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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 hyperactiveTc 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.

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., Base editors can inactivate splice sites or introduce stop codons into a gene sequence. Here the authors present SpliceR to design, rank, and test sgRNAs for efficient gene disruption in T cells.

Background Neoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL. Methods To explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing. Results CISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy. Conclusions These pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted. Trial Registration NCT04426669

Background Engineered immune cells hold tremendous promise for the treatment of advanced cancers. As the scale and complexity of engineered cell therapies increase, reliance on viral vectors for clinical production limits translation of promising new therapies. Here, we present an optimized platform for CRISPR/Cas9-targeted, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches, namely DNA-induced toxicity and low efficiency integration of large genetic cargos. Methods A systematic optimization of nucleic acid delivery, editing reagent composition, and culture protocol was performed to overcome DNA toxicity. Targeted knockin (KI) at AAVS1 and TRAC was compared across multiple vector configurations with genetic cargos ranging from 1 to 3 kilobases (kb) in size. Integration efficiency was measured by flow cytometry and sequencing. Off-target editing and integration were evaluated using GUIDE-seq and targeted locus amplification (TLA), respectively. Phenotype and function of non-virally and lentivirus engineered CAR-T cells was compared using flow cytometry, cytokine profiling and cytotoxicity assays. Results We identified a temporal window following T cell activation where transfection efficiency, cell-cycle-status, and cytosolic DNA sensor expression were optimal for targeted DNA integration and reduced toxicity. Within this window, we targeted a 1kb GFP reporter to the AAVS1 locus with an efficiency of ~45% using homologous recombination (HR). Efficiency was reduced to ~11% with a larger ~3kb TCR cassette targeted to the TRAC locus, consistent with previous reports.1–3 To improve large cargo integration we employed homology mediated end-joining (HMEJ) and short homology design (48bp vs. ~1kb for traditional HR).4 Using HMEJ, knockin of the 1kb GFP cassette at AAVS1 reached ~70%. Strikingly, integration of the 3kb TCR at TRAC reached ~50% using HMEJ. Additional optimization of the culture protocol doubled post-engineering survival and proliferation (up to ~35-fold expansion in 7 days). Non-virally engineered TRAC KI CAR-T cells were phenotypically and functionally equivalent to lentivirally engineered T cells in vitro. In vivo assays in xenograft models are underway and results will be presented. Conclusions Comprehensive, orthogonal optimization of parameters impacting nucleic acid delivery and DNA-toxicity in combination with novel modalities for integration achieved knockin of TCR and CAR cargo at efficiencies equivalent to that of current viral vector platforms without compromising expansion or function. Our protocol is suitable for clinical scale production under GMP conditions and offers an improved methodology over previous methods for non-viral engineering of human T cells. References Roth TL, Puig-Saus C, Yu R, Shifrut E, Carnevale J, Li PJ, Hiatt J, Saco J, Krystofinski P, Li H, Tobin V, Nguyen DN, Lee MR, Putnam AL, Ferris AL, Chen JW, Schickel J-N, Pellerin L, Carmody D, Alkorta-Aranburu G, Del Gaudio D, Matsumoto H, Morell M, Mao Y, Cho M, Quadros M, Gurumurthy CB, Smith, B, Haugwitz M, Hughes SH, Weissman JS, Schumann K., Esensten JH., May AP, Ashworth A., Kupfer G. M., Atma S., Greeley W. & Marson A. Reprogramming human T cell function and specificity with non-viral genome targeting. Naturedoi:10.1038/s41586-018-0326-5 Parker Autoimmune SN, Zuckerberg Biohub C, Francisco S. & Helen U. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat. Biotechnol. doi:10.1038/s41587-019-0325-6 Schober K, Muller TR, Gokmen F, Grassmann S, Effenberger M, Poltorak M, Stemberger C, Schumann K, Roth TL, Marson A. & Busch DH. Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Nature Biomedical Engineering 3, 974–984 ( 2019). Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, Weiss TJ, Kambakam S, Vollbrecht MK, Lan M, McKeighan KC, Levey J, Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien C-B, Balciunas D, Ekker SC, Clark KJ, Webber, BR, Moriarity BS, Solin SL, Carlson DF, Dobbs DL, McGrail M & Essner J. Efficient targeted integration directed by short homology in zebrafish and mammalian cells. Elife9, ( 2020).

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 PLCγ-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.

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.

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 PLCγ-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.

The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types., Multiplexed genome engineering with Cas9 can increase efficiency but also the risk of unintended alterations. Here the authors demonstrate the use of multiplexed base editors on primary T cells with reduced translocation frequency.

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.

Since the advent of large-scale, detailed descriptive cancer genomics studies at the beginning of the century, such as The Cancer Genome Atlas (TCGA), labs around the world have been working to make this data useful. Data like these can be made more useful by comparison with complementary functional genomic data. One new example is the application of CRISPR/Cas9-based library screening for cancer-related traits in cell lines. Such screens can reveal genome-wide suppressors of tumorigenesis and metastasis. Here we describe the use of widely available lentiviral libraries for such screens in cultured cell lines.

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 α constant (TRAC) locus, β-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.

MicroRNAs (miRNAs) are important players of post-transcriptional gene regulation. Individual miRNAs can target multiple mRNAs and a single mRNA can be targeted by many miRNAs. We hypothesized that miRNAs select and regulate their targets based on their own expression levels, those of their target mRNAs and triggered feedback loops. We studied the effects of varying concentrations of let-7a-7f and the miR-17-92 cluster plasmids on the reporter genes carrying either DICER- or cMYC -3′UTR in Huh-7 cells. We showed that let-7 significantly downregulated expression of DICER 3′UTR reporter at lower concentrations, but selectively downregulated expression of a cMYC 3′UTR reporter at higher dose. This miRNA dose-dependent target selection was also confirmed in other target genes, including CCND1, CDKN1 and E2F1. After overexpressing let-7a-7f or the miR-17-92 clusters at wide-ranging doses, the target genes displayed a nonlinear correlation to the transfected miRNA. Further, by comparing the expression levels of let-7a and miR-17-5p, along with their selected target genes in 3 different cell lines, we found that the knockdown dose of each miRNA was directly related to their baseline expression level, that of the target gene and feedback loops. These findings were supported by gene modulation studies using endogenous levels of miR-29, -1 and -206 and a luciferase reporter system in multiple cell lines. Finally, we determined that the miR-17-92 cluster affected cell viability in a dose-dependent manner. In conclusion, we have shown that miRNAs potentially select their targets in a dose-dependent and nonlinear fashion that affects biological function; and this represents a novel mechanism by which miRNAs orchestrate the finely tuned balance of cell function.

Osteosarcoma is the most common primary bone malignancy that affects adolescents. Around 30% of patients with localized osteosarcoma and 70% of patients with metastasis will experience treatment failure within 5 years of diagnosis. The complex biology of osteosarcoma and astounding genetic heterogeneity has made it challenging to identify effective new gene targets and therapeutic agents. Our studies found an overall overexpression of a microRNA cluster, miR-17-92 microRNAs in human primary osteosarcoma compared to normal bone. We learned that upregulation of this miR-17-92 cluster simultaneously silences a suite of key tumor suppressors. Using data from a novel spontaneous osteosarcoma mouse model and genetic screen, we discovered miR-17-92 targets, such as PTEN, PTRPD and SRGAP2, which are potential tumor suppressor genes. Specifically blocking miR-17-92 function in osteosarcoma cells reduced their migration and ability to grow larger tumors in immunodeficient mice compared to the controls. Also, knockdown of miR-17-92 cluster microRNAs led to increase in the levels of PTPRD and SRGAP2 in cells as well as tumors; further suggesting that miR-17-92 is targeting these genes. We also performed gain-of-function of miR-17-92 studies in a poorly aggressive osteosarcoma cell line and found that overexpression of miR-17-92 leads to ability to grow in an anchorage independent manner and form tumors in immunodeficient mice, both features that are lacking in the parental line. Ongoing RNA sequencing studies on miR-17-92 target transcripts in osteosarcoma cells, and functional analyses of miR-17-92 deletion mutants, will be presented. In an attempt to target miR-17-92 miRNA expression, we have tested small molecules. Our data suggests that triptolide, a diterpenoid epoxide, inhibits MYC expression and downregulates miR-17-92 miRNAs resulting in upregulation of several tumor suppressor driver proteins including PTEN, PTPRD, and SRGAP2. Together, our data suggests that upregulation of miR-17-92 miRNAs contributes to osteosarcoma progression and triptolide inhibits miR-17-92 expression. These data have implications for how sarcomas develop in general and suggest a new way to treat cancer by targeting microRNAs using small molecules. Citation Format: Jyotika Varshney, Nicholas J. Slipek, John Osborne, Adrian Chang, Anne E. Sarver, Ingrid Cornax, Gerry M. O’ Sullivan, Subbaya Subramanian, David A. Largaespada. The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1097.

Patients with sickle cell disease have unrelenting hemolysis leading to the release of hemoglobin and heme into the vasculature that promote oxidative stress, inflammation, vaso-occlusive pain crises, ischemia-reperfusion injury and organ damage. Induction of the cytoprotective heme metabolizing enzyme heme oxygenase-1 (HO-1), the iron-binding protein ferritin heavy chain or administration of the HO-1 metabolite CO induces cytoprotective responses that inhibit oxidative stress, inflammation, vaso-occlusion and organ damage in transgenic sickle mice expressing human βS globins. The master regulator of these anti-oxidative and cytoprotective responses is nuclear factor erythroid 2-related factor (Nrf2). Nrf2 activity is controlled, in part, by the cytosolic protein, kelch-like ECH-associated protein 1 (Keap1). Nrf2 is anchored in the cytoplasm through binding with Keap1 which results in its ubiquitination and subsequent proteosomal degradation. Upon exposure to stress stimuli, such as reactive oxygen species and electrophiles, Nrf2 is stabilized and able to translocate to the nucleus where it trans-activates target genes that possess an antioxidant responsive element (ARE) in their promoter regions. Recently the FDA and their European and Canadian counterparts approved dimethyl fumarate (Tecfidera) for the treatment of relapsing multiple sclerosis. Dimethyl fumarate (DMF) and its metabolite monomethyl fumarate alkylate a critical reactive thiol Cys-151 on Keap1 causing release of Nrf2, nuclear localization and activation of cellular anti-oxidant and cytoprotective responses. Based on our previous results showing cytoprotection through HO-1 and it products, we evaluated DMF responses in NY1DD transgenic sickle mice. DMF (15mg/kg) or vehicle (0.08% methyl cellulose) was administered by oral gavage BID X 3 days to NY1DD mice. On the first day of treatment, mice were implanted with dorsal skin-fold chambers. One hour after the last treatment, 20-30 flowing subcutaneous venules were selected and mapped in the dorsal skin-fold chamber window followed by infusion of heme (3.2 µmols/kg) into the tail vein. All of the selected venules were re-examined at 1 and 4 hours post-infusion and the number of static (no flow) venules were counted and expressed as percent stasis. After the 4 hour stasis measurement, blood was collected and organs were removed and flash frozen. In sickle mice treated with vehicle, microvascular stasis was 29% and 24% at 1 and 4 hours, respectively ( Fig. 1 ). In contrast, in sickle mice treated with DMF, stasis was 6% and 4% at 1 and 4 hours (p