Biology subjects

Moriarity, B.

Publications and source records attributed to Moriarity, B..

13 recordsLinked to original sources

bioRxiv2026

Modeling CISH-KO TIL therapy: T-cell persistence and endogenous competition determine response in gastrointestinal cancer

BackgroundTumor-infiltrating lymphocyte (TIL) therapies have shown promise in murine models; however, early-phase clinical trials reveal substantial heterogeneity in treatment response across patients. Elucidating the biological drivers of this variability is critical for improving patient selection and therapeutic design. MethodsWe developed a mechanistic mathematical model of Cish-inactivated TIL therapy capturing interactions among tumor cells, endogenous TILs, and infused CISH-KO (Cish gene knockout) T-cells. The model was calibrated to longitudinal tumor size data from two murine studies and also fitted to clinical data from a first-in-human phase 1 trial enrolling 22 and treating 12 patients with metastatic gastrointestinal cancers (27 independently tracked tumor lesions). We used the calibrated model to identify patient-level determinants of response and to simulate combination and fractionation treatment strategies in silico. FindingsFour mechanistically distinct parameters emerged as dominant determinants of therapeutic outcome: CISH-KO T-cell persistence (r = 0.827, p < 10-4) was the single strongest predictor of therapeutic failure; other major determinants included endogenous T-cell proliferation rate (r = 0.648, p < 10-4) , CISH-KO T-cell killing rate, and intrinsic tumor growth rate. In silico simulations predicted that the addition of anti-PD-1 checkpoint blockade to standard-dose CISH-KO TIL therapy achieved comparable or superior tumor control relative to dose doubling, while maintaining lower peak T-cell level. Additionally, fractionating the same total dose across multiple delayed infusions further improved predicted tumor control by prolonging CISH-KO T-cell persistence, again without raising peak effector burden. InterpretationThe efficacy of current CISH-KO TIL protocols is influenced not only by the cytotoxic potency of infused T-cells, but also by their persistence within the tumor microenvironment and competitive pressure from the reconstituting host immune compartment. These findings support strategies that enhance the functional efficiency and prolong the availability of infused T-cells--such as concurrent PD-1 blockade or fractionated dosing of the same total cell product--rather than dose escalation alone and identify CISH-KO T-cell persistence and endogenous immune competition as actionable targets for improving TIL-based immunotherapies.

Cancer Biology↗

bioRxiv2026

Multiplex engineering of rhesus macaque NK cells enhances homing to sites of HIV replication in B cell follicles.

One barrier to developing an HIV-1 cure is viral reservoirs persisting within B cell follicles of lymphatic tissues, partly due to failure of HIV-specific cytotoxic cells to express the follicular-homing receptor CXCR5. Our group explores CAR cell therapies which also express CXCR5 as a potential cure strategy for HIV. Although previous studies have mostly explored CAR T cell therapies, CAR NK cells may be an attractive alternative as they can be used in allogeneic settings and are naturally cytotoxic towards HIV-infected cells. Here, we developed a novel and innovative multiplex engineering method for rhesus macaque NK cells to create virus-specific CAR NK cells multiplexed (MP) with CAR/CXCR5/IL-15/PD-1 KO/transient-CCR7. We first evaluated MP NK cells in vitro for functionality. MP NK cells were then infused into one chronically SIV-infected rhesus macaque to observe tolerance and localization of therapeutic cells. Finally, we performed a larger primate study in which SIV-infected rhesus macaques were infused with two doses of MP NK cells to study long-term localization, safety, and efficacy. In vitro, MP NK cells were expanded to clinically relevant numbers, migrated to chemokine signaling, and secreted cytotoxic cytokines in response to SIV-Env-expressing cells. In the preliminary rhesus macaque study, the therapy caused no adverse reactions, and CAR+ NK cells localized to sites of SIV replication within the spleen and lymph nodes. In the larger primate study, two doses of MP NK cells at 1.2 x 108 cells/kg were safe and increased the levels of NK cells and CAR+ NK cells found within lymphatic tissues. Importantly, the CAR+ NK cells detected in lymph nodes were predominantly CCR7+, demonstrating the importance of CCR7 and CXCR5 in combination for migration to SIV viral reservoirs in follicles of lymphatic tissues. This study is the first to demonstrate this type of complexity and combination of engineering techniques in NK cells. With further optimization, these techniques could lead to the development of novel NK cell therapies to treat HIV and other diseases.

bioengineering↗

bioRxiv2026

Engineering physically optimized T cells for increased sampling of complex tumor microenvironments

Pancreatic ductal adenocarcinoma (PDA) remains highly lethal, in part, because its dense fibroinflammatory stroma restricts therapy distribution, including adoptive T cell immunotherapies where direct interactions between T and carcinoma cells are essential for effective therapy. While T cell function must be maintained once effector-target engagement occurs, without inducing co-localization subsequent cytotoxic function steps cannot be undertaken. We therefore developed a strategy to "physically optimize" T cells to more effectively sample complex tumor volumes. Informed by pharmacologic perturbations and mathematical modeling we shifted T cell phenotype through expression of constitutively activated RhoA to increase cortical contractility, activation, migration, and sampling in PDA, while showing decreases in exhaustion markers. In CAR T cells this results in more efficient targeting through decreased sampling time and increased engagement with carcinoma cells, consistent with modeling predictions. This significantly increases T cell infiltration and distribution in PDA, resulting in improved tumor control in vivo, suggesting that this is an effective strategy to overcome stromal constraints, improve tumor engagement, and enhance the therapeutic performance of engineered T cell therapies in solid tumors.

bioengineering↗

bioRxiv2025

Bcl-2 protein Noxa is required for metabolic reprogramming to glutamine dependence and for apoptosis in stimulated human CD8+ T cells

Naive or memory T cells reprogram their metabolism upon antigenic stimulation. They increase their glucose uptake, relying on aerobic glycolysis for generating biomass while switching to glutamine to fuel energy production. Here we have identified a requirement for human Bcl-2 family, Noxa, in the metabolic switch to glutamine dependence in activated CD8+ T cells, that is independent of its canonical role in apoptosis at the end of the immune response. Using an in vitro co-stimulation model, we demonstrate that Noxa is induced in CD8+ T cells and remains elevated during the proliferative and differentiation phases of the response and through the onset of apoptosis. Noxa protein induction requires glutamine, is mediated via mTOR, and is independent of glutaminolysis. Glutamine, in turn, requires Noxa to facilitate its conversion to glutamate. CD8+ T cells lacking Noxa showed reduced levels of intracellular glutamate but no impairment of mitochondrial or effector function, and decreased dependence on glutamine for both respiration and growth during the proliferative phase. NOXA knockout CD8 T cells also displayed significantly higher viability in the apoptotic phase of the immune response. CD8+ T cells from a human NOXA gene-replacement mouse responded normally to in vitro stimulation and in vivo acute infection. However, human Noxa-expressing murine CD8+ T cells displayed a distinctly proliferative gene signature in their transcriptome following activation, supporting an early growth-promoting role for this BH3-only protein. Our studies suggest that knocking out NOXA in human CD8+ T cells to increase their lifespan as well as their ability to survive and function in glutamine-poor microenvironments could be a promising immunotherapeutic strategy.

immunology↗

bioRxiv2025

NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control.

One barrier to developing an HIV-1 cure is viral reservoirs persisting within B cell follicles of lymphatic tissues, partly due to failure of HIV-specific cytotoxic cells to express the follicular-homing receptor CXCR5. We evaluated HIV-specific CAR/CXCR5 NK cells armored with IL-15 and a PD-1 knockout for in vitro functionality. The safety and efficacy of these NK cells were assessed in vivo by treating ART-suppressed, HIV-infected-humanized-DRAGA mice following ART interruption. In vitro, CAR and control NK cells proliferated and killed HIV-Envelope-expressing cells. In vivo, there were no adverse health outcomes associated with cell infusions. CAR NK-treated animals had, on average, 1.88 times higher NK cell levels, and cells persisted up to 2 weeks longer than control NK-treated animals. At 56 days post-treatment, 62.5% of CAR NK-treated and 50% of control NK-treated groups had viral loads below the detection limit compared to 0% of the saline control group. Importantly, animals that had undetectable viral loads at 56 days post-treatment had earlier viral rebound post-ART interruption that coincided with high levels of NK cells, suggesting that timing of treatment with viral recrudescence was key to efficacy. This study demonstrates the promise of NK therapies for treating HIV infections.

bioengineering↗

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↗

bioRxiv2025

Mathematical model of CISH knockout predicts increased efficacy in tumor-infiltrating lymphocyte activation with synergistic gene editing

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cell therapy, where the lymphocytes of a cancer patients tumor are harvested, expanded in vitro using IL-2 stimulation, and then infused back into the patient[1], [2]. However, even with the use of TIL therapy, cancer cells can survive for various reasons, such as poor lymphocyte infiltration into tumors, chronic activation of the T cell receptor and the immunosuppressive tumor microenvironment[3]. Cytokine-inducible SH2-containing (CISH) protein is a negative regulator of T cell activation, and in a recent clinical trial was knocked out in TILs to improve TIL therapy efficacy[4]. A mechanistic signaling pathway model was developed to theoretically evaluate the efficacy of CISH knockout (CISH KO) in T cell activation and examine potential alternative target genes that can theoretically be targeted using multiplex gene-editing or drugs to further improve T cell activation and function[5]. Based on the results, CISH knockout increases the transcription of activation biomarkers IL-2 and TNF-, but also inhibitory biomarkers such as PD1 and FasL. Using global sensitivity analysis, we also found that GSK3B, which is responsible for the deactivation of NFAT, is also predicted to further increase T cell activation when knocked out. In addition, it was predicted that PDCD1, FAS and CTLA4 can be knocked out in combination with CISH to further enhance T cell activation and prevent exhaustion and apoptosis.

immunology↗

bioRxiv2025

Allogeneic Polyclonal CD38KO/CD38-CAR γδT Cells For The Treatment Of T Cell Malignancies

Relapsed and refractory T cell malignancies are associated with poor clinical outcomes. Autologous sources of {beta}T cells have been employed for chimeric antigen receptor (CAR) therapies to eliminate the potential for graft-vs-host disease (GvHD). However, the application of CAR-T therapy for T-ALL has been hindered by an inability to obtain sufficient healthy {beta}T cells from patients combined with fratricide due to concurrent antigen expression on normal T cells. Here, we genetically engineered polyclonal {gamma}{delta}T cells, which do not cause GvHD, as an allogeneic source for cancer immunotherapy targeting the pancancer antigen CD38. Utilizing a novel expansion protocol in combination with CRISPR/AAV gene editing, we developed CD38KO/CD38-CAR polyclonal {gamma}{delta}T cells that target T-ALL. Our editing strategy enabled site-directed, on-target insertion of the CD38-CAR transgene into the CD38 locus, with no evidence of significant random CAR DNA integration (as commonly seen with lentiviral CAR transduction) or chromatin abnormalities resulting from CRISPR editing. This enhanced targeting effectively mitigated fratricide through simultaneous CD38 disruption and CAR expression. We demonstrated the efficacy of the CD38KO/CD38-CAR {gamma}{delta}T cells in vitro across multiple patient-derived T-ALL samples collected at baseline and relapse. In vivo, a single injection of CD38KO/CD38-CAR {gamma}{delta}T cells without exogenous cytokine support resulted in potent anti-leukemic efficacy. Fratricide-resistant CD38KO/CD38-CAR polyclonal {gamma}{delta}T cells thus represent a promising off-the-shelf therapeutic platform for T cell malignancies and other CD38-expressing cancers. Key PointsO_LIHybrid pan-{gamma}{delta}TCR antibody/mbIL21-41BBL feeder expansion yields high-purity, polyclonal {gamma}{delta}T cells suitable for CRISPR/AAV editing. C_LIO_LIOn-target CD38-CAR knock-in with simultaneous CD38 knockout prevents fratricide and enables potent T-ALL killing in vitro and in vivo. C_LI

immunology↗

bioRxiv2020

Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding sanctuaries from antitumor immunity and optimizing T cell-related therapeutic strategies. To enhance T cell migration through complex microenvironments, we engineered nanotextured platforms that allowed us to define how the balance between T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues and characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubules instability, leading to increased Rho pathway-dependent cell contractility, promotes migration while clinically used microtubule-targeting chemotherapies profoundly decrease effective migration. Indeed, we show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. This suggests that engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

cancer biology↗

bioRxiv2019

SEMA4C is a novel target to limit osteosarcoma growth, progression, and metastasis

Semaphorins, specifically type IV, are important regulators of axonal guidance and have been increasingly implicated in poor prognoses in a number of different solid cancers. In conjunction with their cognate PLXNB family receptors, type IV members have been increasingly shown to mediate oncogenic functions necessary for tumor development and malignant spread. In this study, we investigated the role of semaphorin 4C (SEMA4C) in osteosarcoma growth, progression, and metastasis. We investigated the expression and localization of SEMA4C in primary osteosarcoma patient tissues and its tumorigenic functions in these malignancies. We demonstrate that overexpression of SEMA4C promotes properties of cellular transformation, while RNAi knockdown of SEMA4C promotes adhesion and reduces cellular proliferation, colony formation, migration, wound healing, tumor growth, and lung metastasis. These phenotypic changes were accompanied by reductions in activated AKT signaling, G1 cell cycle delay, and decreases in expression of mesenchymal marker genes SNAI1, SNAI2, and TWIST1. Lastly, monoclonal antibody blockade of SEMA4C in vitro mirrored that of the genetic studies. Together, our results indicate a multi-dimensional oncogenic role for SEMA4C in metastatic osteosarcoma and more importantly that SEMA4C has actionable clinical potential.

cancer biology↗

bioRxiv2018

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

genetics↗

bioRxiv2018

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

bioengineering↗

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↗