||Ph.D. (University of Hawaii)
||1960 East-West Rd, Room E108
Honolulu, HI 96822
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.
- 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.
- Li Z, Kawasumi M, Zhao B, Moisyadi S, Yang J. Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra. Mol Reprod Dev. 2010 Nov;77(11):990-7. doi: 10.1002/mrd.21252. PubMed PMID: 21049546; PubMed Central PMCID: PMC3099245.
- Owens JB, Urschitz J, Stoytchev I, Dang NC, Stoytcheva Z, Belcaid M, Maragathavally KJ, Coates CJ, Segal DJ, Moisyadi S. Chimeric piggyBac transposases for genomic targeting in human cells. Nucleic Acids Res. 2012 Aug;40(14):6978-91. doi: 10.1093/nar/gks309. Epub 2012 Apr 9. PubMed PMID: 22492708; PubMed Central PMCID: PMC3413120.
- Marh J, Stoytcheva Z, Urschitz J, Sugawara A, Yamashiro H, Owens JB, Stoytchev I, Pelczar P, Yanagimachi R, Moisyadi S. Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis. Proc
Natl Acad Sci U S A. 2012 Nov 20;109(47):19184-9. doi: 10.1073/pnas.1216473109. Epub 2012 Oct 23. PubMed PMID: 23093669; PubMed Central PMCID: PMC3511126.
- Wu Z, Xu Z, Zou X, Zeng F, Shi J, Liu D, Urschitz J, Moisyadi S, Li Z. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer. Transgenic Res. 2013 Dec;22(6):1107-18. doi: 10.1007/s11248-013-9729-0. Epub 2013 Jul 16. PubMed PMID: 23857557; PubMed Central PMCID: PMC3838457.
- Owens JB, Mauro D, Stoytchev I, Bhakta MS, Kim MS, Segal DJ, Moisyadi S. Transcription activator like effector (TALE)-directed piggyBac transposition in human cells. Nucleic Acids Res. 2013 Oct;41(19):9197-207. doi: 10.1093/nar/gkt677. Epub 2013 Aug 5. PubMed PMID: 23921635; PubMed Central PMCID: PMC3799441.
- Urschitz J, Moisyadi S. Transpositional transgenesis with piggyBac.. Mob Genet Elements. 2013 May 1;3(3):e25167. Epub 2013 May 24. PubMed PMID: 23956948; PubMed Central PMCID: PMC3742596.
- Anderson CD, Urschitz J, Khemmani M, Owens JB, Moisyadi S, Shohet RV, Walton CB. Ultrasound directs a transposase system for durable hepatic gene delivery in mice. Ultrasound Med Biol. 2013 Dec;39(12):2351-61. doi: 10.1016/j.ultrasmedbio.2013.07.002. Epub 2013 Sep 11. PubMed PMID: 24035623; PubMed Central PMCID: PMC3838570.
- Owens JB, Mathews J, Davy P, Stoytchev I, Moisyadi S, Allsopp R. Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System. Mol Ther Nucleic Acids. 2013 Dec 10;2:e137. doi: 10.1038/mtna.2013.61. PubMed PMID: 24326734; PubMed Central PMCID: PMC3894583.
- Bertino P, Urschitz J, Hoffmann FW, You BR, Rose AH, Park WH, Moisyadi S, Hoffmann PR. Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8(+) T cell responses. Vaccine. 2014 Mar 26;32(15):1670-7. doi: 10.1016/j.vaccine.2014.01.063. Epub 2014 Feb 7. PubMed PMID: 24513010; PubMed Central PMCID: PMC3973154.
- Li Z, Zeng F, Meng F, Xu Z, Zhang X, Huang X, Tang F, Gao W, Shi J, He X, Liu D, Wang C, Urschitz J, Moisyadi S, Wu Z. Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids. Biol Reprod. 2014 May 8;90(5):93. doi: 10.1095/biolreprod.113.116905. Print 2014 May. PubMed PMID: 24671876; PubMed Central PMCID: PMC4076374.
- Zhao L, Ng ET, Davidson TL, Longmuss E, Urschitz J, Elston M, Moisyadi S, Bowles J, Koopman P. Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination. Proc Natl Acad Sci U S A. 2014 Aug 12;111(32):11768-73. doi: 10.1073/pnas.1400666111. Epub 2014 Jul 29. PubMed PMID: 25074915; PubMed Central PMCID: PMC4136622.
- Bahrambeigi V, Ahmadi N, Moisyadi S, Urschitz J, Salehi R, Haghjooy Javanmard S. PhiC31/PiggyBac modified stromal stem cells: effect of interferon γ and/or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on murine melanoma. Mol Cancer. 2014 Nov 26;13:255. doi: 10.1186/1476-4598-13-255. PubMed PMID: 25428727; PubMed Central PMCID: PMC4258801.
- Ward WS, Moisyadi S. Medical school hotline: The Institute for Biogenesis Research: a flower in the Pacific. Hawaii J Med Public Health. 2014 Dec;73(12):393-6. PubMed PMID: 25628972; PubMed Central PMCID: PMC4300549.
- Rose AH, Hoffmann FW, Hara JH, Urschitz J, Moisyadi S, Hoffmann PR, Bertino P. Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses. Hum Vaccin immunother. 2015 Sep 2;11(9):2305-11. doi: 10.1080/21645515.2015.1047567. Epub 2015 Jun 19. PubMed PMID: 26091088.
- Link to a complete list of publications
Jesse B. Owens, Ph.D.
IBR COBRE Research Specialist