by the FDA to treat skin and skin structure infections caused by Gram-positive pathogens. Daptomycin is particularly significant in that it represents the first new natural product antibacterial structural class approved for clinical use in three decades. The daptomycin gene cluster contains three very large genes (dptA, dptBC, and dptD) that encode the nonribosomal peptide synthetase (NRPS). The related cyclic lipopeptide A54145 has four NRPS genes (lptA, lptB, lptC, and lptD), and calcium dependent antibiotic (CDA) has three (cdaPS1, cdaPS2, and cdaPS3). Mutants of S. roseosporus containing deletions of one or more of the NRPS genes have been trans-complemented with dptA, dptBC, and dptD by inserting these genes under the control of the ermEp* promoter into separate conjugal cloning vectors containing phiC31 or IS117 attachment (attP int) sites; delivering the plasmids into S. roseosporus by conjugation from Escherichia coli; and inserting the plasmids site-specifically into the chromosome at the corresponding attB sites. This trans-complementation system was used to generate subunit exchanges with lptD and cdaPS3 and the recombinants produced novel hybrid molecules. Module exchanges at positions D: -Ala(8) and D: -Ser(11) in the peptide have produced additional novel derivatives of daptomycin. The approaches of subunit exchanges and module exchanges were combined with amino acid modifications of Glu at position 12 and natural variations in lipid side chain starter units to generate a combinatorial library of antibiotics related to daptomycin. Many of the engineered strains produced levels of novel molecules amenable to isolation and antimicrobial testing, and most of the compounds displayed antibacterial activities.

Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G + C codon optimized integrase for site-specific integration

pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/ int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site ( attP). The integrase gene contained a high frequency of codons rarely used in high G + C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/ int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA Phe gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment ( attB) site contain a 63 bp region of sequence identity overlapping the 3′ end of the tRNA Phe gene. Plasmids comprising the site-specific att/ int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.

Horizontal transfer of Yersinia high‐pathogenicity island by the conjugative RP4 attB target‐presenting shuttle plasmid

The high-pathogenicity island (HPI) encodes a highly efficient yersiniabactin system of iron acquisition responsible for mouse lethality in Yersinia. Although the HPI is widely disseminated among Enterobacteriaceae it lacks functions necessary for its replication and transmission. Therefore, the mechanism of its horizontal transfer and circulation is completely obscure. On the other hand, the HPI is a genetically active island in the bacterial cell. It encodes a functional recombinase and is able to transpose to new targets on the chromosome. Here we report on a possible mechanism of the HPI dissemination based on site-specific recombination of the excised HPI with the attB-presenting (asn tRNA gene) RP4 promiscuous conjugative shuttle plasmid. The resulting cointegrate can be transferred by conjugation to a new host, where it dissociates, and the released HPI integrates into any unoccupied asn tRNA gene target in the genome. This mechanism has been proven both with the 'mini' island carrying only the attP recognition site and genes coding for recombination enzymes and with the complete HPI labelled with an antibiotic resistance marker. After acquisition of the mobilized complete form of the HPI, the ability of the HPI-cured Yersinia enterocolitica WA-TH(-) strain to produce yersiniabactin has been restored. Such 'trapping' of pathogenicity islands and subsequent shuffling to new hosts by a conjugative replicon carrying a suitable attB site could be applied to other functional integrative elements and explain wide dissemination of PAIs.

884. Integration of a Helper-Dependent Adenovirus Mediated by the Phage ΦC31 Integrase

Adenoviral vectors (Ad) deleted of all viral genes, known as helper-dependent Ad (HD) or gutted Ad, possess a linear genomic DNA that remains as a non-integrated episome in the nucleus of the infected cells. Because HD can be grown to very high titers, are weakly immunogenic and can carry up to 35-kb of foreign DNA, they are very attractive vectors for gene therapy applications. Under certain circumstances (for example to stably express large cDNAs such as dystrophin into stem cells or myoblasts) it could be valuable to promote integration of HD into the cell chromosomes. The phage C31 integrase normally mediates integration of a phage DNA containing an attP site into the attB site of the bacterial chromosome. Interestingly, when the C31 integrase is expressed in mammalian cells, it can mediate integration of a circular DNA containing an attB site into pseudo attP sites of the cell chromosomes. In the present study, we tested whether the C31 integrase could promote integration of an HD into mammalian cells. We have thus constructed an HD containing an attB site, which carries as a gene reporter, a green fluorescent protein (GFP) expression cassette. In addition, the GFP expression cassette was flanked by two loxP sites to permit its circularization upon expression of the Cre recombinase. HeLa cells were transfected with plasmids expressing Cre and the C31 integrase. The next day, the cells were transduced with HD-GFPattB and at selected time points, without any selective pressure, the expression of GFP was quantified by flow cytometry. At twelve days post-transduction, the fluorescence index ([% of GFP positive cells] X [intensity of fluorescence]) was significantly higher in cells transfected with Cre and the C31 integrase. This observation suggests these two enzymes are able to promote integration of HD-GFPattB. Experiments are in progress to optimize the integration efficiency and to test if Cre expression is essential for this process to occur.

193. Direct Comparison of Integration Efficiency and Stable Gene Expression Mediated by the Sleeping Beauty Transposon System and by the PhiC31 Phage Integrase System in Cultured Human Fibroblasts and in Various Stem Cell Targets

Top of pageAbstract Significant research efforts are directed toward achieving non-viral, DNA-mediated transfer and stable expression of genes in somatic cells for the treatment of diseases. Plasmid DNA has been successfully used to deliver genes into cells, but its usefulness is restricted by the inability to provide sustained expression. One alternative is to include components of an integrating system in the vector design. The Sleeping Beauty (SB) transposase and phiC31 integrase both bring about insertion of DNA sequences into the genomes of human cells, suggesting an alternative to viral transduction. Because each approach requires its own unique genetic elements and catalytic enzyme to mediate gene insertion into cellular chromosomes, we chose to directly compare the effectiveness of integration and stable expression using a two-component system that is internally controlled for the integrating sequence. The integrating vector (pKT2/NAG) consists of (i) a phiC31 integrase recognition site (attB), (ii) eF1-alpha regulated GFP expression unit, and (iii) neomycin resistance gene regulated by the PGK promoter, all flanked by (iv) T2 transposase binding sites (IR/DRs) separated by (v) a colE1 bacterial origin and kanR gene. Overexpression inhibition of the SB transposase suggests a potential limitation with respect to integration efficiency that can be achieved by this vector. To address this issue, we first studied the integrating efficiency of pKT2/NAG by evaluating the effect of the dose of the co-delivered enzyme-encoding plasmid on integration and stable gene expression as determined by G418-resistant colony formation and GFP fluorescence. In each case, pKT2/NAG (500 ng) was co-transfected with 150 ng, 500 ng, or 1500 ng of either CMV-regulated luciferase (pCMV-Luc), transposase (pCMV-SB), or integrase (pCMV-Int) expression plasmids into cultured human HT1080 cells. G418-resistant colony formation exceeded levels achieved by random integration (+ pCMV-Luc) at all tested concentrations of the SB transposase-encoding plasmid, with diminished efficiency at higher concentrations. The inverse relationship was observed for phiC31 integrase, where gene transfer improved as the concentration of pCMV-Int increased relative to pKT2/NAG. Drug-resistant clones have been isolated for studies into: (i) gene silencing by flow cytometric analysis of GFP fluorescence, (ii) insertional preference by cloning and sequencing vector::chromosome junctions, and (iii) insertion frequency by Southern analysis. We have also co-delivered both components into rat multipotent adult progenitor cells and found both systems to effectively mediate G418-resistant colony formation above background. Studies underway in mouse ES cells and in human CD34+ cells using this bifunctional approach will provide a broad assessment of the relative usefulness of these two major non-viral integrating systems for the purpose of ex vivo gene therapy applications in a range of human diseases.

1097. Complete and Persistent Phenotypic Correction of Phenylketonuria in Mice by Site-Specific Genome Integration of Phenylalanine Hydorxylase cDNA

Top of pageAbstract Phenylketonuria (PKU) is a metabolic disorder that predisposes affected children to severe and irreversible mental retardation, and is secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH), that catalyses the conversion of phenylalanine to tyrosine. In an attempt to correct this metabolic deficiency, we explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine PAH cDNA in the livers of Phenylketonuric mice. The integrase from Streptomyces phage |[Phi]|BT1 mediates efficient recombination between the phage attachment site attP and the bacterial attachment site attB, results in the integration of phage DNA into the bacterial genome in a site-specific manner. Here we showed that this phage enzyme functioned efficiently both in Escherichia coli and mouse cells in vitro. Several naturally occurring |[ldquo]|pseudo|[rdquo]| attP sites in the mouse genome, which contain partial sequence identity to the wild type attP site and are recognizable by the phage integrase, have been identified and molecularly characterized. In the presence of the phage integrase, naked plasmid DNA containing a wild-type attB site can be integrated specifically into these pseudo attP site permanently both in culture mouse cells and in the livers of mice. We further demonstrated that the addition of nuclear localization signal sequences to the C-terminus of the phage integrase enzyme enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system we delivered mouse PAH cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that serum phenylalanine levels in the treated mice decreased significantly and persistently. After 3 applications, serum phenylalanine levels in all treated mice were reduced to the normal range and remained stable for more than 6 weeks. Furthermore, their fur color also changed from gray to black, indicating the reconstitution of melatonin biosynthesis as a result of available tyrosine derived from phenylalanine hydroxylation. Thus the bacterio-phage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in plasmid DNA-mediated gene therapy of a multitude of genetic disorders.

1103. Site-Specific Chromosomal Integration in Human Mesenchymal Stem Cells Mediated by PhiC31 Integrase

Human mesenchymal stem cells (hMSCs) have emerged as a promising new tool in gene therapy/ cell therapy strategies due to their potential use in a wide variety of applications, including both short- and long-term regeneration. With viral vectors being frequently criticized for their immunogenicity, random integration and difficult production, alternatives are desirable. A safe and effective gene delivery method resulting in targeted chromosomal integration with prolonged therapeutic levels of gene expression would be valuable. PhiC31 integrase, a site-specific recombinase from phage phiC31, pairs an attB recognition site on an incoming plasmid with pseudo attP sites in mammalian genomes. This integrase has been used to achieve genomic integration in human keratinocytes, mouse liver, rat retina and rabbit joints. Here we describe non-viral approaches for gene delivery using phiC31 integrase, transfected either as mRNA or as a DNA expression vector, in conjugation with an attB-containing donor plasmid. We evaluated the efficacy of mRNA encoding phiC31 integrase to mediate unidirectional integration into pseudo attP sites when co-transfected with an attB donor plasmid containing a transgene of interest. We also demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the luciferase marker gene and an attB site resulted in 39-fold higher expression of luciferase as assessed by live imaging, compared to control transfections without integrase. Expansion of the integrants and their differentiation into the osteogenic phenotype revealed sustained gene expression, indicating that the integration sites used in undifferentiated hMSCs continued to provide gene expression after differentiation of the cells. Finally, plasmid rescue and DNA sequence analysis of the integration sites showed that the integration events occurred at several pseudo attP sites in the human genome. Summarizing our results, phiC31 integrase, provided either as mRNA or DNA, mediates site-specific integration of transgenes and can provide high levels of gene expression in hMSCs and differentiated osteogenic cells. These findings suggest that phiC31 integrase represents an efficient and practical non-viral approach for genetic modification of hMSCs that could have potential for clinical applications.

195. Non-Viral, Site-Specific Gene Transfer into Murine Hematopoietic Stem Cells

Gene therapy into hematopoietic stem cells (HSC) provides the potential to correct a large range of disorders. To date, the most successful strategy for delivering genes to HSC has been with retroviral vectors. While gene transfer is efficient with these vectors, they possess limitations such as random integration, limited size capacity, low vector titers, and position effect silencing. We are developing the phiC31 integrase as a non-viral and site-specific alternative to retroviral transduction of HSC. The integrase approach involves delivering the gene of interest as a plasmid also containing the integrase recognition site attB. The integrase pairs attB with endogenous sequences similar to the attP recognition site and catalyzes genomic integration. The system has been used successfully in gene therapy experiments in mouse liver and muscle, human skin, rat retina, and rabbit joints.

600. A Novel Gene-Deleted Adenoviral Vector for Somatic Integration and Long-Term Phenotypic Correction of Murine and Canine Hemophilia B

This study describes the development of a novel gene-deleted adenoviral vector system for somatic integration. This system utilizes either a very recently generated hyperactive Sleeping Beauty transposase (HSB5) or a bacteriophage integrase derived from Streptomyces (phiC31). Previous data from our laboratory revealed that integration efficiencies for both recombinases are higher from circular substrates compared to linear substrates. Thus, we generated the gene-deleted adenoviral vector FTC/TcFIX/attB-(FRT)2 in which a canine factor IX (cFIX) expression cassette with the phiC31 recognition site attB and the transposase inverted repeats (IR) for HSB5 mediated integration are flanked by FRT sites for Flp mediated circle formation from the recombinant adenoviral vector.

821. Development of a Site-Specific Integrating Gene-Deleted Adenoviral Vector for Sustained Transgene Expression Levels In Vivo

Integrating vectors have been demonstrated to result in sustained transgene expression levels in vivo, but most are hampered by the risk of insertional mutagenesis. For a safer gene therapy protocol, site-specific integrating vectors are needed. Recently, it was shown that integration mediated by the integrase phiC31 derived from a Streptomyces phage resulted in site-specific integration by recombining the phiC31 recognition site attB in an episomal plasmid with pseudo attP sites in the host chromosome (Olivares et al., Nat Biotechnol 20:1124-8, 2002). In this study, we present a hybrid vector which utilizes the high transduction efficiency of a gene-deleted (HD) adenoviral vector to deliver the site-specific integration machinery of phiC31. Recombinant adenoviral vector genomes mainly persist as linear DNA molecules which motivated us to first compare the phiC31 mediated integration efficiency from circular and linear episomal DNA. We found that the integration efficiency was 14.8-fold higher from circular substrates and only 3.8-fold higher from linear DNA molecules compared to the mutant integrase control group. Thus, we based our system on a two vector system in which the transgene expression cassette is circularized from a HD vector by Flp mediated recombination, followed by phiC31- mediated integration.