Christopher R. Sclimenti

Phage integrases for the construction and manipulation of transgenic mammals

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the φC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the φC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.

Epstein-Barr virus vectors for gene expression and transfer.

Vectors based on components of Epstein-Barr virus (EBV) have found increasingly wide applications in biotechnology. Three areas of recent advancement comprise the use of EBV vectors to improve the convenience of gene expression systems, the development of EBV vectors for gene transfer in gene therapy, and the use of EBV components to generate large vectors carrying sizable regions of genomic DNA.

Epstein‐Barr Virus Vectors Provide Prolonged Robust Factor IX Expression in Mice

We demonstrate that vectors incorporating components from Epstein‐Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high‐pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5′, 3′, and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10‐ to 100‐fold increase in prolonged hFIX expression in mouse liver. A single 25‐μg dose of vector DNA generated normal (>5 μg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.

Assaying extrachromosomal gene therapy vectors that carry replication/persistence elements

Persistence in the cell is a desirable property for most gene therapy vectors. For extrachromosomal vectors, persistence is limited in most cell types. To address this problem, we have developed vectors with the ability to replicate and be retained in the nucleus. These properties are conferred by specific elements present on the vectors and derived from genomic DNA and from Epstein-Barr virus. In order to begin evaluation of these vectors for use in gene therapy, we developed and present here two assays that measure the persistence of vector DNA in tissue culture cells under rapidly dividing and slowly dividing conditions. Our results indicate that inclusion of DNA replication and nuclear retention elements on a vector increases persistence of vector DNA in slowly dividing cells by at least 500%. Further improvement of the system is discussed.