Eric C. Olivares

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

ABSTRACT We previously established that the phage φC31 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 phageattP 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.

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

We used the integrase from phage φC31 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 ∼4 μg/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.

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

In Vivo Correction of Murine Hereditary Tyrosinemia Type I by ϕC31 Integrase-Mediated Gene Delivery

Phage ϕC31 integrase is a site-specific recombinase that mediates efficient integration of circular extrachromosomal DNA into the host genome. Here, the integrase system was used to transfer the fumarylacetoacetate hydrolase (FAH) gene into the liver of mice affected with hereditary tyrosinemia type 1. Approximately 3.6% of transfected hepatocytes experienced an integration event. The absolute frequency of integration was 1/1374. A higher proportion of integrase-transfected FAH+ hepatocytes displayed abnormal morphology (bizarre nuclei, enlarged cells) on day 25 after gene transfer, compared to cells not receiving integrase. The increased frequency of these abnormal cells correlated with the amount of integrase plasmid administered, suggesting some form of integrase toxicity in Fah-/- livers. The abnormal hepatocyte appearance was transient and livers analyzed after longer selection (90 days) showed 60% repopulation with only normal healthy FAH+ hepatocytes. A total of seven different integration sites (accounting for >90% of integration) were identified. Serial transplantation of integrase-corrected hepatocytes to Fah-/- recipients was successful, suggesting long-term viability of corrected cells and persistent gene expression through many rounds of cell division. The stability of transgene expression, relatively high integration frequency, and significant site specificity that characterize the ϕC31 integration system suggest that it may have utility in many gene therapy settings.

Mutational Derivatives of PhiC31 Integrase With Increased Efficiency and Specificity

phiC31 integrase is a sequence-specific phage recombinase that can recombine two short DNA sequences called attB and attP. The enzyme can also promote genomic integration of plasmids carrying attB into native mammalian sequences having partial identity to attP. To increase the efficiency of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in an assay for higher recombination frequency between attB and attP. We report in this article the isolation of a mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a preintegrated chromosomal attP site and at endogenous pseudo attP sequences in cultured human cells. In mouse liver, P2-mediated integration provided therapeutic long-term levels of human factor IX that were double those generated by wild-type phiC31 integrase. We also describe an additional mutant, P3 that combines the mutations of P2 with further changes and possesses an elevated specificity for integration at a chromosomally placed attP site in human cells. Forty-four percent of colonies carrying integration events mediated by P3 have integration at the placed attP site. These mutant integrases are useful for gene therapy and genome modification, and they demonstrate the feasibility of engineering phiC31 integrase toward more desirable properties.

Phage φC31 integrase-mediated site-specific integration for gene therapy

For most genetic disorders, long-term correction is necessary. Integration of a therapeutic gene into a patients genome is an obvious route to achieving such permanent correction. Several technologies have been applied to the goal of achieving integration, including viruses and transposases. While these techniques are effective at some level, they each have drawbacks that can be improved upon. A novel integration system based on a phage integrase can address some of the previous limitations. The integrase from the Streptomyces bacteriophage C31 catalyzes site-specific, unidirectional integration into the genomes of higher eukaryotes. This integrase has the ability to recognize a limited number of native genomic sequences and integrate introduced plasmid DNA into them. These native sequences, termed pseudo att sites, resemble the wild-type phage attachment site enough to support integrase-mediated integration. Molecular evolution holds the promise of creating custom integrases that preferentially recombine at particular pseudo att sites. Furthermore, the system has no apparent size limit on carrying capacity. These features make the C31 integrase system extremely appealing for gene therapy applications. The system has been successfully employed in several model gene therapy studies to date. Here we review the development of this novel integration system and its current and potential applications to gene therapy.

810. Site-Specific Integration Enhances Expression of DNA Introduced into Skeletal Muscle

Non-viral approaches to gene therapy have often been limited by transient expression of delivered genes. Permanent integration of therapeutic genes into the host genome holds promise for lasting expression of the deficient or defective protein. Integration that is site-specific can limit the hazards of insertional mutagenesis. Here we demonstrate a novel non-viral approach to gene therapy for muscle disease. We used phage phiC31 integrase to catalyze unidirectional, site-specific integration of incoming plasmid DNA carrying the luciferase gene and an attB site into pseudo attP sites in the genomes of muscle fibers. In vivo bioluminescent imaging was used to assess the levels of luciferase expression, as a measure of gene transfer and integration into mouse leg skeletal muscle. in vivo imaging demonstrated that electroporation following intramuscular injection of plasmid DNA increased initial luciferase activity by 1-2 orders of magnitude. Intramuscular injection of the luciferase-attB plasmid together with a plasmid expressing the phiC31 integrase produced persistent levels of expression that were 5-10 fold higher than the levels seen without integrase. Genomic integration was verified by performing PCR on muscle DNA using primers spanning a known hotspot pseudo attP site for phiC31–mediated integration in mouse liver cells. Four out of four muscles that received integrase and the attB plasmid were positive for integration events at this site. Application of this site-specific integration technology to gene therapy for muscular dystrophies may provide long-term, sustained amelioration of the disease phenotype. Delivery of wild type dystrophin cDNA into dystrophin-deficient muscles using the phiC31 integrase system is currently under investigation.