non-viral-engineering-primary-human-t-cells-via-homology-mediated-end

Non-Viral Engineering of Primary Human T Cells <em>via</em> Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) ...

University of Minnesota

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.

CRISPR/Cas9-Based Positive Screens for Cancer-Related Traits.

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.

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

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

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.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma.

We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas (OSAs) using the Sleeping Beauty (SB) transposon system. Here, we followed up investigating the genetic role of ZNF217 in OSA initiation and progression through establishment of a novel genetically engineered mouse model (GEMM), in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of OSA transformation including proliferation, cell motility, anchorage independent growth, and ultimately promoting OSA growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling. Pharmacological blockade of AKT signaling with nucleoside analogue triciribine (TCN) in ZNF217+ orthotopically-injected OSA cell lines reduced tumor growth and metastasis. Our data demonstrate that TCN treatment may be a relevant and efficacious therapeutic strategy for OSA patients with ZNF217+ and p-AKT rich tumors. With the recent revitalization of TCN for clinical studies in other solid cancers (PTX-200, Prescient Therapeutics), our study provides rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ OSA.

Implication of ZNF217 in accelerating tumor development and therapeutically targeting ZNF217-induced PI3K-AKT signaling for the treatment of metastatic osteosarcoma.

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.

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

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.

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

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.

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

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.

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

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  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  and . 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.

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

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.

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

Enzymopathy disorders are the result of missing or defective enzymes. Among these enzymopathies, mucopolysaccharidosis type I is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), which ultimately causes toxic buildup 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 (Tm) cells migrate throughout the body's 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 cells as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm cells leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm cells 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, although only minimal improved cognition was observed. Our study indicates that genetically engineered Tm cells hold 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.

Engineering memory T cells as a platform for long-term enzyme replacement therapy in lysosomal storage disorders.

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.

Article	Year
CRISPR/Cas9-Based Positive Screens for Cancer-Related Traits. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30542997	2019
SEMA4C is a novel target to limit osteosarcoma growth, progression, and metastasis. Oncogene\| PubMed ID: 31582836	2020
Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors. Nature communications\| PubMed ID: 31745080	2019
Author Correction: Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors. Nature communications\| PubMed ID: 31811147	2019
PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma. Bone\| PubMed ID: 32251854	2020
Implication of ZNF217 in accelerating tumor development and therapeutically targeting ZNF217-induced PI3K-AKT signaling for the treatment of metastatic osteosarcoma. Molecular cancer therapeutics\| PubMed ID: 32999043	2020
CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells. Nature communications\| PubMed ID: 33893286	2021
Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade. Med (New York, N.Y.)\| PubMed ID: 36007524	2022
A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes. International journal of molecular sciences\| PubMed ID: 36077152	2022
Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair. Nature biomedical engineering\| PubMed ID: 38092857	2024
Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing. bioRxiv : the preprint server for biology\| PubMed ID: 38496503	2024
Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 38627969	2024
Engineering memory T cells as a platform for long-term enzyme replacement therapy in lysosomal storage disorders. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 39367605	2024
Precision enhancement of CAR-NK cells through non-viral engineering and highly multiplexed base editing. Journal for immunotherapy of cancer\| PubMed ID: 40341025	2025

Nicholas J. Slipek

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