A robust platform for expansion and genome editing of primary human natural killer cells

Huang, Rih-Sheng; Lai, Min-Chi; Shih, Hsin-An; Lin, Steven

doi:10.1084/jem.20201529

Cited by 49 publications

(75 citation statements)

References 34 publications

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“…Recently another charge-based chemical method has been tried successfully to deliver CAR mRNA into non-dividing NK cells using a nucleofection approach that showed high efficiency ( 128 ). A specific advantage of using mRNA delivery system is the transient expression of protein by mRNA, thus avoids the risk of genome integration, least expensive to manufacture and savings of time ( 129 ).…”

Section: Delivery Systems To Engineer Nk Cellsmentioning

confidence: 99%

Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

et al. 2021

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Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

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Section: Delivery Systems To Engineer Nk Cellsmentioning

confidence: 99%

Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

et al. 2021

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“…CRISPR-Cas9 is being used as a gene editing tool to ameliorate the efficiency of CAR cell products [ 228 , 229 ] Based on this technology, a programmable single guide RNA (sgRNA) brings a Cas9 nuclease to a specific site of the genome for inducing double-strand breaks followed by integration of the desired gene cassette via endogenous DNA repair mechanisms, homologous recombination (HR) or non-homologous end-joining (NHEJ). The productivity of CRISPR-Cas9 depends on the applied format of CRISPR-Cas9 components, transfection method, and cell type [ 230 ]. The CRISPR-Cas9 elements can be introduced into cells in multiple formats such as DNA, RNA, and ribonucleoproteins (RNPs).…”

Section: Car Transfer Methodology Into Nk Cellsmentioning

confidence: 99%

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development

Dezfouli

Yazdi

Pockley

et al. 2021

Cells

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In recent years, cell-based immunotherapies have demonstrated promising results in the treatment of cancer. Chimeric antigen receptors (CARs) arm effector cells with a weapon for targeting tumor antigens, licensing engineered cells to recognize and kill cancer cells. The quality of the CAR-antigen interaction strongly depends on the selected tumor antigen and its expression density on cancer cells. CD19 CAR-engineered T cells approved by the Food and Drug Administration have been most frequently applied in the treatment of hematological malignancies. Clinical challenges in their application primarily include cytokine release syndrome, neurological symptoms, severe inflammatory responses, and/or other off-target effects most likely mediated by cytotoxic T cells. As a consequence, there remains a significant medical need for more potent technology platforms leveraging cell-based approaches with enhanced safety profiles. A promising population that has been advanced is the natural killer (NK) cell, which can also be engineered with CARs. NK cells which belong to the innate arm of the immune system recognize and kill virally infected cells as well as (stressed) cancer cells in a major histocompatibility complex I independent manner. NK cells play an important role in the host’s immune defense against cancer due to their specialized lytic mechanisms which include death receptor (i.e., Fas)/death receptor ligand (i.e., Fas ligand) and granzyme B/perforin-mediated apoptosis, and antibody-dependent cellular cytotoxicity, as well as their immunoregulatory potential via cytokine/chemokine release. To develop and implement a highly effective CAR NK cell-based therapy with low side effects, the following three principles which are specifically addressed in this review have to be considered: unique target selection, well-designed CAR, and optimized gene delivery.

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“…However, multiplexed gene editing poses a risk of inducing chromosomal translocation due to misligation of the Cas9-induced DSBs by the nonhomologous end-joining pathway. A simple PCR assay can help detect evidence of chromosomal translocation (Huang et al, 2021).…”

Section: Preparation Of Genome Editing Reagentsmentioning

confidence: 99%

“…Alternatively, Cas9 RNP can be electroporated into NK cells using a transfection system to also achieve high editing efficiency using a protocol described in Huang et al (2021).…”

Section: Preparation Of Natural Killer Cells For Nucleofectionmentioning

confidence: 99%

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

2021

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Natural killer (NK) cells are potent innate immune cells that provide the surveillance and elimination of infected, stressed, and malignant cells. The unique immune recognition mechanisms and functions of NK cells make them an attractive cell type for immunology research and adoptive immunotherapy. However, primary NK cells are challenging to culture ex vivo and lack efficient genetic tools, hindering the research of NK cells and the development of NK cell therapeutics. Here we describe methods for the freeze-thaw process, feeder-free ex vivo expansion, CRISPR-Cas9 genome editing, and functional characterizations of primary human NK cells. Our protocol enables ∼30-fold and ∼2000fold average expansion rates from 1 × 10 7 cryopreserved NK cells in 14 and 28 days, respectively. We also detail methods for CRISPR gene knockout and knockin by nucleofection of Cas9 ribonucleoproteins (RNP) and DNA repair templates. Gene knockout by Cas9 RNP nucleofection can be multiplexed to simultaneously target three genes. The CRISPR-edited cells can be cryopreserved and rethawed with high viability for future studies.

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A robust platform for expansion and genome editing of primary human natural killer cells

Cited by 49 publications

References 34 publications

Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development

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

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