phiC31 integrase in liver  
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                                  Michele and

Michele Pamela Calos

Department of Genetics

Research Career Summary To Date – Michele Calos


My research over the course of my career has focused on creating and utilizing innovative genetic systems involving chromosomes and plasmids. The body of work has encompassed studies elucidating gene expression, transposable elements, mutation and repair, DNA replication, DNA recombination, genomic integration, gene therapy, and stem cell therapy.

This review concentrates on my work over the past ten years, which has focused on creating sequence-specific integration systems and using them, predominantly to advance the science and clinical application of gene therapy and stem cell therapy. I will briefly recap my earlier research, which laid the groundwork for the present activities and demonstrates my general approach.

Earlier research

At Oxford, I received a broad undergraduate education in biology, ranging from biochemistry and biophysics to genetics and evolution. As a graduate student with Nobel laureate Prof. Walter Gilbert at Harvard, I took advantage of newly available cloning and sequencing technology to be the first to clone the classic lac repressor gene, determine the DNA sequence of its promoter, and determine the DNA sequences at the junctions between transposable elements and their chromosomal targets. During a brief postdoc in Jeffrey Miller's lab in Geneva, Switzerland, I made the transition from working with E. coli to mammalian cells, creating a forerunner of the first shuttle vector system for analyzing mutation in human cells.

I obtained a Stanford Assistant Professor position at this time and began a program in human genetics utilizing the innovative shuttle vector approach. I established a laboratory in the Department of Genetics, where we were able to demonstrate the utility of the shuttle vector approach for the analysis of mutation in mammalian cells. This approach soon made its mark on the mutation and repair field and provided the first sequence-level analysis of mutation in mammalian cells that was of sufficient power to define the mutational spectra produced by a variety of mutagens.

I was fascinated by the chromosome-like behavior of the shuttle vectors developed by my lab. We studied plasmids based on Epstein-Barr virus and from these EBV vectors, my lab developed the first autonomous replication system for mammalian cells that used human genomic sequences as replication origins. The data we produced indicated that mammalian cells lacked the precisely defined replication origins of viruses and prokaryotes, and instead used more diffuse signals spread over a large area. This view was controversial in the field, but has stood the test of time and is now accepted as correct.

The field of gene therapy was developing during this period, based largely on the use of viral vectors for introduction of DNA into cells. I realized that the vectors my lab had created to study replication, with their ability to replicate and be retained in mammalian cells, might represent a viable alternative that could help solve the problem of transient expression plaguing non-viral approaches to gene therapy that used conventional plasmid DNA. We thus made the transition into the gene therapy field. The extrachromosomal vectors we developed were successful in numerous animal studies and have provided curative levels of therapeutic proteins such as a1 anti-trypsin and factor IX. On the other hand, the extrachromosomal vectors had the drawbacks for gene therapy of being not completely stable in dividing cells and of requiring the expression of a viral protein, EBNA1.

Site-specific integration

To overcome these limitations, we sought to create an integrating vector system. Integration offers the most reliable route to stability, because a vector then acquires the impressive stability of the chromosomes. This feature is critical in dividing cells, such as stem cells. Moving beyond previous gene therapy vectors, which integrate randomly, we became committed to creating sequence-specific integration systems. We saw the need for such technology, not only for gene therapy, but also for creation of cell lines, transgenic organisms, protein production, and many other applications. Thus, my lab accepted the challenge of creating such technology, which had previously been largely missing from the genetics toolkit.

Recombinant DNA technology of the 1970s provided the methodology to manipulate DNA molecules precisely in a test tube. However, the field of genetics had been largely unable to create precise and efficient genomic integration in living cells of higher organisms. The contribution of methodologies that would permit efficient integration at native sequences in the chromosomes became a central goal of my lab's research program.

The solution we devised to achieve sequence-specific integration was original and relied on use of microbial site-specific recombinases that in theory would recognize sequences in mammalian chromosomes having degenerate homology to their actual recognition sites. This hypothesis, based on the statistics of large genomes and the nature of protein-DNA interactions, proved to be correct. We pioneered this idea using the prokaryotic resolvase Cre, demonstrating for the first time that the enzyme can recognize "pseudo" lox sequences in the human and mouse genomes that depart significantly from the native 34-bp loxP site that Cre normally recognizes. However, Cre was not a good enzyme for integration, because it also performed excision efficiently, which reversed integration events.

We found a complete solution to this problem by using a different recombinase, the phage φC31 integrase. This enzyme from a Streptomyces phage could perform unidirectional recombination at its attachment (att) sites without host co-factors. We recognized that these were the properties needed to create a useful integration tool and were the first to use φC31 integrase in eukaryotic cells. My lab rapidly and successfully constructed expression and assay systems for φC31 integrase and demonstrated the ability of the enzyme to function on extrachromosomal plasmids in human cells in a landmark paper in 2000 (Groth et al., 2000).

We extended this study the next year with the first demonstration that the φC31 integrase could indeed find native sequences in the human and mouse genomes where it could carry out site-specific integration (Thyagarajan et al, 2001). The enzyme recognized a number of "pseudo" att sites in the human genome that had partial identity to attP. Integration generally occurred at a single copy per cell. In order to characterize the chromosomal sequences where integration took place, we carried out a large study of integration sites in human cell lines (Chalberg et al., 2006). The study revealed a 28-bp consensus sequence present at the sites of integration and a hierarchy of preferred integration sites in the genome having sequences related to this consensus. Chromatin context features also played a role in creating favorable integration sites. Statistical analysis suggested that φC31 integrase could potentially integrate at approximately 370 genomic locations in human cell lines. It is probable, based on our studies in animals summarized below, that a considerably narrower set of target sites is actually available in vivo, which provides greater site-specificity.

This level of site-specificity was an improvement of several orders of magnitude over the quasi-random integration exhibited by previously available integrating vectors, such as those based on oncoretroviruses, lentiviruses, and transposases. The tighter specificity of φC31 integrase substantially reduced the insertional mutagenesis risk during gene therapy. Control over integration location is considered critically important, especially in stem cell populations such as hematopoietic stem cells. The need for site-specific integration was illustrated by a gene therapy clinical trial in Paris. Leukemia occurred in several patients as a result of expression of an oncogene, activated by random integration of the retroviral gene therapy vector.

Research tools

As research tools, our site-specific integration systems are of widespread utility. One area of intense activity is use of φC31 integrase for generation of transgenic organisms. We initiated this research area by demonstrating the utility of our system for generating transgenic Drosophila in a collaboration with Roel Nusse of the Dept. of Developmental Biology. By injecting φC31 integrase mRNA into fly embryos, we catalyzed genomic integration of an injected attB plasmid, producing transgenic flies at an unprecedented level of efficiency (50%) and precision (100%) (Groth et al., 2004). This methodology has been widely adopted by the Drosophila research community.

The φC31 system has also found utility in the community of researchers seeking to modify the genome of mosquitoes in order to combat malaria and other diseases. Success has been achieved as well by using φC31 integrase to generate transgenic Xenopus tadpoles at a frequency of 30-40%. Moreover, φC31 integrase has been used to develop methodologies for the generation of transgenic zebrafish, chickens, and other important research and industrial organisms. We have also demonstrated the utility of the system for robust protein production in mammalian cells and for construction of cell lines with integrations at the same position, which are useful for the systematic analysis, for example, of gene control regions in a constant chromosomal context. These types of achievements demonstrate that we have created a system of broad utility for genetics research.

Gene therapy
A key goal of my laboratory is to use the integration systems we create to cure disease. To further this goal, we completed several studies in animal models that illustrated the utility of the φC31 integrase system for gene therapy.

In our first animal study, we demonstrated that we could introduce normal levels of human factor IX to mice by site-specifically integrating in liver a vector carrying the human factor IX gene. We co-delivered a factor IX-attB plasmid with a plasmid encoding the φC31 integrase. DNA was delivered by high-pressure tail vein injection of the plasmids, in a manner that directed DNA to the liver. We observed that the majority of integrations in hepatocytes occurred at a position on chromosome 2 and that we obtained high levels of factor IX that would be sufficient to cure hemophilia in patients. More recently, we carried out a similar study in disease model mice with factor IX (Keravala et al., 2011) and factor VIII deficiency (Chavez et al., 2012).

We also demonstrated the utility of the site-specific integration method in many other gene therapy applications, including skin, retina, joints, and neural stem cells. In collaboration with Tom Rando in Neurology, we showed that integrase provides greater, long-lasting expression of dystrophin in skeletal muscle in a mouse model of muscular dystrophy (Bertoni et al., 2006).

Stem Cell Therapy

Most recently, we have become interested in applying the power of our genome engineering systems to the creation and modification of stem cells for therapeutic uses. For example, we demonstrated the use of φC31 integrase for reprogramming adult cells into induced pluripotent stem cells (iPSC; Karow et al, 2011). After iPSC from patients with genetic diseases such as muscular dystrophy are corrected with genetic engineering methods, the corrected cells can be transplanted back to the patient for engraftment and tissue repair. Using the latest, evolving methods for genome engineering, we are currently pursuing these types of approaches to develop novel strategies to treat Duchenne and limb girdle muscular dystrophies.

Selected References

  • Bertoni, C., Jarrahian, S., Wheeler, T.M., Li, Y., Olivares, E.C., Calos, M.P., and Rando, T.A. (2006). Enhancement of plasmid-mediated gene therapy for Duchenne muscular dystrophy by directed plasmid integration. Proc. Natl. Acad. Sci. USA 103, 419-424.
  • Chalberg, T.W., Portlock, J.L., Olivares, E.C., Thyagarajan, B., Kirby, P.J., Hillman, R.T., Hoelters, J., and Calos, M.P. (2006). Integration specificity of phage φC31 integrase in the human genome. J. Mol. Biol. 357, 28 - 48.
  • Chavez, C.L., Keravala, A., Chu, J.N., Farruggio, A.P., Gabrovsky, V.E., Voorberg, J., and Calos, M.P. (2012). Long-term expression of human coagulation factor VIII in a tolerant mouse model using the phiC31 integrase system. Human Gene Therapy 23, 390-398
  • Groth, A.C., Olivares, E.C., Thyagarajan, B., and Calos, M.P. (2000). A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. 97, 5995-6000.
  • Groth, A.C., Fish, M., Nusse, R., and Calos, M.P. (2004). Construction of transgenic Drosophila by using the site-specific integrase from phage φC31. Genetics 166, 1775-1782.
  • Karow, M., Chavez, C.L., Farruggio, A.P., Geisinger, J.M., Keravala, A., Jung, W.E., Lan, F., Wu, J.C., Chen-Tsai, Y., and Calos, M.P. (2011) Site-specific recombinase strategy to create iPS cells efficiently with plasmid DNA. Stem Cells 29, 1696 - 1704.
  • Keravala, A., Chavez, C.L., Hu, G., Woodard, L.E., Monahan, P.E., and Calos, M.P. (2011) Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy. Gene Therapy 18, 842-848.
  • Thyagarajan, B., Olivares, E.C., Hollis, R.P., Ginsburg, D.S., & Calos, M.P. (2001). Site-specific genomic integration in mammalian. cells mediated by phage φC31 integrase. Mol. Cell. Biol. 21, 3926-3934.

Maintained by Tawny Neal
Last updated on July 19th