Site-specific Genomic Integration Research Articles

Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma

A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well-controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild-type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi-strategy mediates specific knock-down of gene expression. Site-specific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down-regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site-specific shRNA-strategy circumvents interference by IFN-response induced when using other RNAi gene knock-down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL-derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease-related genetic defects.

RETRACTED: Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA

We explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine phenylalanine hydroxylase cDNA in the livers of phenylketonuric (PKU) mice. The phiBT1 phage integrase is an enzyme that catalyses the efficient recombination between unique sequences in the phage and bacterial genomes, leading to the site-specific integration of the former into the latter in a unidirectional manner. Here we showed that this phage integrase functions efficiently in mouse cells, and several naturally occurring pseudo-attP sites located in the intergenic regions of the mouse genome have been identified and molecularly characterized. We further demonstrated that the addition of nuclear localization signal sequences to the C terminus of the phage integrase enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system, we delivered mouse phenylalanine hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperphenylalaninemic phenotype in the treated mice decreased significantly. After three applications, serum phenylalanine levels in all treated PKU mice were reduced to the normal range and remained stable thereafter. Their fur color also changed from gray to black, indicating the reconstitution of melanin biosynthesis as a result of available tyrosine derived from reconstituted phenylalanine hydroxylation in the liver. Thus, the phiBT1 bacteriophage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in DNA-mediated gene therapy for a multitude of genetic disorders.

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.

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells.

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

1057. Successful Gene Therapy for Retinal Degenerations by Lentiviral Gene Transfer

Our goal is to perform gene therapy by efficient integration into safe positions in the host genome. The integrase enzyme from the phage phiC31 directs site-specific integration into a small subset of native genomic sequences 1. Recent gene therapy studies using phiC31 integrase in mice have demonstrated site-specific integration of transgenes and long-term gene expression in liver, skin, and muscle (e.g., 2). Here, we demonstrate use of the phiC31 integrase as a simple and effective method for non-viral long-term gene transfer in the eye. We developed a method to achieve efficient non-viral delivery of plasmid DNA to rat retinal epithelium (RPE) in vivo. The method involves subretinal injection of plasmid DNA, followed by in situ electroporation (adapted from 3). Delivery of GFP by this method was evaluated in frozen sections using fluorescence microscopy. In subsequent experiments, a luciferase plasmid, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. Subretinal DNA injection followed by electroporation yielded abundant transgene delivery in rat RPE, as assayed by both GFP and luciferase expression. Expression was strongest 48 hours after delivery. In the absence of integrase, luciferase expression declined to near-background levels within 3-4 weeks after treatment. Co-injection of the integrase plasmid led to long-term stable transgene expression at all time points tested, up to three months post-procedure. Animals receiving integrase showed ~64-fold higher long-term expression than control animals. We conclude that subretinal injection followed by electroporation affords abundant transfer of plasmid DNA in mammalian RPE. Addition of phiC31 integrase confers robust long-term transgene expression, presumably by providing site-specific integration of the gene of interest into the genome of RPE cells. Having established non-viral permanent retinal gene transfer by site-specific genomic integration, we now plan to apply the technology to rodent models of retinal degeneration. Because site-specific integration provides strong, stable gene expression while reducing the probability of insertional mutagenesis, the phiC31 integrase system has great potential as a novel approach to non-viral gene therapy in the eye.

809. PhiC31 Integrase Mediates Site-Specific Genomic Integration in Rabbit Cells and Rabbit Knee Joints

Top of pageAbstract A safe and effective gene delivery vector is vital to successful gene therapy. A major limitation of non-viral gene delivery has been transient gene expression. Site-specific integration of vector DNA into the host genome could confer prolonged, therapeutic levels of gene expression with limited toxicity. Additionally, site-specific integration may mitigate safety concerns regarding integration of the vector DNA into genomic sites that could induce malignancies by activation of proto-oncogenes or inactivation of tumor-suppressor genes. Previous studies have demonstrated the successful use of the phage phiC31 integrase for achieving genomic integration of plasmids bearing an attB site in human and mouse cells. In this report we evaluate the potential of phiC31 integrase in rabbit cells and tissue. PhiC31 integrase is a unidirectional site-specific recombinase belonging to the serine family. We demonstrate that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene of interest and an attB site increases the level of gene expression by increasing the frequency of stable gene integration in cultured rabbit cells. Plasmid rescue and DNA sequence analysis reveals specific integration sites at pseudo attP sequences in the rabbit genome. Lastly, we show significantly higher levels of transgene expression in vivo in rabbit knee joints of an attB-reporter gene plasmid when co-introduced with a plasmid expressing phiC31 integrase. These data suggest that phiC31 integrase-mediated integration of plasmid DNA can provide high-level, persistent gene expression in rabbit, as has been observed in several other mammalian species. Furthermore, the ability of the phiC31 integrase to facilitate long-term gene delivery to the synovium in the rabbit knee suggests that this integrase system could be appropriate for intra-articular delivery of therapeutic genes for treatment of rheumatoid arthritis, osteoarthritis, and other joint diseases.

165. Site-Specific Genomic Integration with a Phage Integrase

165. Site-Specific Genomic Integration with a Phage Integrase

Requirements for Adeno-Associated Virus-Derived Non-Viral Vectors to Achieve Stable and Site-Specific Integration of Plasmid DNA in Liver Carcinoma Cells

Background and Aims: Adeno-associated virus (AAV) is the only known virus capable of site-specific genomic integration in human cells. Thus, AAV-based vectors may be an attractive option to achieve prolonged transgene expression in human cells. We therefore studied the minimal elements of gene therapy vectors necessary for stable integration and tested the effectiveness of this approach in hepatoma cells. Methods: Plasmids were constructed that contained a GFPneo fusion transgene with or without the AAV-inverted terminal repeats (ITRs). In addition, Rep protein was either encoded in cis or supplied in trans by co-transfections. Stable clones were analyzed by Southern blotting for site-specific integration. Results: The ITRs alone conferred neither stable nor site-specific transgene integration. Expression of Rep protein in cis or trans resulted in an increased frequency of integration regardless of the presence of ITRs. It was shown that in the absence of the ITRs, other Rep-binding site (RBS) like sequences such as the ColE1 sequence present in plasmid backbones can function as RBS. Site-specific integration was achieved in up to 26% of clones derived from hepatoma cells. Conclusion: Both expression of Rep proteins and inclusion of a RBS are necessary for enhanced and stable integration of AAV-based non-viral vectors. A novel two-plasmid system capable of achieving stable and site-specific gene transfer in hepatoma cells is introduced.

Site-specific genomic integration produces therapeutic Factor IX levels in mice.

We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

Gene Therapy for Human Hemoglobinopathies

Gene transfer of human globin genes into human pluripotent stem cells via viral vectors may soon be realized. The high level of globin gene expression believed to be required for the treatment of severe hemoglobinopathies necessitated the inclusion of cis-acting sequences (LCR). Retroviral vectors containing the LCR elements are prone to rearrangement, low titer, and poor expression. Inclusion of a "minilocus" containing four HS sites linked to a globin gene resulted in higher expression in transplanted mice, but rearrangement of the provirus still occurs, and it is unclear what significance these experiments have with regard to human marrow stem cell transduction. Recombinant AAV is among the newest of genetic transfer vectors. This once obscure virus possesses unique properties that distinguish it from all other vectors. Its major advantage is the lack of pathogenicity in humans. Wild-type AAV has the unusual ability to selectively integrate into the mammalian genome at a specific region, thus reducing the concern for genomic disruption and insertional mutagenesis. The ability of AAV to carry regulatory elements without interference from the viral template may enable greater control of transferred gene expression. Disadvantages currently include the inferior packaging systems which yield low numbers of recombinant virions which are contaminated with wild-type adenovirus. The small AAV genome that can be packaged (approximately 5 kb) rules out its use for transfer of larger genes. Recombinant AAV viruses do not appear to demonstrate the same site-specific genomic integration as wild-type viruses. Elucidation of the mechanism of site-specific integration should prove useful in the development of safe vectors for gene transfer as well as provide insight into the nature of DNA recombination in humans.