Project Background
Glioblastoma multiforme is one of the most aggressive types of brain tumors and originates from glial cells of the central nervous system known as astrocytes. These cells possess a star-shaped morphology (hence the name astrocyte) and their primary function is to support neurons with the appropriate nutrients and environment necessary for normal functioning. Although there is a higher prevalence of GBM occurrences in adults, this type of cancer does not discriminate against younger age groups. In recent years, there has been an increasing number of reported cases of GBM in pediatric populations.
These tumors can form as a result of various genetic mutations; however, one which was of keen interest to our team is the epidermal growth factor receptor variant iii (EGFRvIII). The EGFR receptor is primarily involved in cellular signaling cascades that dictate replication and overall survival. In many cases, the EGFR gene can be subject to extensive amplification and thus overexpression. With this, however, there is an increased likelihood for genetic rearrangements such as the deletion of EGFR’s exons 2-7, which result in the EGFRvIII mutation. The resultant EGFR protein no longer displays binding capability to any ligands and has been scientifically identified to encourage tumor formation (i.e., GBM), typically resulting in a cumbersome prognosis for patients diagnosed with a resultant cancer.
Our team proposed to generate a lentiviral organism packaged with a CRISPR-cas9 complex that would identify the target EGFRvIII sequence, excise it, and replace it with the five missing exons. Our hypothesis is that, by using our lentiviral organism to reverse this in-frame deletion of the EGFR gene in glioblastoma cells, wild-type EGFR proteins would then be synthesized, thus restoring the cell’s normal function and hindering tumor progression.
Project Inspiration
Personal
One of our team members, Eden Goldenberg, first developed this hypothesis for a science fair project during her junior year of high school. After being invited to CRISPR-Con, an international CRISPR conference that took place in her hometown of Madison, Wisconsin, she collaborated with Ph.D. student Katie Mueller, and Dr. Krishnau Saha, head of a molecular genetics/oncology research lab at UW-Madison. Through collaborative efforts with these individuals, Eden continued to develop her hypothesis and research procedure. This process was then refined and tailored for purposes of team UFlorida’s iGEM project for the 2022 cycle.
Academia
Furthermore, researchers at the University of Texas conducted a study investigating genes that could be engineered to target the specific mechanisms that make GBM formations so deadly. Originally, they sought to induce apoptosis in infected glioma cells using wild-type p53 gene cDNA; however, the results across the different cell lines used presented much variation. In three cell lines that exhibited an internal mutated p53 gene, the addition of the wild-type p53 gene induced apoptosis, as expected. On the other hand, three cell lines that were found to have the wild-type p53 displayed overexpression of the gene and resulted in a dramatic reduction of cell proliferation (Gomez-Manzano, et. al). These results introduce two novel ways to induce either cell death or reduce tumor growth using one gene. This research directly relates to our project, as the p53 gene has also been a focus in our investigation into viral vectors.
iGEM Teams
In 2021, there were three iGEM teams whose projects directly related with our current research. Team Duke 2021 utilized a two-phase process to develop a novel organoid-dependent drug efficacy system, (NODES) that served as an improved economical method of therapeutic drug screening that minimized animal testing. Their team transfected glioma cells with their own plasmids as part of their proof of concept, giving us insight into various intracellular mechanisms specific to this cell line.
In addition, Team Korea HS 2021 engineered a cancer-specific cell-penetrating peptide for the efficient delivery of siRNA into cancerous cells, providing us with an in-depth look into the mechanisms one can approach when trying to combat different types of cancer. This is very important to our team given that we aim to one day generalize the treatment we are developing to other types of cancer, as opposed to strictly targeting GBM.
Lastly, Team ZJU-China 2021 developed a new oncolytic virus to treat hepatocellular carcinoma (HCC) via RNAi to kill malignant cells and eliminate possible virulence. Their project granted us information regarding the feasibility behind the types of viruses that can be used in cancer treatment.
Our Vision
Once a GBM patient is diagnosed, their median survival rate hovers around 5%. Not only do we want to collaborate with other iGEM teams to design and successfully create a novel treatment method for targeting GBM’s, but we want to use the creation of our project to raise awareness and honor those fighting their battle against it, bringing us one step closer towards finding a cure. In the future, we aim to work alongside the Pediatric Brain Tumor Foundation within our local area. By working with the patients they support, we hope to give these patients the opportunity to share their inspiring stories with the world. We believe these efforts will help give a face to this deadly disease, thus raising awareness and bringing more attention to it in the field of research. Although the current median survival rate may be 5%, our efforts are placed in increasing those odds via our novel treatment. Ultimately, since GBM primarily targets pediatric demographics, we are trying to give children diagnosed with this disease a shot at a life, which they very well deserve.
We hope to eventually publish our findings in an official paper format in various medical journals. Through obtaining statistically significant results and the support from proper educational and financial outlets, we will proceed to in vivo trials. The competition allows for a platform for these ideas to be shared and hopefully cause more people to do research on this topic. The long term goal would be to improve the quality of life for these patients.
References
Fassler, M., Weissberg, I., Levy, N., Diaz-Griffero, F., Monsonego, A., Friedman, A., & Taube, R. (2013). Preferential lentiviral targeting of astrocytes in the central nervous system. PloS one, 8(10), e76092. https://doi.org/10.1371/journal.pone.0076092
Merienne, N., Le Douce, J., Faivre, E., Déglon, N., & Bonvento, G. (2013). Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors. Frontiers in cellular neuroscience, 7, 106. https://doi.org/10.3389/fncel.2013.00106
Humbel, M., Ramosaj, M., Zimmer, V. et al. Maximizing lentiviral vector gene transfer in the CNS. Gene Ther 28, 75–88 (2021). https://doi.org/10.1038/s41434-020-0172-6
106. Tropism Changes of HIV-1 Based Vectors Pseudotyped with Various Lyssavirus Envelopes in the Central Nervous System, Molecular Therapy, Volume 5, Issue 5, Supplement, 2002, Page S37, ISSN 1525-0016, https://doi.org/10.1016/S1525-0016(16)42936-9.
Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies Christina L. Parker , Timothy M. Jacobs , Justin T. Huckaby , Dimple Harit , and Samuel K. Lai Eleftherios T. Papoutsakis , Editor
Lee, Y., Messing, A., Su, M., & Brenner, M. (2008). GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia, 56(5), 481–493. https://doi.org/10.1002/glia.20622
Lee, Y., Su, M., Messing, A., & Brenner, M. (2006). Astrocyte heterogeneity revealed by expression of a GFAP-LacZ transgene. Glia, 53(7), 677–687. https://doi.org/10.1002/glia.20320
Hicks, Martin J et al. “Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma.” PloS one vol. 11,10 e0162978. 6 Oct. 2016, doi:10.1371/journal.pone.0162978
Candelaria Gomez-Manzano, Juan Fueyo, Athanassios P. Kyritsis, Peter A. Steck, Victor A. Levin, W. K. Alfred Yung, Timothy J. McDonnell, Characterization of p53 and p21 Functional Interactions in Glioma Cells en Route to Apoptosis, JNCI: Journal of the National Cancer Institute, Volume 89, Issue 14, 16 July 1997, Pages 1036–1044, https://doi.org/10.1093/jnci/89.14.1036
Gomez-Manzano C, Fueyo J, Kyritsis AP, Steck PA, Roth JA, McDonnell TJ, Steck KD, Levin VA, Yung WK. Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. Cancer Res. 1996 Feb 15;56(4):694-9. PMID: 8630997.
McCarty, N. (2020, August 15). CRISPR - how it works, top applications and how to use it yourself. Medium. Retrieved May 22, 2022, from https://nikomccarty.medium.com/almost-everything-you-should-know-about-crispr-how-it-works-top-applications-and-how-to-use-it-61e27b04bea6
Faria MH, Patrocínio RM, Moraes Filho MO, Rabenhorst SH. Immunoexpression of tumor suppressor genes p53, p21 WAF1/CIP1 and p27 KIP1 in humam astrocystic tumors. Arq Neuropsiquiatr. 2007 Dec;65(4B):1114-22. doi: 10.1590/s0004-282x2007000700004. PMID: 18345413.
Parker, C. L., Jacobs, T. M., Huckaby, J. T., Harit, D., & Lai, S. K. (2020). Efficient and highly specific gene transfer using mutated lentiviral vectors redirected with bispecific antibodies. MBio, 11(1). https://doi.org/10.1128/mbio.02990-19
Kato S, Kobayashi K, Inoue K, Kuramochi M, Okada T, Yaginuma H, Morimoto K, Shimada T, Takada M, Kobayashi K. A lentiviral strategy for highly efficient retrograde gene transfer by pseudotyping with fusion envelope glycoprotein. Hum Gene Ther. 2011 Feb;22(2):197-206. doi: 10.1089/hum.2009.179. Epub 2011 Jan 27. PMID: 20954846.
Martin, Petra & Kelly, Catherine & Carney, Desmond. (2006). Epidermal Growth Factor Receptor-Targeted Agents for Lung Cancer. Cancer control : journal of the Moffitt Cancer Center. 13. 129-40. 10.1177/107327480601300207.
Comprehensive Genomic Profiling of Circulating Cell-Free DNA Distinguishes Focal MET Amplification from Aneuploidy in Diverse Advanced Cancers - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Gene-location-on-chromosome-7-In-chromosome-7-the-EGFR-gene-is-located-in-7p-112_fig1_354865346 [accessed 25 May, 2022]
“Glioma.” Mayo Clinic, Mayo Foundation for Medical Education and Research, 4 Apr. 2020, https://www.mayoclinic.org/diseases-conditions/glioblastoma/cdc-20350148#:~:text=Glioblastoma%20is%20also%20known%20as,more%20often%20in%20older%20adults.
Gan, Hui K et al. “The epidermal growth factor receptor variant III (EGFRvIII): where wild things are altered.” The FEBS journal vol. 280,21 (2013): 5350-70. doi:10.1111/febs.12393