Dataset Information

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

ABSTRACT: Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

SUBMITTER: Naeimi Kararoudi M

PROVIDER: S-EPMC9243630 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Naeimi Kararoudi Meisam M Likhite Shibi S Elmas Ezgi E Yamamoto Kenta K Schwartz Maura M Sorathia Kinnari K de Souza Fernandes Pereira Marcelo M Sezgin Yasemin Y Devine Raymond D RD Lyberger Justin M JM Behbehani Gregory K GK Chakravarti Nitin N Moriarity Branden S BS Meyer Kathrin K Lee Dean A DA

Cell reports methods 20220613 6

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus con ...[more]

PMID: 35784645

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Project description:Chimeric antigen receptor (CAR) T and CAR NK cell therapies opened new avenues for cancer treatment. Although original successes of CAR T and CAR NK cells for the treatment of hematological malignancies were extraordinary, several obstacles have since been revealed, in particular their use for the treatment of solid cancers. The tumor microenvironment (TME) is competing for nutrients with T and NK cells and their CAR-expressing counterparts, paralyzing their metabolic effective and active states. Consequently, this can lead to alterations in their anti-tumoral capacity and persistence in vivo. High glucose uptake and the depletion of key amino acids by the TME can deprive T and NK cells of energy and building blocks, which turns them into a state of anergy, where they are unable to exert cytotoxic activity against cancer cells. This is especially true in the context of an immune-suppressive TME. In order to re-invigorate the T, NK, CAR T and CAR NK cell-mediated antitumor response, the field is now attempting to understand how metabolic pathways might change T and NK responses and functions, as well as those from their CAR-expressing partners. This revealed ways to metabolically rewire these cells by using metabolic enhancers or optimizing pre-infusion in vitro cultures of these cells. Importantly, next-generation CAR T and CAR NK products might include in the future the necessary metabolic requirements by improving their design, manufacturing process and other parameters. This will allow the overcoming of current limitations due to their interaction with the suppressive TME. In a clinical setting, this might improve their anti-cancer effector activity in synergy with immunotherapies. In this review, we discuss how the tumor cells and TME interfere with T and NK cell metabolic requirements. This may potentially lead to therapeutic approaches that enhance the metabolic fitness of CAR T and CAR NK cells, with the objective to improve their anti-cancer capacity.

Dataset Information

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Publications

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Similar Datasets

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