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Stanford UniversitySchool of MedicineDepartment of GeneticsCancer Biology Program |
Our current research is focused on development of novel vectors for gene therapy. Our initial approach was development of extrachromosomal replicating vectors to carry the gene of interest. More recently, we have developed a system for site-specific integration of gene therapy vectors into the chromosomes. Extrachromosomal vectors Our extrachromosomal vectors are based on technology developed in the lab over the past several years. We isolated sequences from the human genome that can mediate autonomous replication. We paired these sequences with elements derived from Epstein-Barr virus that can mediate nuclear retention of the vectors in mammalian cells. We have shown that gene expression from these vectors is greatly prolonged compared to expression from conventional plasmid vectors. This was first shown in tissue culture cells:
We were also successful in delivering normal levels of human factor IX to mice using similar extrachromosomal vectors:
Site-specific recombinases for gene therapy and genetic engineering While our extra-chromosomal vectors provide high level, long-term transgene expression, the approach has two potential drawbacks. First, it requires continuous expression of a viral protein, the EBNA1 protein from Epstein-Barr virus. It is conceivable that expression of this protein could have undesirable effects on some cells. In addition, while the vectors are relatively stable, they lack a true centromere and are gradually lost over time in dividing cells. Therefore, extrachromosomal vectors are not expected to provide permanence in some cell types of interest in gene therapy, such as stem cells.In order to achieve permanent expression, the lab began exploring the use of site-specific recombinases for genomic integration in mammalian cells. Site-specific recombinases exist in nature to perform functions such as the resolution of concatenated DNA or the integration of phage DNA into bacterial chromosomes. These enzymes usually recognize relatively short (~30-300 bp) DNA sequences and mediate precise recombination between them. The first enzyme we looked at was the Cre recombinase from phage P1. This enzyme recombines two identical 34 bp loxP sequences. Cre recombinase functions at a high frequency in mammalian cells. We demonstrated that there are sequences that already exist in the human and mouse genomes that are similar enough to loxP that Cre can recognize them and mediate recombination between them:
However, we did not see a high frequency of integration at such "pseudo" loxP sites. The Cre recombination reaction is reversible. Once a loxP-containing plasmid is integrated into a loxP site, the integrated DNA is flanked by identical loxP sites and may be easily excised out again. Therefore, while Cre is highly efficient at making deletions between loxP sites, it is poor at stable integration. In order to avoid the problem of the reverse excision reaction, we focused on a different recombinase, the integrase from phage PhiC31. This integrase recognizes two different sequences, the phage attP and the bacterial attB. Once integrated, DNA cannot be excised without the expression of an excisionase, so the integration is permanent. This type of unidirectional recombinase is ideal for obtaining a high net frequency of stable integration. In our first study with the PhiC31 integrase, we showed that the minimal size att sites for 100% recombination in bacteria are 34 bp for attB and 39 bp for attP. In addition, we showed that the integrase functions well in mammalian cells, even on the minimal sites. In addition, we demonstrated integration of an attP-containing plasmid into a chromatinized EBV vector in human cells at a frequency of 1.7% for a 35 bp attB and 7.5% with a full length attB.
Once it was established that the PhiC31 integrase could function in human cells at high frequencies on extrachromosomal plasmid substrates, we began to explore integration into the chromosomes. We determined that integration occurred into attP sites pre-placed in the chromsomes. In addition, integration into naturally occurring pseudo attP sites was readily observed. Many of these pseudo attP sites were isolated and sequenced. The pseudo sites displayed a percent identity of a 25 bp region to the wild-type attP ranging from 28 - 56%.
Because the PhiC31 enzyme worked well in mammalian cells, we studied other closely related enzymes from the serine recombinase family. Another enzyme from this family is the integrase from phage R4. We determined that R4 functioned in human tissue culture cells, and R4 pseudo attP sites were identified.
We also showed that another serine recombinase, the integrase from phage TP901-1, functioned well in a mammalian intramolecular recombination assay.
Serine recombinases are the subject of ongoing research in the lab. All three of the enzymes studied to date show promise as tools for gene therapy and genetic engineering, though the PhiC31 integrase appears to be the most useful. We have also explored the potential of directed evolution to increase the efficiency of the PhiC31 integrase and to modify its site specificity. We demonstrated that two cycles of DNA shuffling and screening in E. coli were sufficient to produce enzymes with enhanced properties in human cells.
We have begun developing the PhiC31 integrase as a tool for gene therapy, starting with experiments in mice. We showed that high-pressure naked DNA injection of a plasmid expressing integrase along with a plasmid carrying an attB site and the gene for human blood clotting factor IX, deficient in hemophilia, produced therapeutic levels of factor IX in the blood. This effect was due to stable integration of the factor IX plasmid into liver cells and robust expression of factor IX from its chromosomal integration sites. We demonstrated that >99% of the integrations occurred at a pseudo attP site on chromosome 2.
The system was also used to deliver the collagen VII gene, defective in recessive dystrophic epidermolysis bullosa (RDEB), to human skin cells from RDEB patients. A collagen VII-attB plasmid and an integrase expression plasmid were transfected into the skin cells. Corrected cells were selected and used to make a graft, which was placed onto immune-deficient mice. The graft produced human skin with normal levels and localization of collagen VII, completely correcting the RDEB defect.
We are currently testing the PhiC31 integrase system in many further gene therapy applications. The site-specific integration mediated by this system is likely to greatly reduce the risk of insertional mutagenesis typical of randomly integrating gene therapy vectors such as retroviruses. In addition, the PhiC31 integrase appears to direct integration to sites of robust gene expression, thus providing high level, long-term expression of the introduced gene. These advantageous features suggest that the PhiC31 integrase system will produce successful gene therapies across a broad range of targets. Go to the Top of the Page. |