1. Development of p53 knockout U87MG cell line for unbiased drug delivery testing system using CRISPR-Cas9 and transcriptomic analysis
- Author
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Erik Arner, Hidefumi Mukai, Takahiro Arakawa, Bogumil Kaczkowski, Maiko Takahashi, Kohta Mohri, Harukazu Suzuki, Andrew T. Kwon, Shunsuke Tagami, Satoshi Takizawa, and Masaaki Furuno
- Subjects
0106 biological sciences ,0301 basic medicine ,Cell type ,Mutant ,Bioengineering ,Computational biology ,Biology ,Cell morphology ,01 natural sciences ,Applied Microbiology and Biotechnology ,Cell Line ,Transcriptome ,03 medical and health sciences ,In vivo ,010608 biotechnology ,CRISPR ,General Medicine ,Genes, p53 ,030104 developmental biology ,Targeted Mutation ,Pharmaceutical Preparations ,Drug delivery ,CRISPR-Cas Systems ,Tumor Suppressor Protein p53 ,Biotechnology - Abstract
Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.
- Published
- 2020