Biology subjects

Urnov, F. D.

Publications and source records attributed to Urnov, F. D..

4 recordsLinked to original sources

bioRxiv2026

A Toolbox for Binary and Rheostat-like Modulation of TOX Expression via Genome and Epigenome Editing in Primary Human T cells

T cells, which are central mediators of the adaptive immune response, can become dysfunctional when faced with persistent antigen stimulation, such as in chronic infections and cancer. This dysfunctional state, known as T cell exhaustion, limits pro-inflammatory T cell function, dampens cytotoxicity and proliferative capacity, and promotes expression of inhibitory receptors. Thymocyte Selection-Associated High Mobility Group Box (TOX) has been proposed as a master regulator of T cell exhaustion due to its necessity for survival of exhausted T cells as well as its role in shaping chromatin accessibility in murine models. Interestingly, partial Tox deficiency may improve control of murine tumors. In human tumor infiltrating lymphocytes, high TOX expression is associated with poor disease prognosis. However, the mechanisms by which TOX expression is regulated and its importance to human T cell exhaustion remain poorly understood. We report here a robust strategy for generating a genetic knockout of TOX via base editing or a knockout phenocopy via epigenome editing in primary human T cells ex vivo, with each approach resulting in near-complete elimination of TOX mRNA. Guided by enhancer prediction data, we use epigenome editing to identify several human cis-regulatory regions which function to silence TOX expression to varying levels when targeted with CRISPRoff. TOX deficiency had no measurable impact on survival or exhaustion marker levels in human CD8+ T cells in a model of anti-CD3/anti-CD28 stimulation in vitro. In agreement with these data, expression profiling revealed that TOX knockout effects on the transcriptome are limited to TOX itself, with no observable downstream effects. These studies show that a complete TOX knockout or silencing has no effect on exhaustion marker expression levels or the transcriptome in repeat-anti-CD3/anti-CD28-stimulated primary human T cells in vitro. Taken together, we developed a powerful toolkit of genome and epigenome editing strategies to modify expression of a gene of interest in primary human T cells and study its function. We propose that this framework can be applied to additional genes of interest both to gain mechanistic information about T cell function, as well as develop strategies for improvement of T cell immunotherapies.

molecular biology↗

bioRxiv2024

Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

BackgroundProgressive multifocal leukoencephalopathy (PML) is a frequently fatal disease of the central nervous system caused by JC virus (JCV). Survival is dependent on early diagnosis and ability to re-establish anti-viral T cell immunity. Adoptive transfer of polyomavirus-specific T cells has shown promise; however, there are no readily available HLA-matched anti-viral T cells to facilitate rapid treatment. ObjectiveIdentify epitopes of the JCV major capsid protein VP1 that elicit an immune response in the context of human leukocyte antigen allele A*02:01 (HLA-A2) and isolate cognate T cell receptors (TCRs) from healthy donors. Evaluate individual VP1-specific TCRs for their capacity to be expressed in T cells and clear JCV in vitro. MethodsPBMCs from HLA-A2+ healthy donors were stimulated with peptide libraries tiled across the JCV VP1 protein. Multiple rounds of stimulation were performed to identify the antigens that induced the largest expansion and CD8+ T cell response (measured as INF{gamma}, TNF, CD137, and CD69 expression). High-affinity, antigen-specific CD8+ T cells were isolated based on intensity of tetramer binding for downstream single-cell TCR sequencing. Candidate TCRs were selected based on tetramer binding affinity and activation assays. Promising TCRs were introduced into the T cell genome via viral transduction for in vitro validation including peptide-pulsed K562 cells and astrocyte cells, and JCV-infected astrocytes. ResultsFour conserved JCV VP1 epitopes (amino acids 100-108, 251-259, 253-262, and 274-283) presented by HLA-A2 were identified. VP1(100-108) consistently elicited the highest level of IFN-{gamma} production from multiple donors and this peptide is in a highly conserved region of VP1. We next identified fourteen high avidity TCRs specific for VP1(100-108). When virally transduced into primary human T cells, seven of these TCRs demonstrated specific binding to VP1(100-108):HLA-A2 tetramers, and four showed increased IFN-{gamma} response when incubated with peptide. Primary CD8+ T cells expressing two of these TCRs cleared both HLA-A2 positive K562 cells and HLA-A2 positive SVG astrocyte cell line presenting exogenously added VP1 peptide at a range of E:T ratios. In addition, both TCR-transduced T cell populations effectively lysed JCV-infected astrocytes. ConclusionsWe identified JCV VP1 epitopes that are immunogenic in the context of HLA-A2 MHC-I, including epitopes that have not been previously described. The VP1(100-108) epitope was used to isolate HLA-A2-restricted TCRs. When cloned into primary human CD8+ T cells, these TCRs recognized VP1 (100-108)-presenting targets, and the transduced T cells conferred cytotoxic activity and eliminated K562 and astrocyte cells displaying the VP1(100-108) peptide and not sham peptide, as well as JCV-infected astrocytes. Taken together, these data suggest that JCV VP1-specific TCRs could be appealing therapeutics for HLA-A2+ individuals with PML in whom intrinsic T cell immunity cannot be rescued.

neuroscience↗

bioRxiv2021

Uncoupling of Chromatin Assembly from DNA Replication in Sciara Reveals a Domain of Postreplicative Immature Chromatin

DNA replication in dividing eukaryotic cells imposes a requirement for the faithful recreation on the newly synthesized chromatids of the nucleoprotein architecture of parent chromosomes. Practically nothing is known about the structure of postreplicative immature chromatin--a very short-lived entity (< 30 min.). We report here the unexpected discovery that during DNA amplification of locus II/9A in salivary gland polytene chromosomes of the fungus fly Sciara coprophila, DNA replication fork passage is uncoupled from postreplicative chromatin assembly; this enables visualization and analysis of chromatin fibers disassembled by DNA replication. We used electron microscopy to visualize a wealth of low nucleosome density immature chromatin fibers in preparations of Sciara chromatin from amplification-stage tissue. Remarkably, as gauged by high sensitivity to micrococcal nuclease and an unusually short length of DNA associated with each histone octamer, we found that locus II/9A which undergoes amplification and is replicated once every 4-6 hrs.--but not the bulk genome or a replicatively quiescent DNA stretch--was maintained in such an ummature fiber for ca. 24 hrs. Following amplification, locus II/9A assumed conventional chromatin organization, indicating that the epigenetic mark targeting nascent DNA to the chromatin assembly machinery is stable for several hours. We propose that this very unusual prolonged maintenance of a segment of the genome in immature chromatin facilitates access by the basal transcriptional machinery to the amplified DNA, and thus is an evolutionary adaptation to the demand for high transcription from genes that reside in the amplified loci.

molecular biology↗

bioRxiv2020

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Human genes are regulated quantitatively, yet the ability to specify the expression level of a native gene accurately and specifically using a defined reagent has remained elusive. Here we show that precise targeting of KRAB repressive domain within regulatory DNA unlocks an endogenous quantitative dial that can be engaged at nucleotide resolution to program expression levels across a wide physiologic range, with single-gene specificity and high reproducibly in primary cells.

synthetic biology↗