Toward next-generation allogeneic CAR-T cells by adenine base editor-mediated epitope editing and protein disruption to avoid fratricide and GvHD in pan-hematopoietic cancer patients
Board Location: #13
Discipline: Biological Sciences
Subcategory: Cancer Research
Session: 3
Joaquin Parrilla-Garcia - University of Puerto Rico - Mayaguez
Co-Author(s): Gozde Karakus; University of Pennsylvania Perelman School of MedicineElahe Kamali Ghahe; University of Pennsylvania Perelman School of MedicineSoon-Keat Ooi; University of Pennsylvania Perelman School of MedicineNils Wellhausen; University of Pennsylvania Perelman School of MedicineSaar Gill; University of Pennsylvania Perelman School of MedicineCarl June; University of Pennsylvania Perelman School of MedicineFriederike Herbst-Nowrouzi; University of Pennsylvania Perelman School of Medicine
Chimeric antigen receptor (CAR)-T cell-based immunotherapeutic approaches, targeting others than B cell-derived neoplasms, may require the modification of benign hematopoietic stem and progenitor cells (HSPCs) due to the lack of tumor cell-specific surface proteins. Therefore, genetic engineering of donor HSPCs will be used to deplete the targeted antigen and thereby preventing CAR-directed toxicity in benign hematopoietic cells. As an example, Kim et al. showed that knockout of CD33 in human HSPCs leads to restoration of the hematopoietic system after transplantation in mice and non-human primates and prevents CAR-T cell-mediated cytotoxicity, which is then directed against CD33-positive acute myeloid leukemia cells only. To expedite clinical applications, our goal was to target a range of blood cancers by focusing on CD45, a pan-hematologic antigen. Recently, Wellhausen et al. introduced a next-generation engineering strategy that involves inducing a single amino acid substitution in the CAR-targeted epitope of human CD34 cells. This was accomplished using an adenine base editor, which prevents CAR binding while preserving the CD45 protein and its function (Wellhausen et al., Sci Transl Med 2023). Epitope editing is also necessary in CAR45-T cells, which express CD45, to prevent fratricide. To mitigate potential side effects such as Graft-versus-Host Disease (GvHD) that may be induced by the allogeneic CAR45-T cell product, we hypothesize that depleting CD3ε could disrupt the T-cell receptor (TCR) and prevent GvHD. Here, our aim was to evaluate the efficiency of a dual genomic modification strategy to achieve two goals: a) efficient editing of the CD45 epitope and b) effective protein disruption of CD3ε to abolish TCR expression. We validated this dual genetic engineering approach by demonstrating a high efficiency of adenine base editor-mediated gene editing, resulting in the simultaneous disruption of translated protein generation of CD3e [77%] and CD45 epitope editing [98%] in primary T cells. Our findings indicate that simultaneous mutation of the splice donor site of CD3ε leads to effective TCR disruption, making it a potential candidate for inclusion in clinical allogeneic CAR T cell products to mitigate the risk of Graft-versus-Host Disease.
Funder Acknowledgement(s): National Institutes of Health (NIH)University of Pennsylvania Perelman School of Medicine
Faculty Advisor: Friederike Herbst-Nowrouzi, friederike.herbst-nowrouzi@pennmedicine.upenn.edu
Role: My involvement was marked by designing and executing an appropriate methodology to address our research aims focused on evaluating the efficiency of a dual genomic modification strategy. As the data was collected, I took the lead in conducting data analysis. This step involved performing various statistical analyses, employing software tools like GraphPad Prism, as well as resources for RNA Sequencing like EditR and SnapGene. After the data analysis, I was responsible for synthesizing our findings with the relevant academic literature. This process required a comprehensive review of relevant research studies and a critical evaluation of how our results contributed to related genetic engineering approaches.