Model Organism Introduction And Clinical Trial Design Using Mice (Proof-Of-Concept Experiment 5)

Hello! Today, we will be discussing my design of a mouse clinical trial as a proof-of-concept experiment that is part of my theoretical approach. This week, I researched what model organism to use in testing CRISPR gene therapies, how to obtain different mouse models, and how the clinical trial could potentially be designed. I have added this experiment to the methods section of my paper, and I am working on finishing the discussion section. I have also been looking for journals to publish my paper as well. 

Why Mice? 

One of the main reasons why I decided to use mice is because the genome of mice has been sequenced already, and the genome of mice has very striking similarities to that of humans. All of the genes in mice share the same functions as the genes in humans. They also have the same kinds of organs, organ systems, and development as well. This makes it easier to study mice physiology and study growth of cells, particularly tumors in living organisms.  

In addition to this, the life expectancy of mice in labs is about 2 years., it also makes it easier to study the progression of chronic diseases and cancers over a lifetime. It helps scientists to analyze the impact of aging on tumor growth as well. Finally, mice are also small and relatively economical organisms to maintain, which makes mice the ideal model organism for this experiment. 

IDLV-Mediated Gene Therapy in a Murine Model of HNPCC 

To design this model, 200 mice divided into 4 groups of 50 will be used. The 2 groups of 50 mice will bear mutations in exon 2 of MLH1, of which one group receives the treatment and the other group doesn’t receive the treatment. Although Jackson Laboratories does not have mice with this mutation, transgenic mice can be created using the following protocol: https://www.cell.com/fulltext/S0092-8674(00)81312-4#%20. The next 50 mice will bear mutations in exon 12 of MSH2, which is present in the Jackson Laboratories database: https://www.jax.org/strain/016231, of which one group receives the treatment and the other group doesn’t receive the treatment. 

One-thousand nanograms of IDLVs containing the Cas9n, gRNAs, and the HDR template or IDLVs containing the pLentiCRISPR/v2 and psPAX2 backbones will be intrarectally administered into mice via lipophilic enemas (Matsumoto et al., 2010). These mice will be monitored for changes in the following metrics: body weight, food consumption, stool composition, blood pressure, complete blood cell count, and electrolytes. These mice will be treated for 21 days, starting from when they are 2 months old, and they will be euthanized at 6 months old. The colonic epithelia will be harvested and analyzed by H&E (hematoxylin/eosin) histology and immunohistochemical stains for carcinoembryonic antigens, carbohydrate antigens, tissue polypeptide-specific antigens, and tumor-associated glycoprotein-72 [biomarkers of HNPCC] (Jelski and Mroczko, 2020). 

Results of HNPCC Treatment in Mice Colon 

The expected results of the treatment for 2 of the four groups of mice are described in Figure 1 below. Upon injection into the rectum, the IDLVs with the CRISPR-Cas9n system and template enter the crypt cells and the colonic epithelium. The plasmids are then released into the cells, and cells then use their native machinery for repairing DNA to cut out the old copy of DNA and replace it with the new copy of DNA. The expected goal of this treatment is that normal MLH1 and MSH2 protein function is restored and mice exhibit improved symptoms such as more appetite, less vomiting, less blood in stools, and an increase in body weight.  

In addition to this, we also expect the results of immunohistochemical staining for colon cancer to be similar to Figure 2 below. For H&E staining, I expect that there will be more nuclei than cytoplasm and that the shape of the colonic villi will not be as clear in the mice that have not received the treatment. For the mice that have received the treatment, there will be a clearer shape for the colonic villi, and there will also be an equal balance of nuclei and cytoplasm throughout the sample.  

Although this experiment with mice will show that the treatment does work effectively, it is not enough to end pre-clinical analysis. I am planning to do experimentation on human colonic explants, which will be coming up in the next blog. Thank you for reading and stay tuned for more information!  

Figure 1: Expected treatment administration and result of gene therapy. 

Figure 2: Expected results of HNPCC biomarker staining. CEA stands for carcinoembryonic antigen. TAG-72 stands for tumor-associated glycoprotein-72. The top two squares show normal mice colon with biomarker stains. The bottom two squares show biomarker staining results of mice colon with tumors. Source: https://www.nature.com/articles/bjc2012605     

Figure 3: Expected results of H&E staining of mice colon at the 10x and 60x magnification. Image A and Image B show H&E staining results of normal mice colon. Image C and Image D show H&E staining results of mutated mice colon. Source: https://www.nature.com/articles/s41598-019-47743-y  

Sources: 

1. Matsumoto, Hiroshi, et al. “Effective in Vivo and Ex Vivogene Transfer to Intestinal Mucosa by VSV-G-Pseudotyped Lentiviral Vectors.” BMC Gastroenterology, vol. 10, no. 1, 11 May 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC2881878/, https://doi.org/10.1186/1471-230x-10-44. Accessed 24 Apr. 2023. ‌
2. Wojciech Jelski, and Barbara Mroczko. “Biochemical Markers of Colorectal Cancer – Present and Future.” Cancer Management and Research, vol. Volume 12, 22 June 2020, pp. 4789–4797, www.ncbi.nlm.nih.gov/pmc/articles/PMC7319530/,   https://doi.org/10.2147/cmar.s253369.  Accessed 22 Apr. 2023.