dystrophin in dysferlin-deficient A/J skeletal muscle  
Stanford University  
line decor
    HOME  ::  
line decor
 
 
 
 
 
 
Research Interests
 
The Calos Lab is interested in developing novel gene and cell therapy approaches to address human diseases.

We use genes as therapeutics, for example to develop strategies to treat genetic diseases like hemophilia and muscular dystrophy.

Our favorite tool for addition of genes to cells is a recombinase enzyme from a bacteriophage. This enzyme is called phiC31 integrase, and it can add genes to mammalian chromosomes at specific sequences.

PhiC31 integrase pairs two short recognition sites, called attB and attP, and catalyzes recombination that results in chromosomal integration.

This figure shows how we use phiC31 integrase. We place an attB site on the plasmid we wish to integrate into the chromosome. The enzyme finds a site similar to attP in the chromosome and carries out recombination. The net result is integration of the plasmid into the chromosome:
integration reaction

  • Groth AC, Olivares EC, Thyagarajan B, Calos MP. (2000). A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci U S A. 97(11):5995-6000. [Abstract]
  • Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP. (2001). Site-specific genomic integration in mammalian cells mediated by phage φC31 integrase. Mol Cell Biol. 21(12):3926-34. [Abstract]

This short movie created by Alfonso Farruggio, a Genetics graduate student in the lab, illustrates how the reaction works:


We found that phiC31 integrase recognizes a hierarchy of sites within the human genome, influenced both by DNA sequence and chromosomal context. Bioinformatics analysis indicates that the integration sites appear to be safe.

  • Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP. (2006). Integration specificity of phage φC31 integrase in the human genome. J Mol Biol. 357(1):28-48. [Abstract]

Application to gene therapy

The integrase system is a powerful tool for gene addition. However, it takes much more than a gene addition system to formulate an effective gene therapy. Over the past several years, we have carried out numerous collaborative studies in animals with several labs to illustrate how phiC31 integrase can be used to create gene therapies. These studies employ various strategies for gene delivery to different tissues and organs.

For example, we showed long-term expression of human factor IX after hydrodynamic delivery of factor IX-attB and integrase plasmids to mouse liver:

  • Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP. (2002). Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol. 20(11):1124-8. [Abstract]

Similarly, integrase was used to transfer therapeutic genes to skin, retina, and muscle, among others:

  • Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Calos MP, Khavari PA. (2003). φC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa. Hum Gene Ther. 14(9):923-8. [Abstract]
  • Chalberg TW, Genise HL, Vollrath D, Calos MP. (2005). phiC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest Ophthalmol Vis Sci. 46(6):2140-6. [Abstract]
  • Bertoni C, Jarrahian S, Wheeler TM, Li Y, Olivares EC, Calos MP, Rando TA. (2006). Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration. Proc Natl Acad Sci U S A. 103(2):419-24. [Abstract]

We are now attempting to translate some of these gene therapies to the clinic through work in large animals.

Use of phiC31 integrase in stem cells

Another area of intense interest in the lab is the use of integrase technology in stem cells. For example, stem cells derived from patients with genetic diseases like hemophilia and muscular dystrophy can be corrected with phiC31 integrase, then engrafted back to the patient.

Integrase can also be used to add genes to reprogram cells into pluripotent stem cells or into different types of differentiated cells.

  • Keravala A, Ormerod BK, Palmer TD, Calos MP. (2008). Long-term transgene expression in mouse neural progenitor cells modified with φC31 integrase. J Neurosci Methods. 173(2):299-305. [Abstract]
  • Jung WE. Calos MP. (2009). PhiC31 integrase for modification of stem cells. In Emerging Technology Platforms for Stem Cells, Chapter 20, (U. Lakshmipathy, J.D. Chesnut, and B. Thyagaragan, eds.) John Wiley and Sons, Inc.

Other applications of integrase

Another important application of phiC31 integrase is to create transgenic animals and plants.

We pioneered this area by showing that integrase could be used to target gene addition in Drosophila embryos at high efficiency and specificity. This method is now popular in the Drosophila community, and related strategies have been successful in many other organisms:

  • Groth AC, Fish M, Nusse R, Calos MP. (2004). Construction of transgenic Drosophila by using the site-specific integrase from phage φC31. Genetics. 166(4):1775-82. [Abstract]
  • Fish MP, Groth AC, Calos MP, Nusse R. (2007). Creating transgenic Drosophila by microinjecting the site-specific φC31 integrase mRNA and a transgene-containing donor plasmid. Nat Protoc. 2(10):2325-31. [Abstract]
 
 
  
Maintained by Jackie Chu
Last updated on Aug 30, 2010