Institute for Biogenesis Research
University of Hawaii John A. Burns School of Medicine

Supported by IBR-COBRE (Center of Biomedical Research Excellence) IDeA Program
NCRR 5P20RR024206-01, NIGMS 8P20GM103457-05

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Name Stefan Moisyadi
Affiliation JABSOM
Position Associate Professor
Degree Ph.D. (University of Hawaii)
Phone 808-956-3118
Fax 808-956-7316
Email moisyadi@hawaii.edu
Address 1960 East-West Rd, Room E108
Honolulu, HI 96822
 

Research projects:

Transgenesis, Transfection and Gene Therapy with Transposase Enzymes

Description of research:

My laboratory’s collaborative efforts with researchers at several domestic and international institutes were the first to propose modification of a transposon for site-specific integration and/or enhancing site-specificity. Additionally, the collaboration was first to suggest that these methods would allow for site-selective, efficient integration into an organism’s genome, whether for gene therapy or transgenic organisms. For example, transposons, such as Sleeping Beauty, and bacteriophage integrases, such as φC31, have been developed that can transform a variety of cell lines and organisms; however, they integrate the transgene they carry randomly into host genomes and we know that non-specific integration can induce cancer and also disrupt functional genes, unrelated to cancer that affect the fitness of the organism.

We and our collaborators have constructed chimeric transposases and other integrating enzymes for targeted integration and/or to enhance site-specificity. The ability to customize zinc-finger DNA binding modules will enable us to target integration events to specific locations in the genome. Such targeted integration of transgenes is essential for proper and stable levels of protein expression and to prevent integration into unwanted areas of the genome.

Why DNA based integrating vectors?

DNA constructs have many advantages over retroviruses. They are frequently in plasmid form and can be packaged non-virally, thus allowing increased stability. Furthermore, a DNA construct is preferred because the reverse transcriptase of a retrovirus has high error rates in copying both RNA and DNA due to its lacks the 3' to 5' exonuclease activity necessary for proofreading during DNA synthesis.

Adding new genes to mice to create new experimental models, or to human tissues for gene therapy, is an important new technology that has evolved many different ways in the past 30 years. My research focuses on a new methodology that does not employ viruses, and has the potential for site-specific integration. This means that we can not only add new genes, but replace defective ones. We have developed a new method we term Active Transgenesis that uses enzymes to facilitate the integration of new DNA into genomes. Previously, we directly compared the genomic integration efficiencies of four enzymes that integrate DNA into chromosomes, called transposases; piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1, in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines, while Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA binding domain retained trasposition activity, whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of utilizing it routinely for mammalian transgenesis and eventually gene therapy.

Much of previous gene therapy research had focused on different viral modes of gene delivery. While viruses seem well suited to deliver therapeutic genetic material, they are also effective at causing mutations on the host organism’s DNA. A significant drawback in using this form of gene therapy has been that it can trigger immune reactions or activate cancer-causing genes, with lethal consequences to the patient. The theoretical advantage of using a piggyBac, in contrast, is that the gene can be delivered safely to a specific location in the genome, where it can achieve the desired therapeutic response.


Selected Publications:

  • Stefan Moisyadi and H.M. Harrington. Characterization of the Heat Shock Response in Cultured Sugarcane Cells (1989). Plant Physiol. 90:1156-1162.
  • Harrington H.M., S. Moisyadi, Y.T. Lu. Structural and functional analysis of heat shock proteins (1990). In: Leonard R.T. and Hepler P.K. (eds). Calcium in Plant Growth and Development. The American Society of Plant Physiologists Symposium Series, Vol 4 pp 161-167.
  • Harrington, H.M., Y.-T. Lu, S. Moisyadi and S. Dharmasiri. Functional Analysis of Heat Shock Proteins (1994). In: Davenprot, T.L. and H.M. Harrington (eds). Plant Stress in the Tropical Environment, pp 112-117.
  • Stefan Moisyadi, S. Dharmasiri, H.M. Harrington and T.J. Lukas. Characterization of a Low Molecular Mass Autophosphorylating Protein in Cultured Sugarcane Cells and Its Identification as a Nucleoside Diphosphate Kinase1.Plant Physiol. 1994;104:1401-1409.
  • Stefan Moisyadi and J.I. Stiles. A cDNA Encoding a Metallothionein I-Like Protein from Coffee Leaves (Coffea arabica)1. Plant Physiol, 1995;107:295-296.
  • Stefan Moisyadi, K. R. Neupane and J.I. Stiles. Cloning and Characterisation of a cDNA Encoding Xanthosine-N7-Methyltransferase from Coffee (Coffea arabica). Acta Horticulturae. 1998;461:367-377.
  • Perry, A.C.F., R. Jones, S. Moisyadi, J. Coadwell, and L. Hall. The Novel Epididymal Secretory Protein, ESP13.2 in Macaca fascicularis1 . Biol Reprod. 1999;61(4):965-972.
  • Monika A. Szczygiel, Stefan Moisyadi, and W. Steven Ward. Expression of Foreign DNA Is Associated with Paternal Chromosome Degradation in Intracytoplasmic Sperm Injection-Mediated Transgenesis in the Mouse. Biol Reprod. 2003;68:1903-1910.
  • Tomoharu Osada, Atsushi Toyoda, Stefan Moisyadi, Hidenori Akutsu, Masahira Hattori, Yoshiyuke Sakaki and Ryuzo Yanagimachi. Production of Inbred and Hybrid Transgenic Mice Carrying Large (>200 kb) Foreign DNA Fragments by Intracytoplasmic Sperm Injection. Mol Rep Dev.2005; Nov;72 (3):329-335.
  • Takehito Kaneko, Stefan Moisyadi, Ryota Suganuma, Barbara Hohn, Ryuzo Yanagimachi and Pawel Pelczar. Theriogenology. Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection. Nov 2005;64(8):1704-1715.
  • Ryota Suganuma, Pawel Pelczar, Jean Francois Spetz, Barbara Hohn, Ryuzo Yanagimachi and Stefan Moisyadi. Tn5 Transposase-Mediated Mouse Transgenesis. Biol Reprod 2005;73:1157-1163.
  • Sareina Chiung-Yuan Wu, Yaa-Jyuhn James Meir, Craig J. Coates, Alfred M. Handler, Pawel Pelczar, Stefan Moisyadi and Joseph M. Kaminski. piggyBac: A Flexible and Highly Active Transposon as Compared to Sleeping Beauty, Tol2, and Mos1 in Mammalian Cells Proc. Natl. Acad. Sci (USA) 2006;103 (41):15008-15013.
  • Matthew Coussens, Yukiko Yamazaki, Stefan Moisyadi, Ryota Suganuma, Ryuzo Yanagimachi, and Richard Allsopp. Regulation and effects of modulation of telomerase reverse transcriptase expression in primordial germ cells during development. Biol Reprod 2006;75:785-791.
  • Shinohara ET, Kaminski JM, Segal DJ, Pelczar P, Kolhe R, Ryan T, Coates CC, Fraser MJ, Handler AM, Yanagimachi R, Moisyadi S. Active integration: new strategies for transgenesis. Transgenic Res 2007;16:333-339.
  • Moisyadi S, Kaminski JM, Yanagimachi R. Use of Intracytoplasmic Sperm Injection (ICSI) to generate transgenic animals. Comparative Immunology, Microbiology and Infectious Diseases. 2009 Mar;32(2):47-60.
  • Yamashiro H, Toyomizu M, Kikusato M, Toyama N, Sugimura S, Hoshino Y, Abe H, Moisyadi S, and Sato E. Exogenous lactate and adenosine triphosphate in the extender regulated motility, oxidative activity and enhanced cryosurvival of rat epididymal sperm. J. Am. Assoc. Lab. Anim. Sci. 2010;49(2):160-166.
  • Yamashiro H, Toyomizu M, Toyama N, Aono N, Sakurai M, Hiradate Y, Yokoo M, Moisyadi S, and Sato E. Extracellular ATP and dibutyryl cAMP confer freezability on and enhanced the fertilizing ability of rat epididymal sperm. J. Am. Assoc. Lab. Anim. Sci. 2010;49(2):167-172.
  • Sabine Heuter, Miyuri Kawasumi, Igor Asner, Urszula Brykczynska, Paolo Cinelli, Stefan Moisyadi, Kurt Burki, Antoine H.F.M. Peters and Pawel Pelczar. Genetic Vasectomy - Overexpression of Prm1-EGFP fusion protein in Elongating Spermatids Causes Dominant Male Sterility in Mice . Genesis 2010; 48(3):151-160.
  • Johann Urschitz, Miyuri Kawasumi, Jesse Owens, Kazuto Morozumi, Hideaki Yamashiro, Ilko Stoytchev, Joel Marh, James A. Dee, Kris Kawamoto, Craig J. Coates, Joseph M. Kaminski, Pawel Pelczar, Ryuzo Yanagimachi and Stefan Moisyadi Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects. Proc. Natl. Acad. Sci (USA) 2010;107 (18):8117-8122.

Lab members

Johann Urschitz
Assistant Researcher
johann@hawaii.edu
OFFICE 956-7417
Ilko Stoytchev
Junior Researcher
ilko@hawaii.edu
OFFICE 956-9684
Jesse B. Owens
Graduate Student
jbowens@hawaii.edu
OFFICE 956-2678
Hideaki Yamashiro
Junior Researcher
hideaki.yamashiro@hawaii.edu
OFFICE 956-2145