Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Meta-Analysis of Incidence and Mortality of Firefighter Cancer: An Update on Emerging Science.

BACKGROUND: Firefighters are faced with a broad range of toxic exposures during their work, including known and suspected carcinogens. The current study is an update to the previously published meta-analysis of cancer risk among firefighters by Soteriades and colleagues, and focuses on studies published from 2008 to 2020. METHODS: A comprehensive search of the literature was conducted, including electronic databases and bibliographies of recently published papers. Analyses include stratification of studies conducted in the United States (US) versus other countries. Cancer incidence and mortality rates were compared to the relevant general population. Random effects models were used to calculate summary risk estimates and their 95% confidence intervals. RESULTS: A total of 24 studies were included in the meta-analysis. Among the 42 cancer types covered, incidence was associated with firefighting in US samples for colon, kidney, large intestine, pleura, and prostate cancer, as well as malignant melanoma. There was an increased incidence of Hodgkin's Disease and malignant melanoma and a significantly lower risk of kidney cancer for non-US samples. Significant cancer mortality estimates for US samples included oral/buccal/mouth, other parts of the buccal cavity, pharynx, colon, esophagus, large intestine, lung, Non-Hodgkin's Lymphoma, pancreas, pleura, rectum, and soft tissue sarcoma. No cancer had a significantly higher rate of mortality among non-US samples. CONCLUSIONS: The findings underscore the global cancer burden among firefighters, and indicate that geographically stratifying studies afford a more nuanced risk perspective. Further research should investigate why US firefighters exhibit higher cancer mortality rates compared to international counterparts.

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 Burkitt's 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.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Germline T cell receptor exchange results in physiological T cell development and function.

T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin (Msln), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac-targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. Together, our results support that the TRex methodology is an advanced tool to study physiological antigen-specific T cell behavior.

CRISPR-Cas9 base editors and their current role in human therapeutics.

BACKGROUND: Consistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification. METHODS: This review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes. RESULTS: CRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction. CONCLUSION: As such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.

Cerebellar granule neurons induce Cyclin D1 before the onset of motor symptoms in Huntington's disease mice.

Although Huntington's disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

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

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using 'digital' genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Clearance of variant Creutzfeldt-Jakob disease prions in vivo by the Hsp70 disaggregase system.

The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. Here we have tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so we used prion protein (PrP) transgenic Drosophila that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was downregulated in prion-exposed PrP Drosophila. We demonstrated that RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas overexpression mitigated toxicity. Strikingly, prion seeding activity in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 overexpression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease.

A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers.

B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.

A qualitative study to assess perceptions, barriers, and motivators supporting smokeless tobacco cessation in the US fire service.

While firefighters currently have low smoking rates, rates of smokeless tobacco (SLT) use among this population are remarkably high and substantially greater than similar occupational groups, and the general population. This study explored determinants associated with SLT use, barriers to cessation, and motivators for SLT cessation in the fire service. Key informant interviews were conducted in 23 career firefighters who were current (n = 14) and former (n = 9) SLT users from across the U.S. Discussions were recorded and independently coded according to questions and themes. Major themes that developed among firefighters regarding SLT use determinants included positive perceptions of SLT products, social influences from their peers and family members, acceptability of SLT use in the fire service, and a coping resource for job stress. Firefighters discussed several barriers to SLT cessation, including intrapersonal barriers such as SLT use habits and its dependency, concerns about withdrawal symptoms; and social-environmental barriers including lack of support from health and other services providers, and lack of enforcement of existing tobacco policies regarding SLT use. Firefighters also mentioned both internal and external motivators for cessation. Internal motivators included self-motivation and their health concerns while external motivators included friends and family support, incentives or rewards, and price of SLT products. Findings provide unique perspectives from firefighters on factors that influence SLT use and barriers and motivators to SLT cessation. These are insufficiently assessed and considered by the fire service organizations and their health care providers. Thus, the organizations must understand these issues in order to mitigate barriers and motivate the personnel to quit using SLT. Information gained from firefighters who were current and former SLT users can be used to develop an effective, culturally-tailored intervention that is acceptable to fire service personnel.

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 escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we 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 microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. 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. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

Development and beta test of a smokeless tobacco cessation program for firefighters.

INTRODUCTION: The prevalence of smokeless tobacco (SLT) use among firefighters is substantially higher than the general population and similar occupational groups. Despite the significant health risks associated with SLT and its impact on occupational readiness, there are no occupationally-tailored SLT education or treatment programs for the fire service. The purpose of this study was to beta test QUIT SPIT!, a self-help SLT cessation program that is culturally tailored for the US fire service and firefighters who are interested in quitting. METHODS: After development and tailoring the QUIT SPIT! SLT cessation program for firefighters, the feasibility and acceptability of the program were evaluated in a sample of eleven SLT-using firefighters who wanted to quit. The primary outcome was a 7-day point prevalence of SLT abstinence measured at 4 and 12 weeks post-enrollment follow-up assessments. RESULTS: Four firefighters reported having quit SLT (7-days point prevalence) at follow-up at 12 weeks. Those who did not achieve SLT abstinence reported reductions in frequency and quantity in SLT use and demonstrated a decrease in nicotine dependence. Firefighters also reported being satisfied with the QUIT SPIT! cessation program. CONCLUSIONS: The results provide strong support for the feasibility and acceptability of the QUIT SPIT! in SLT-using firefighters interested in quitting. The findings provide critical information about the next steps for further development and evaluation of the QUIT SPIT! program.

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

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.