Biology subjects

Moriarity, B. S.

Publications and source records attributed to Moriarity, B. S..

At least 19 recordsLinked to original sources

bioRxiv2025

A Singular Base Editing Platform for Polyfunctional Multiplex Engineering of Immune Cells

Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand break induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knock in (KI), we expand the use of cytosine and adenine base editor (ABE) nickase activity to stimulate homology-directed repair (HDR) and insert clinically relevant chimeric antigen receptors (CARs) into specific loci. Through a novel sgRNA design strategy and a recombinant adeno-associated virus (rAAV) delivered DNA template, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19-, CD33-, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3{varepsilon}/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines, and a suppressive 3D spheroid culture model. This efficient engineering process of Iterative Nicking for Synchronous Engineered Reprogramming of T cells (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.

bioengineering↗

bioRxiv2025

CISH, a novel intracellular immune checkpoint, in comparison and combination to existing and emerging cancer immune checkpoints

Over the past decade, Immuno-Oncology has largely focused on blocking inhibitory surface receptors like PD-1 to enhance T cell anti-tumor activity. However, intracellular immune checkpoints such as CISH, which function independently of tumor-expressed ligands, offer powerful and previously untapped therapeutic potential. As a downstream regulator of TCR signaling, CISH controls T cell activation, expansion, and neoantigen reactivity. Though historically considered undruggable, recent advances in CRISPR engineering have enabled functional interrogation of these targets. We demonstrate that CISH deletion enhances T cell activation and anti-cancer functions more effectively than other emerging intracellular checkpoints. In CAR-T cells, CISH inactivation significantly increased sensitivity to tumor antigen, enabling robust recognition and killing even at low antigen levels, conditions that often lead to treatment failure with conventional T cell therapies, mirroring antigen escape scenarios seen in solid tumors. Our findings further validate CISH as a potent and druggable intracellular checkpoint capable of boosting anti-tumor T cell responses across diverse cancer types, independent of PD-L1 status. The underlying mechanisms of CISH inhibition may help explain the positive outcomes reported in recent clinical studies of this approach in solid tumor immunotherapy.

cancer biology↗

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↗

bioRxiv2024

Engineering Memory T Cells as a platform for Long-Term Enzyme Replacement Therapy in Lysosomal Storage Disorders

Enzymopathy disorders are the result of missing or defective enzymes. Amongst these enzymopathies, mucopolysaccharidosis type I, is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), ultimately causes toxic build-up of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS). Human memory T cells (Tm) migrate throughout the bodys tissues and can persist for years, making them an attractive approach for cellular-based, systemic enzyme replacement therapy. Here, we tested genetically engineered, IDUA-expressing Tm as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm take up residence in nearly all tested tissues, producing IDUA and leading to metabolic correction of GAG levels in the heart, lung, liver, spleen, kidney, bone marrow, and the CNS. Our study indicates that genetically engineered Tm holds great promise as a platform for cellular-based enzyme replacement therapy for the treatment of mucopolysaccharidosis type I and potentially many other enzymopathies and protein deficiencies.

bioengineering↗

bioRxiv2024

Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing

Natural killer (NK) cells unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate IL-15 armored CD19 CAR-NK cells with significantly improved functionality in a highly suppressive model of Burkitts lymphoma both in vitro and in vivo. The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

bioengineering↗

bioRxiv2022

In vivo functional genomics identifies essentiality of potassium homeostasis in medulloblastoma

The identification of cancer maintenance genes--driver genes essential to tumor survival--is fundamental for developing effective cancer therapy. Transposon-based insertional mutagenesis screens can identify cancer driver genes broadly but not discriminate maintenance from progression or initiation drivers, which contribute to cancer phenotypes and tumorigenesis, respectively. We engineered a nested, double-jumping transposon system to first dysregulate gene expression during tumorigenesis and then restore gene expression following tumor induction, allowing for genome-wide screening of maintenance essentiality in vivo. In a mouse model of medulloblastoma, the most common pediatric malignancy, insertion and remobilization of this nested transposon uncovers potassium channel genes as recurrent maintenance drivers. In human medulloblastoma, KCNB2 is the most overexpressed potassium channel across Group 3, Group 4, and SHH subgroups, and Kcnb2 knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives proliferative expansion of medulloblastoma-propagating cells. Thus, our novel transposon system reveals potassium homeostasis as essential to tumor maintenance through biomechanical modulation of membrane signaling.

cancer biology↗

bioRxiv2022

Indirect CRISPR screening with photoconversion revealed key factors of drug resistance with cell-cell interactions

Comprehensive screenings to clarify indirect cell-cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell-cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to determine the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell- cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell-cell interactions. This screening method could reveal new mechanisms in all kinds of cell-cell interactions by revealing live phenotype-inducible cells, and it could be a new platform for discovering new targets of drugs for conventional chemotherapies.

cancer biology↗

bioRxiv2022

Ablation of SYK kinase from primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production

Cytomegalovirus (CMV) infection alters natural killer (NK) cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the Fc receptor {gamma} chain (Gene: FCER1G, FcR{gamma}), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (Fc{gamma}RIIIA) signaling apparatus, which includes FcR{gamma}, CD3{zeta}, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcR{gamma} chain caused a modest increase in TNF production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcR{gamma} or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3{zeta} chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.

immunology↗

bioRxiv2022

Myosin Heavy Chain Converter Domain Mutations Drive Early-Stage Changes in Extracellular Matrix Dynamics in Hypertrophic Cardiomyopathy

More than 60% of hypertrophic cardiomyopathy (HCM)-causing mutations are found in the gene loci encoding cardiac myosin-associated proteins including myosin heavy chain (MHC) and myosin binding protein C (MyBP-C). Moreover, patients with more than one independent HCM mutation may be at increased risk for more severe disease expression and adverse outcomes. However detailed mechanistic understanding, especially at early stages of disease progression, is limited. To identify early-stage HCM triggers, we generated single (MYH7 c.2167C>T [R723C] with a known pathogenic significance in the MHC converter domain) and double (MYH7 c.2167C>T [R723C]; MYH6 c.2173C>T [R725C] with unknown significance) myosin gene mutations in human induced pluripotent stem cells (hiPSCs) using a base-editing strategy. Cardiomyocytes (CMs) derived from hiPSCs with either single or double mutation exhibited phenotypic characteristics consistent with later-stage HCM including hypertrophy, multinucleation, altered calcium handling, metabolism and arrhythmia. We then probed mutant CMs at time points prior to the detection of known HCM characteristics. We found MYH7/MYH6 dual mutation dysregulated extracellular matrix (ECM) remodeling, altered integrin expression, and interrupted cell-ECM adhesion by limiting the formation of focal adhesions. These results point to a new phenotypic feature of early-stage HCM and reveal novel therapeutic avenues aimed to delay or prohibit disease onset.

bioengineering↗

bioRxiv2021

Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a significant bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to cGMP compliant manufacturing and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.

bioengineering↗

bioRxiv2021

Genetic editing of CISH enhances T cell effector programs independently of immune checkpoint cell surface ligand expression

PD-1 acts as a negative regulator of T cell-mediated immune responses in the setting of persistent antigen expression, including cancer and chronic pathogen infections. Antibody-mediated blockade of the PD-1/PD-L1 axis benefits a subset of patients with highly immunogenic malignancies; however, many patients fail to respond due to a requirement for expression of the cell surface ligand PD-L1 within the tumor microenvironment. CISH is a member of a new class of intra-cellular immune checkpoint molecules that function downstream of the T cell receptor to regulate antigen-specific effector functions, including reactivity to cancer neoantigens. Herein, we employed multiplex CRISPR editing of primary human T cells to systematically compare the function of CISH deletion relative to PDCD1 (the gene encoding PD-1) and/or VSIG9 (the gene encoding TIGIT) in a model of neoantigen-mediated cancer cell cytolysis. PD-1 and TIGIT disruption enhanced cytolytic activity exclusively in the setting of high PD-L1 expression. In contrast, CISH inactivation enhanced antigen-specific cytolysis of tumor cells regardless of PD-L1 expression, including outperforming PD-1 and TIGIT disruption even in the presence of high PD-L1 tumor cells. Furthermore, we observed a synergistic increase in tumor cell killing when CISH and PD-1 or TIGIT are inactivated in combination, supporting the notion that these immune checkpoints regulate non-redundant pathways of T cell activation. Together, these data demonstrate that the intra-cellular immune checkpoint protein CISH can potentially enhance anti-tumor responses against a broad range of cancer types regardless of PD-L1 biomarker status.

cancer biology↗

bioRxiv2021

Non-Viral Engineering of CAR-NK and CAR-T cells using the Tc Buster Transposon System™

Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome. Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function. Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

bioengineering↗

bioRxiv2021

Splice-specific lyn knockout mice reveal a dominant function of LynB in preventing autoimmunity

The unique roles of the Src-family kinases LynA and LynB in immune activating and inhibitory signaling have eluded definition. Here we report that LynB, the shorter splice product of lyn, carries the dominant immunosuppressive function. We used CRISPR/Cas9 gene editing to constrain lyn splicing and expression to a single product: LynAKO or LynBKO mice. While activities of both isoforms regulate homeostatic Lyn expression, only LynB protects against autoimmune disease. LynBKO monocytes and dendritic cells are TLR4-hyper-responsive, and TLR4 expression increases with age in LynBKO myeloid and B cells. These changes are accompanied by the development of an inflammatory disease that resembles human systemic lupus erythematosus (SLE). The interplay between LynB and TLR4 likely underlies the autoimmunity risk associated with LYN hypomorph and TLR4 hypermorph alleles.

immunology↗

bioRxiv2020

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLC{gamma}-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

immunology↗

bioRxiv2020

CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary cells

Base editors allow for precise nucleotide editing without the need for genotoxic double-stranded breaks. Prior work has used base editors to knockout genes by introducing premature stop codons or by disrupting conserved splice-sites, but no direct comparison exists between these methods. Additionally, while base editor mediated disruption of splice sites has been used to shift the functional isoform pool, its utility for gene knockout requires further validation. To address these needs, we developed the program SpliceR (z.umn.edu/spliceR) to design cytidine-deaminase base editor (CBE) and adenosine-deaminase base editor (ABE) splice-site targeting guides. We compared the splice-site targeting and premature stop codon introduction in a knockout screen against the TCR-CD3 immune synapse in primary human T-cells. Our data suggests that 1) the CBE, BE4 is more reliable than the ABE, ABE7.10 for splice-site targeting knockout and 2) for both CBEs and ABEs, splice-donor targeting is the most reliable approach for base editing induced knockout.

bioengineering↗

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↗

bioRxiv2018

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

Chimeric antigen receptor engineered T cell (CAR-T) immunotherapy has shown efficacy against a subset of hematological malignancies1,2, yet its autologous nature and ineffectiveness against epithelial and solid cancers limit widespread application. To overcome these limitations, targeted nucleases have been used to disrupt checkpoint inhibitors and genes involved in alloreactivity3-6. However, the production of allogeneic, \"off-the-shelf\" T cells with enhanced function requires multiplex genome editing strategies that risk off-target effects, chromosomal rearrangements, and genotoxicity due to simultaneous double-strand break (DSB) induction at multiple loci7-10. Moreover, it has been well documented that DSBs are toxic lesions that can drive genetic instability11,12. Alternatively, CRISPR/Cas9 base editors afford programmable enzymatic nucleotide conversion at targeted loci without induction of DSBs13,14. We reasoned this technology could be used to knockout gene function in human T cells while minimizing safety concerns associated with current nuclease platforms. Through systematic reagent and dose optimization, we demonstrate highly efficient multiplex base editing and consequent protein knockout in primary human T cells at loci relevant to the generation of allogeneic CAR-T cells including the T cell receptor constant (TRAC) locus, {beta}-2 microglobulin (B2M), and programmed cell death 1 (PDCD1). Multiplex base edited T cells equipped with a CD19 CAR killed target cells more efficiently; and importantly, both DSB induction and translocation frequency were greatly reduced compared to cells engineered with Cas9 nuclease. Collectively, our results establish a novel multiplex gene editing platform to enhance both the safety and efficacy of engineered T cell-based immunotherapies.

genetics↗

bioRxiv2018

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Tumors can evade natural killer (NK) cells by activating inhibitory pathways. We therefore have developed a highly efficient CRISPR/Cas9-based method for editing the genome of peripheral blood human NK cells (PB-NKs) to knock out ADAM17 and PD1 or knock-in genes using recombinant AAV6. Our method allows editing of PB-NKs at efficiencies reaching 90%, equivalent to methods reported for primary human T cells. Moreover, we demonstrate that ADAM17 and PD1 KO PB-NKs have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Our platform represents a feasible method for generating engineered NK cells as a universal therapeutic for cancer immunotherapy.

immunology↗