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Harnessing CRISPR and AAV for CAR transduction into human primary NK cells leads to improved anti-AML activity

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4 min read

Natural killer (NK) cells are known to kill virus-infected cells as well as detect and control early signs of cancer. Thanks to these innate abilities, this type of white blood cell (WBC) has garnered a lot of interest - from the biomedical community - as a serious candidate for cancer immunotherapy. In fact, human primary NK cells have shown great promise in clinical trials by revealing a high safety profile. They have also demonstrated evidence of clinical benefit especially for patients suffering from acute myelogenous leukemia (AML), a type of cancer in which the bone marrow produces a large amount of abnormal blood cells.

Unlike successes seen with other immune effector cells, genetically modifying NK cells proves to be a rather challenging task. Methods previously explored - for engineering these cells - often led to relatively low transductions rates, high cell death and/or loss of transgene expression after expansion. Now, thanks to investigators at Nationwide Children’s Hospital and co-workers, the secret to facilitating a precise and highly controllable manipulation of NK cell genetic expressions has finally been uncovered!

This first-of-its-kind study was led by Dean Lee, MD, PhD (Director of the Cellular Therapy and Cancer Immunology Program at Nationwide Children’s and The Ohio State University Comprehensive Cancer Center) and Meisam Naeimi Kararoudi, DVM, PhD (Director of the CRISPR/Gene Editing Core and Principal Investigator in the Center for Childhood Cancer and Blood Diseases at Nationwide Children’s). The results were published in Cell Reports Methods.


Graphical abstract Nationwide paper

Graphical abstract retrieved from publication.



Targeting a safe harbor-locus for gene insertion in human primary NK cells by coupling CRISPR and AAV technique

In this publication, the authors describe a highly efficient approach that combines CRISPR (Cas9/RNP) and adeno-associated viral vectors (AAV) to allow site-directed gene insertion of CD33-specific CARs (i.e., an AML-specific CAR) into human primary NK cells.

By leveraging AAV as a therapeutic delivery vehicle, the researchers tested out different DNA repair mechanisms, homology arm lengths and virus concentrations. Of note, Cas9/RNP electroporation was accompanied by a DNA template encoding a transgene (with or without homology arms for Cas9 targeting) that was delivered using a single-stranded or self-complementary AAV6. In the end, the approach was applied and validated in primary human NK cells.

Integration site analysis of the transgene in AAVS1 locus via TLA-based solutions

Our proprietary genomic-identity assays found that the vector has integrated as intended in human chromosome 19: 55,115,754–55,115,767, which is in intron 1 of PPP1R12C. (Fig. 1)


Figure 1. Genome-wide coverage plot generated by TLA.
The chromosomes are displayed on the y-axis and the chromosomal position are shown on the x-axis.
Here, a single integration site was identified above background (encircled in blue).


Other integration sites were also observed between chr19: 55,115,155–55,116,371, which is also in intron 1 of PPP1R12C (data not shown here, please refer to the paper). Our experts also found no indication of a dominant secondary off-target integration site.

Furthermore, 1 sequence variant and 4 structural variants were picked up. The frequency of detection suggests that this variant was present within the AAV6 vector itself.

In sum, TLA-data revealed high prevalence of vector integration at the targeted location, with low-level random integrations identified throughout the genome.

CRISPR and AAV open the door to a wide range of CAR-NK cell therapies

In short, Dr. Kararoudi et al. optimized and validated a new approach for site-directed gene insertion of CD33-specific CARs into primary human NK cells. Taken together, their experimental results showed that:

  1. CAR transduction was highly efficient
  2. Stable expression was retained even after expansion
  3. These transgene-modified human primary NK cells improved efficacy against AML targets compared to unmodified NK cells
Therefore, this novel method has wide potential applications in cancer immunotherapy and this platform has high utility for studying NK cell biology.

Reprogramming human T cells via a viral or non-viral approach

To date, various strategies have been resorted to in order to reprogram human T cells. In fact, DNA template can be provided via either a viral (e.g. AAV) and non-viral approach (e.g. single-stranded or double-stranded DNA template electroporated along with Cas9/RNP). With over a decade of experience, our in-house experts have accumulated substantial know-how when it comes to performing comprehensive genetic characterization and QC on a variety of gene editing tools.

If you’re working in the area of Cell & Gene Therapy, make sure to also check out these 2 blogs:

A rapid and versatile non-viral CRISPR-based strategy to reprogram T cells

This study was led by Alexander Marson, MD, PhD, Director of the Gladstone-UCSF Institute of Genomic Immunology, and Associate Professor in the UCSF Department of Medicine


Memorial Sloan Kettering Cancer Center targets a CAR to the TRAC locus to  enhance tumor rejection

This study was led by Justin Eyquem, PhD, Assistant Professor at UCSF and Michel Sadelain, MD, PhD, Director of the Center for Cell Engineering at MSK.


If you would like to know more about how we could support your cell therapy work, please contact us


Story source:

Naeimi Kararoudi M, Likhite S, Elmas E, Yamamoto K, Schwartz M, Sorathia K, de Souza Fernandes Pereira M, Sezgin Y, Devine RD, Lyberger JM, Behbehani GK, Chakravarti N, Moriarity BS, Meyer K, Lee DA. Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV. Cell Rep Methods. 2022 Jun 13;2(6):100236. doi: 10.1016/j.crmeth.2022.100236. PMID: 35784645; PMCID: PMC9243630.

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