* a brief project overview * sufficient background information for everyone to understand your proposal * a statement of the research problem and goals * project details and methods * predicted outcomes if everything goes according to plan and if nothing does * needed resources to complete the work * societal impact if all goes well
Stem Cell Population Control: The Big Picture
- Stem cell science holds tremendous promise for advancement in medicine and primary research from the regeneration of tissues and whole organs to the elucidation of human development.
- Unfortunately, current attempts to introduce pluripotent stem cells to model animals are almost always followed by the development of cancer; this is due to the apparently intrinsic tumorigenic potential of pluripotent stem cells.
- Various methods for reducing the risk of cancer from nondifferentiated cells in the context of stem cell-based cytotherapy exist, including pre-introductory screening of cells, addition of an agent toxic only to stem cells, and inducible gene suicide.
- While sorting and stemotoxic agents are perhaps best suited to chemical methods of selection, inducible gene suicide is a problem uniquely suited to the production of synthetic genetic circuits.
- We propose to make use of such synthetic genetic circuits to selectively remove cells that have not differentiated after an arbitrary time period in order to obviate chances of stem-cell-facilitated tumorigenesis.
- These background papers cover a variety of topics, divided into five overarching categories: Transgenic circuitry, gene circuit delivery, cell suicide mechanisms, the nature of pluripotency, and potential applications.
Hosoda H, Miyao T, Uchida S, Sakai S, Kida S. Development of a tightly-regulated tetracycline-dependent transcriptional activator and repressor co-expression system for the strong induction of transgene expression. Cytotechnology. 2011 May;63(3):211-216. Epub 2011 Feb 20.
This second article is a paper concerning the development of a new and improved dual activation/repression system for transgene expression. The authors discuss how the use of rtTA2-M2, the activator, and TetR-KRAB, the repressor, results in a transgene expression that is significantly less leaky in the absence of doxycycline and significantly more activated in its presence than any traditional rtTA-based system or previous iterations. Our current circuit design proposes to make use of this activation system.
Diester I, Kaufman MT, Mogri M, Pashaie R, Goo W, Yizhar O, Ramakrishnan C, Deisseroth K, Shenoy KV. An optogenetic toolbox designed for primates. Nat Neurosci. 2011 Mar;14(3):387-97. Epub 2011 Jan 30.
Gene Circuit Delivery
Pincha M, Salguero G, Wedekind D, Sundarasetty BS, Lin A, Kasahara N, Brugman MH, Jirmo AC, Modlich U, Gutzmer R, Büsche G, Ganser A, Stripecke R. Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells. Gene Ther. 2011 Mar 17.
Ngo MC, Rooney CM, Howard JM, Heslop HE. Ex vivo gene transfer for improved adoptive immunotherapy of cancer. Hum Mol Genet. 2011 Mar 25.
Cell Suicide Mechanisms
Amano S, Gu C, Koizumi S, Tokuyama T, Namba H. Tumoricidal bystander effect in the suicide gene therapy using mesenchymal stem cells does not injure normal brain tissues. Cancer Lett. 2011 Mar 28.
Johnson AJ, Ardiani A, Sanchez-Bonilla M, Black ME. Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications. Cancer Gene Ther. 2011 Mar 11.
Schmidt M, Gruensfelder P, Roller J, Hagen R. Suicide gene therapy in head and neck carcinoma cells: an in vitro study. Int J Mol Med. 2011 Apr;27(4):591-7. doi: 10.3892/ijmm.2011.610. Epub 2011 Feb 3.
Leveille S, Samuel S, Goulet ML, Hiscott J. Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy. Cancer Gene Ther. 2011 Mar 11.
5. http://www.ncbi.nlm.nih.gov/pubmed/21418659 Ma J, Li M, Mei L, Zhou Q, Liu L, Yu X, Che G. Double suicide genes driven by kinase domain insert containing receptor promoter selectively kill human lung cancer cells. Genet Vaccines Ther. 2011 Mar 22;9:6.
=== The Nature of Pluripotency ==-
Knoepfler PS. Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine. Stem Cells. 2009 May;27(5):1050-6.
This first article is a discussion about current issues with and potential solutions to pluripotent stem cell technology. The article opens with a discussion about the striking similarity between pluripotent stem cells and cancer stem cells and continues to discuss empirical studies of tumorigenicity in stem cells. The article concludes with a set of four potential pathways for eliminating or controlling for tumorigenic behavior in stem cells. The most critical for our project idea is the use of suicide genes.
Tang Y, Lin CJ, Tian XC. Functionality and Transduction Condition Evaluation of Recombinant Klf4 for Improved Reprogramming of iPS Cells. Cell Reprogram. 2011 Apr;13(2):99-112.
Wang Y, Mah N, Prigione A, Wolfrum K, Andrade-Navarro MA, Adjaye J. A transcriptional roadmap to the induction of pluripotency in somatic cells. Stem Cell Rev. 2010 Jun;6(2):282-96.
Zhang C, Wang QT, Liu H, Zhang ZZ, Huang WL. Advancement and prospects of tumor gene therapy. Chin J Cancer. 2011 Mar;30(3):182-8.
Stem Cell Population Control: A Potential Circuit
- The current implementation we envision would be constructed as follows:
- hTERT Promoter - KillerRed
- Oct4/Sox2/Nanog Transcription Sequences - KillerRed
- tetO - Caspase / Other Suicide Genes
- In short, the hTERT promoter is active only in cells that express factors necessary for activating telomerase production, which are probably like Nanog, Oct-3/4, and/or Sox-2. These cells would be either pluripotent cells, stem cells, or cancer cells. So the activator for a cell-death gene is made only when the cell is one of these three types. Then there's also a component where the repressor is normally made unless the optogenetic element is repressed by light. So, if the cell is a stem cell but the repressor is still active, no cell death occurs and the population can proliferate. Only if both light hits the cell and it's a stem cell (or cancer cell) will apoptosis be triggered. (This all unfortunately would have to happen in the presence of Doxycycline, but I'll look to see if there are other good systems.)