Ex Vivo Expansion and CRISPR‐Cas9 Genome Editing of Primary Human Natural Killer Cells

Huang, Rih-Sheng; Lai, Min-Chi; Lin, Steven

doi:10.1002/cpz1.246

Cited by 3 publications

(1 citation statement)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nowadays, with the rapid progress of available remedies for cancer treatments, patients are adequate to live with the consequences of cancer. NK cells have been regarded as the first line of innate immune cells, which are involved in providing the surveillance and elimination of the stressed, infected, damaged, and malignant cells [90,91]. NK cell-based cancer immunotherapy has largely benefited the treatments of patients for longer living and better outcomes, which also supplies new preferences for cancer administration and helps improve decisions about optimal treatment.…”

Section: Discussionmentioning

confidence: 99%

Natural Killer Cells for Cancer Immunotherapy: Opportunities and Challenges

Zhang¹,

Feng²,

Han³

et al. 2023

Natural Killer Cells - Lessons and Challenges

View full text Add to dashboard Cite

Natural killer (NK) cells are advantaged immune cells and play a pivotal role in both innate and adaptive immune responses. To date, autogenous and allogenic NK cells have been generated from a variety of origins, including perinatal blood (e.g., umbilical cord blood and placental blood), peripheral blood, and even stem cells (hematopoietic stem cells and pluripotent stem cells). NK cells function mainly via antibody-dependent cell-mediated cytotoxicity (ADCC), direct cytolytic effect, and paracrine effects (e.g., IFN-γ, GM-CSF, granzyme, and perforin). Distinguishing from the adaptive immunizing cells (e.g., T and B lymphocytes), NK cells, and chimeric antigen receptor-transduced NK (CAR-NK), cell-based cytotherapy is adequate to fulfill the biofunction of eliminating pathogenic infection, combating hematological malignancies and metastatic solid tumors, and delaying aging. In this chapter, we mainly focus on the state-of-the-art renewal of NK cell-based cytotherapy for cancer immunosurveillance and immunotherapy from the view of high-efficient in vitro preparation (e.g., candidate cell sources and ex vivo cultivation) and preclinical and clinical investigation. Furthermore, we also figure out the promising prospects and the concomitant challenges of NK cell-based remedies for cancer management in future, which will collectively benefit the development of NK cell-based cancer immunotherapy in future.

show abstract

Section: Discussionmentioning

confidence: 99%

Natural Killer Cells for Cancer Immunotherapy: Opportunities and Challenges

Zhang¹,

Feng²,

Han³

et al. 2023

Natural Killer Cells - Lessons and Challenges

View full text Add to dashboard Cite

show abstract

C-JUN overexpressing CAR-T cells in acute myeloid leukemia: preclinical characterization and phase I trial

Zuo,

Li,

Sun

et al. 2024

Nat Commun

View full text Add to dashboard Cite

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function, accompanied by impaired antigen receptor downstream calcium, ZAP70, ERK, and C-JUN signaling, compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN, but not other antigen receptor downstream components, maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation, independent of anti-exhaustion. We conduct an open-label, non-randomized, single-arm, phase I trial of C-JUN-overexpressing CAR-T in AML (NCT04835519) with safety and efficacy as primary and secondary endpoints, respectively. Of the four patients treated, one has grade 4 (dose-limiting toxicity) and three have grade 1–2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus, overexpressing C-JUN endows CAR-T efficacy in AML.

show abstract

Interrogation of human microglial phagocytosis by CRISPR genome editing

2023

Self Cite

View full text Add to dashboard Cite

BackgroundMicroglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3.MethodsWe performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aβ and glioblastoma phagocytosis assays.ResultsOur platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aβ1-42 peptides. Confocal microscopy further confirmed the localization of Aβ1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells.ConclusionCRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aβ and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

show abstract

Ex Vivo Expansion and CRISPR‐Cas9 Genome Editing of Primary Human Natural Killer Cells

Cited by 3 publications

References 20 publications

Natural Killer Cells for Cancer Immunotherapy: Opportunities and Challenges

Natural Killer Cells for Cancer Immunotherapy: Opportunities and Challenges

C-JUN overexpressing CAR-T cells in acute myeloid leukemia: preclinical characterization and phase I trial

Interrogation of human microglial phagocytosis by CRISPR genome editing

Contact Info

Product

Resources

About