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.

The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/ leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system could be used to identify new genes involved in lymphomagenesis/leukemogenesis. [Cancer Res 2009;69(21):8429-37]

Chimeric antigen receptor engineered T cell (CAR-T) immunotherapy has shownefficacy against a subset of hematological malignancies 1,2 , 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 alloreactivity 3-6 . 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 loci 7-10 . Moreover, it has been well documented that DSBs are toxic lesions that can drive genetic instability 11,12 .Alternatively, CRISPR/Cas9 base editors afford programmable enzymatic nucleotide conversion at targeted loci without induction of DSBs 13,14 . 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 ɑ constant (TRAC) locus, β-2 microglobulin (B2M), and programmed cell death 1 (PDCD1). Multiplex base edited T cells equipped with a CD19CAR 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.Base editing has been previously used to induce premature stop (pmSTOP) codons for gene knockout in mice and in mammalian cells [15][16][17][18] . However, we reasoned that splice site disruption could have several advantages over induction of pmSTOP codons (Supplemental Data 1). For instance, stop codon readthrough has been shown to occur at frequencies up to 31% in some genes, and can be promoted under conditions of cellular stress 19,20 . Splice site editing mitigates this concern as it alters gene processing at the RNA level 21 , which is less likely to be bypassed at the translational level. Additionally, current base editors do not produce strict C to T edits, with even the most recent base editors producing up to 25% non-target editing (C to G/A) 22 .

To study the oncogenic role of the NRAS oncogene (NRAS G12V ) in the context of acute myeloid leukemia (AML), we used a Vav promoter-tetracycline transactivator (Vav-tTA)-driven repressible TRE-NRAS G12V transgene system in Mll-AF9 knock-in mice developing AML. Conditional repression of NRAS G12V expression greatly reduced peripheral white blood cell (WBC) counts in leukemia recipient mice and induced apoptosis in the transplanted AML cells correlated with reduced Ras/Erk signaling. After marked decrease of AML blast cells, myeloproliferative disease (MPD)-like AML relapsed characterized by cells that did not express NRAS G12V . In comparison with primary AML, the MPD-like AML showed significantly reduced aggressiveness, reduced myelosuppression, and a more differentiated phenotype. We conclude that, in AML induced by an Mll-AF9 transgene, NRAS G12V expression contributes to acute leukemia maintenance by suppressing apoptosis and reducing differentiation of leukemia cells. Moreover, NRAS G12V oncogene has a cell nonautonomous role in suppressing erythropoiesis that results in the MPD-like AML show significantly reduced ability to induce anemia. Our results imply that targeting NRAS or RAS oncogene-activated pathways is a good therapeutic strategy for AML and attenuating aggressiveness of relapsed AML. IntroductionKelly and Gilliand have proposed that acute myeloid leukemia (AML) is induced by the cooperation of 2 general classes of mutations. 1 Class I mutations confer cell survival and proliferation advantages and generally result from mutations in genes encoding cell-signaling molecules like NRAS and FLT3. Class II mutations impair differentiation and subsequent apoptosis and, in general, result from mutations in genes encoding transcription factors or chromatin-modifying proteins such as AML1 or mixed lineage leukemia (MLL). Most studies to develop new anticancer drugs have focused on finding efficient inhibitors against the mutant products of class I oncogenes such as ABL, c-KIT, and FLT3. [2][3][4][5][6][7][8] Although previous studies showed that FLT3 inhibition effectively suppresses the cell growth of leukemia induced by the cooperation of an FLT3-activating mutation and an MLL translocation, 9,10 the appropriateness of class I oncogene inhibitors for AML therapy has not been well studied, particularly in leukemia induced in cooperation with a class II oncogene. Conditionally expressed transgenes in mice provide an ideal setting in which to address this issue. Studies have shown that continued expression of oncogenes such as c-Myc or Bcr/Abl is required for maintaining leukemia in transgenic mouse models. [11][12][13] Cancer cells in these models are said to have "oncogene addiction," because they die, differentiate, or become quiescent upon shutting off oncogene expression. 13 To study the functions of a class I mutation in AML, we investigated the role of an NRAS-activating mutation, NRAS G12V , in murine AML induced in combination with the Mll-AF9 oncogene.RAS mutants, which abnormally stimulate RAF/...

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Key Points• NRAS G12V maintains leukemia self-renewal in a genetically engineered murine model of AML.• NRAS G12V differentially regulates transcription and signaling among leukemic subpopulations.Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS G12V ). Using computational approaches to explore our gene-expression data sets, we found that NRAS G12V enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9-and Myb-dependent leukemia self-renewal geneexpression program. NRAS G12V was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS G12V -mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1 Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS G12V withdrawal. NRAS G12V maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction. (Blood. 2014;124(22):3274-3283)

Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.

BackgroundNeoantigen-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.MethodsTo 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.ResultsCISH 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.ConclusionsThese 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 RegistrationNCT04426669