IDLV Transduction Of Human Colonic Epithelium And Assessing Cellular And Morphologic Effects Of IDLV Transduction
Hello! Today, we will discuss my design of a pre-clinical study involving human colonic explants as a proof-of-concept experiment that is part of my theoretical approach. This week, I researched how to transduce organoids with IDLVs (integrase-deficient lentiviruses) and how to analyze them in the bigger scope of my project. I have added this experiment to the methods section of my paper, and I have fine-tuned the abstract. I have also settled on the Journal of Emerging Investigators to publish my paper.
IDLV Transduction of Epithelial Cells from Human Colonic Explants
As discussed in prior proof-of-concept experiments, IDLVs will be concentrated into pellets after being grown in HEK293 cells and then resuspended in fresh media. The viral pellet concentration will be calculated by counting plaque-forming units and adjusting them to concentrations differing by factors of 10 (Andersson and Lood. JoVE, 2019), as seen in Figure 1. Four groups of 50 patient-derived organoids (PDOs) will be collected from 200 HNPCC patients through either colonoscopic polypectomies or prophylactic colectomies (Okamoto et al., 2021).
Figure 1: Counting viral pellet concentration through plaque forming units (“Viability”).
To grow PDOs, conditioned media is necessary. To make this media, L1-cells expressing Noggin protein will be grown in Dulbecco Modified Eagle’s Medium and incubated for 24 hours to reach 80% confluency. The media of these cells will be collected and used for PDOs. The PDOs will be grown in Noggin-conditioned media for 3-6 days, with the medium being changed every 3 days. The gene treatment will be administered by injecting IDLVs into the conditioned media the PDOs are in (Figure 2). Each group of PDOs will be in their separate dish, and two groups will be injected with the treatment. Two groups will be injected with a placebo. To determine optimal transduction conditions, the viral transduction amount must be tested over multiple periods (3 days, 7 days, 14 days, and 21 days) with 1 injection per day.
Figure 2: Schematic diagram of PDO experiment with analyzed results (Okamoto et al., 2021).
After injection of the treatment, we will be able to verify that the organoids received the treatment because of the presence of GFP in the organoids. The organoids that glow with GFP fluorescence received the treatment, and the organoids that don’t glow did not receive the treatment. After 5 weeks, the organoids that glow will be picked and genome editing will be verified in them by Western blot experiments.
Assessing Cellular and Morphologic Effects of IDLV Transduction
After viral transduction, PDOs will be observed to see any cellular and morphologic changes. Three assays will be conducted to analyze metrics like cell survival, cell proliferation, and cell morphology. For cell survival assays, Western blotting will be used to quantify markers of apoptosis, like Annexin V, and necrosis like miR-122, FK18, and HMCB1. For cell proliferation assays, Western blotting and fluorescent microscopy will be used to assess decreases in cell count and restoration of normal phosphorylation of cyclin-dependent kinases. Microscopy will be used in cell morphology assays to see the restoration of density-dependent inhibition before and after treatment injection. We hope to see a return to a non-tumorigenic phenotype in the PDOs after injection of the treatment compared to the placebo groups.
- Andersson, Tilde, and Rolf Lood. Determining Viral Titer as Plaque Forming Units (PFU) | Microbiology | JoVE. https://www.jove.com/v/10514/plaque-assay-method-to-determine-viral-titer-as-plaque-forming-units. Accessed 9 May 2023.
- Okamoto, Takuya, et al. “A Protocol for Efficient CRISPR-Cas9-Mediated Knock-in in Colorectal Cancer Patient-Derived Organoids.” STAR Protocols, vol. 2, no. 4, Sept. 2021, p. 100780, doi:10.1016/j.xpro.2021.100780.
- “Viability.” Biology Articles, Tutorials & Dictionary Online, 7 Oct. 2019, https://www.biologyonline.com/dictionary/viability.