Tropoism- virus developed to preferentially target specific host speciesAnother viral vector used for genetic transfer is the herpesvirus. ItsdsDNA and large size make it a viable candidate for use in mammalian cells. The high specificity and transgenic capacity allow for efficient transport. It expresses immune suppressant factors for long lasting gene expression in vivo. However, the viral proteins can be toxic and replication control is required.
Nuclear-replicating: DNA transcribed to RNA within the cell’s nucleaus
Key Properties of Viral Vector• Safety – original virus particle is manipulated to minimize risk handling them (i.e. replication, pathogenicity)• Low Toxicity – vector should have minimal physiological effects on target cell• Stability – genes that confer viral genome instability are depleted from vector• Cell Type Specificity – viral receptor modified to target specific kind of cell
Use of Viral Vectors Gene Transfer ®Kimmelman• Applications – Cancer - e.g. transfer tumor suppressor genes – Immunology – e.g. transduce hematopoietic stem cells (HSC) – Stem Cell Biology – e.g. transduce human ES cells, create induced pluripotent stem cells (iPS) from human fibroblasts
Resources• Definition and Key Properties – Wikipedia.org “Vector” and “Viral Vector”• 1st image of viral vector – Hampson DR, Gholizadeh S, Pacey LKK. 2012. Pathways to Drug Development for Autism Spectrum Disorders. Clinical Pharmacology & Therapeutics 91:189-200.• 2nd image – Kimmelman J. 2005. Recent developments in gene transfer: risks and ethics. British Medical Journal 330:79-82.• Applications – Cancer – Seth P. 2005. Vector-mediated cancer gene therapy: an overview. Cancer Biology & Therapy 4:512-7. – Immunology – Chen W, et al. 2000. Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally radiated mice. Stem Cells 18:352-9. – Stem Cell – Koch P, et al. 2006. Transduction of human embryonic stem cells by ecotropic retroviral vectors. Nucleic Acids Research 34: e120. – Stem Cell – Takahashi K, et al. 2007. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 5:861-872.
Herpesvirus Advantages Disadvantages • Viral proteins can be toxic to• dsDNA (linear) host cells• Large (150-200 nm in diameter) • Requires replication control• Deliver genes with high specificity• High transgenic capacity• Non-integrating• Infects dividing and non-dividing cells• Long lasting gene expression (life-long) in vivo Breakefield, X., Pechan, P., Johnston, K., Jacoby, D.. Herpes Virus Vectors. Stem Cell Biology and Gene Therapy. 1998, 8, 1-32.
Primary Means of Application• Recombinant virus vectors – 30kb transgene capacity – Virus genome disrupted via recombination - renders virus nontoxic/replication defective• Amplicon vectors – 15kb transgene capacity – DNA can be replicated/ packaged into virions through helper viruses • Way to package plasmid DNA without viral coding regionsBreakefield, X., Pechan, P., Johnston, K., Jacoby, D.. Herpes Virus Vectors. Stem Cell Biology and Gene Therapy. 1998, 8, 1-32.
High-efficiency Transient Transduction of Human Embryonic Stem Cell–derived Neurons With Baculoviral Vectors Molecular Therapy vol. 17 no. 9, 1585–1593, Sep. 2009Original article By: Jieming Zeng, Juan Du, Jiakai Lin, Xiao Ying Bak, Chunxiao Wu and Shu Wang
Outline• Purpose of the experiment• Approaches• Details• Results• Conclusion
Purpose of the experiment• Obtaining human neurons from hESC’s and using them as a potential treatment for CNS neurological disorders.• Studying the efficiency of Baculoviral vectors as a tool for basic gene function studies and therapeutic applications of the human neuronal cells.• To bring about transient transduction of stem cells without chromosomal integration• Increase the survival rate and boost the functional recovery of the grafted hESC derived neurons. (increasing transplantation efficiency)
Approaches (1) Problems Transduction followed by differentiation Stable expression ofApproaches to certain genes. bring about 1) anti-apoptosis genesmanipulation of 2) Genes encoding transcription factors hESC’s into Neurons (2) Differentiation followed by transduction Preferred Alternative
Details• Baculovirus used: Autographa californica multiple nucleopolyhedrovirus- based vector• Transgene: eGFP reporter gene (enhanced Green Fluorescent protein) under the influence of CMV promoter and WPRE (Woodchuck Hepatitis virus post-transcriptional regulatory element) 5’ 3’LTR LTR CMV E-GFP W LTR
Baculoviral Transduction of hESC derived human neurons(a) Phase contrast image of the live (b) Fluorescence image of the live HES-1 HES-1 derived neurons derived neurons
Expression of neuronal markers in HES-1 βIII tubulin MAP2ab NF200 NCAM
WPRE Sequence prolonged transgeneexpression in hESC derived human neurons w/o WPRE With WPRE
Conclusion• Baculoviral vectors are highly efficient in transducing human neurons.• The WPRE sequence prolonged transgene expression in hESC derived human neurons.• The Baculoviral vectors had little cytotoxicity to hESC derived human neurons.• Baculoviral vector transduction did not affect the immunogenicity of the transplanted neurons
Summary• Introduction to Viral vectors 1) Properties of Viral vectors 2) Uses of Viral vectors 3) Types of viral vectors• Retroviruses• Herpes Simplex Virus• Research paper findings and Conclusion
THE ENDCAST IN THE ORDER OF APPEARANCE- GIOVANNI BOTTEN- Introduction to Viral vectors. KRISTYL FELIX- Retroviruses ROYA MIRILAVASSANI- Herpes Simplex Virus ROMIT BOSE Paper APARNA SUDARSHAN