Biology subjects

Klermund, J.

Publications and source records attributed to Klermund, J..

3 recordsLinked to original sources

bioRxiv2025

Clinical development of gene edited tacrolimus-resistant Treg (FKBP12KO-Treg) to enable simultaneous immunosuppression and support of immune regulation

Background: Unwanted immune responses play a central role in the pathogenesis of solid organ allograft rejection. These are managed by life-long immunosuppression with considerable burden for the patient and society. Adoptive therapy with regulatory T-cells (Treg) is a promising approach to restore sustainable immune balance and avoid long-term adverse effects of immunosuppression. While Treg effectively inhibit activation of unwanted immune responses, they are less effective in controlling pre-existing/activated memory effector T-cells (Teff). Thus, co-administration of Treg with immunosuppressants is required to achieve a sustainable organ acceptance. Calcineurin inhibitors (CNI) are powerful in controlling de novo generated and preformed Teff. However, CNI also dampen Treg immunoregulatory function. Thus, we hypothesize improved results of adoptive Treg therapy in immunosuppressed patients applying tacrolimus-resistant Treg. Methods: While retaining CNI modulation of Teff with tacrolimus, we knocked-out FKBP12 in Treg (FKBP12KO-Treg) by gene-editing using ribonucleoprotein-based CRISPR/Cas9 technology to generate tacrolimus-resistant Treg and characterised them using flow cytometry, functional assays and in-depth phenotyping. Results: This detailed in vitro analysis showed FKBP12KO-Treg were comparable to non-gene edited Treg and impervious to tacrolimus while maintaining immunoregulatory function and sensitivity to alternative CNIs raising no safety concerns. Furthermore, we aligned our methodology to achieve GMP compliance laying the basis for a manufacturing license in preparation of a clinical trial. Conclusion: Based on the presented preclinical dataset implying safety and efficacy of FKBP12KO-Treg, we are now seeking to undertake a proof-of-concept clinical trial to evaluate the co-administrationof FKBP12KO-Treg and tacrolimus to enhance the management of living donor kidney transplant recipients.

immunology↗

bioRxiv2020

Validation and Long-Term Follow Up of CD33 Off-Targets Predicted In Vitro and In Silico Using Error-Corrected Sequencing in Rhesus Macaques

ABSTRACTThe programmable nuclease technology CRISPR/Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR/Cas9 therapeutically. Here, we utilized a rhesus macaque model to ask whether CIRCLE-Seq (CS), an in vitro off-target prediction method, more accurately identifies off-targets compared to in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-Seq and ISP for guide RNAs designed against TET2 and CD33 genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both ISP and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a CD33 gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both ISP and CIRCLE-Seq, however there was poor correlation between the sites predicted by the two methods. When almost 500 sites identified by each method were searched for in hematopoietic cells following transplantation, 19 detectable mutations in off-target sites were detected via error-corrected sequencing. Of these 19 sites, 8 sites were predicted in the top 500 sites by both methods, 8 by CIRCLE-Seq only, and 3 by ISP only. Cells with off-target editing exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In conclusion, neither methodology predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods may be required for optimizing safety of clinical development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2018

Checkpoint adaptation in repair-deficient cells drives aneuploidy and resistance to genotoxic agents

Human cancers frequently harbour mutations in DNA repair genes, rendering the use of DNA damaging agents as an effective therapeutic intervention. As therapy-resistant cells often arise, it is important to better understand the molecular pathways that drive resistance in order to facilitate the eventual targeting of such processes. We employ repair-defective diploid yeast as a model to demonstrate that, in response to genotoxic challenges, nearly all cells eventually undergo checkpoint adaptation, resulting in the generation of aneuploid cells with whole chromosome losses that have acquired resistance to the initial genotoxic challenge. We demonstrate that adaptation inhibition, either pharmacologically, or genetically, drastically reduces the occurrence of resistant cells. Additionally, the aneuploid phenotypes of the resistant cells can be specifically targeted to induce cytotoxicity. We provide evidence that TORC1 inhibition with rapamycin, in combination with DNA damaging agents, can prevent both checkpoint adaptation and the continued growth of aneuploid resistant cells.

cell biology↗