Biology subjects

Kluesner, M.

Publications and source records attributed to Kluesner, M..

4 recordsLinked to original sources

bioRxiv2025

Installation of Dominant-Negative Mutations in FAS and TGFβR2 via Base Editing in Primary T Cells

Adoptive cell transfer (ACT) of engineered T cells is effective against B-cell malignancies but has faltered against solid tumors due to the immunosuppressive tumor microenvironment (TME). FASL and TGF{beta} are key mediators of T cell dysfunction in the TME and overexpressing dominant negative (dn) forms of their receptors in T cells increases anti-tumor efficacy in solid tumor models. However, an approach which directly targets the endogenous genes would be more amenable to multiplex editing and reduce competition with WT alleles. Here, we employ base editing (BE) in primary human T cells to install naturally occurring dominant negative FAS and TGF{beta}R2 mutations. In vitro survival and proliferation assays demonstrate that BE T cells are resistant to pro-apoptotic and anti-proliferative effects of FAS and TGF{beta} signaling. CAR-T cells with BE-installed dn TGF{beta}R2 or dn FAS exhibit improvements in cytotoxicity, while dn TGF{beta}R2 CAR T demonstrate increased persistence and reduced expression of phenotypic markers of exhaustion compared to controls. Moreover, BE-engineered dn CAR T outperform lentiviral-engineered cDNA over expression counterparts in several functional assays. Considering the efficiency of BE and its amenability for multiplex editing, our novel approach lends itself to engineering strategies necessary to overcome T cell dysfunction in solid tumors.

immunology↗

bioRxiv2024

Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Gamma delta ({gamma}{delta}) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, {gamma}{delta} T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of {gamma}{delta} T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-{gamma}{delta} T cells in support of therapeutic application. Engineered CAR-{gamma}{delta} T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-{gamma}{delta} T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-{gamma}{delta} T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-{gamma}{delta} T cell subsets to therapeutic efficacy in vivo.

immunology↗

bioRxiv2019

MultiEditR: An easy validation method for detecting and quantifying RNA editing from Sanger sequencing

RNA editing is the base change that results from RNA deamination by two predominant classes of deaminases; the APOBEC family and the ADAR family. Respectively, deamination of nucleobases by these enzymes are responsible for endogenous editing of cytosine to uracil (C-to-U) and adenosine to inosine (A-to-I). RNA editing is known to play an essential role both in maintaining normal cellular function, as well as altered cellular physiology during oncogenesis and tumour progression. Analysis of RNA editing in these important processes, largely relies on RNA-seq technology for the detection and quantification of RNA editing sites. Despite the power of these technologies, multiple sources of error in detecting and measuring base editing still exist, therefore additional validation and quantification of editing through Sanger sequencing is still required for confirmation of editing. Depending on the number of RNA editing sites that are of interest, this validation step can be both expensive and time-consuming. To address this need we developed the tool MultiEditR which provides a simple, and cost-effective method of detecting and quantifying RNA editing form Sanger sequencing. We expect that MultiEditR will foster further discoveries in this rapidly expanding field.

bioinformatics↗

bioRxiv2017

EditR: A novel base editing quantification software using Sanger sequencing

CRISPR/Cas9-Cytidine deaminase fusion enzymes - termed Base Editors - allow targeted editing of genomic deoxcytidine to deoxthymidine (C[->]T) without the need for double stranded break induction. Base editors represent a paradigm-shift in gene editing technology, due to their unprecedented efficiency to mediate targeted, single-base conversion; however, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time consuming. To overcome these limitations, we developed a simple, cost effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed EditR. We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence (baseeditr.com). EditR is more accurate than enzymatic assays, and provides added insight to the position, type and efficiency of base editing. Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.

genetics↗