scholarly journals Highly efficient site-directed gene insertion in primary human natural killer cells using homologous recombination and CRISPaint delivered by AAV

2019 ◽  
Author(s):  
Meisam Naeimi Kararoudi ◽  
Shibi Likhite ◽  
Ezgi Elmas ◽  
Maura Schwartz ◽  
Kathrin Meyer ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Human Peripheral Blood ◽  
Genetic Material ◽  
Gene Insertion ◽  
Ribonucleoprotein Complexes ◽  
Large Numbers ◽  
Human Natural Killer Cells ◽  
Efficient Vector ◽  
Strong Antitumor Activity

AbstractHuman peripheral blood natural killer (NK) cells have strong antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. However, genetic modification of NK cells has been challenging due to the high expression of innate sensing mechanisms for viral nucleic acids. Recently, we described an efficient vector-free method using Cas9/ribonucleoprotein complexes for gene deletion in NK cells. Here, we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a user-defined locus using homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. Using these approaches, we identified scAAV6 as the superior serotype for successful generation of stable mCherry-expressing primary NK cells (up to 89%). To maximize transgene packaging in HR-directed gene insertion, we identified minimum optimal homology arm lengths of 300bp for the flanking region of the Cas9-targeting site. Lastly, we demonstrate that mCherry positive NK cells can be expanded to large numbers using feeder cells expressing membrane-bound IL-21. This efficient method for site-directed insertion of genetic material into NK cells has broad potential for basic discovery and therapeutic applications for primary NK cells.

scholarly journals CD33 Targeting Primary CAR-NK Cells Generated By CRISPR Mediated Gene Insertion Show Enhanced Anti-AML Activity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3 ◽  
Author(s):  
Meisam Naeimi Kararoudi ◽  
Shibi Likhite ◽  
Ezgi Elmas ◽  
Maura Schwartz ◽  
Kinnari Sorathia ◽  
...  
Keyword(s):  
Nk Cells ◽  
Efficient Method ◽  
Human Peripheral Blood ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Adeno Associated Virus ◽  
Gene Insertion ◽  
Ribonucleoprotein Complexes ◽  
Membrane Bound ◽  
Signaling Domains

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein complexes (PMID: 29985369 and 32603414). Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. We demonstrated that expansion of NK cells on feeder cells (CSTX002) expressing membrane-bound IL21 increases expression of HDR-related genes and provides optimum biological condition for targeted gene insertion. For proof-of-concept, we successfully generated stable mCherry-expressing primary NK cells (up to 89% mCherry+) and determined that sc vectors with 300bp homology arms were optimal. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ), which continued to show robust expansion on CSTX002 and stably maintained their CAR expression. This resulted in CAR-NK-cells of high number and purity (mean 68% CAR+) that demonstrated enhanced antileukemic activity against acute myeloid leukemia (AML) cell lines. This efficient method for site-directed insertion of genetic materials into primary NK cells has broad potential for fundamental discovery and therapeutic applications. Keywords: CRISPR, NK, Cas9/RNP, AAV6, CRISPaint, HR, CD33CAR-NK Figure Disclosures Naeimi Kararoudi: Kiadis Pharma Netherlans B.V: Patents & Royalties. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3489
Author(s):  
Takayuki Morimoto ◽  
Tsutomu Nakazawa ◽  
Ryosuke Matsuda ◽  
Fumihiko Nishimura ◽  
Mitsutoshi Nakamura ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Protein Complexes ◽  
Lymphocyte Activation ◽  
Exon 2 ◽  
Effector Cells ◽  
Guide Rna ◽  
Human Natural Killer Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.

scholarly journals CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

2021 ◽  
Author(s):  
Meisam Naeimi Kararoudi ◽  
Shibi Likhite ◽  
Ezgi Elmas ◽  
Kenta Yamamoto ◽  
Maura Schwartz ◽  
...  
Keyword(s):  
Nk Cells ◽  
Efficient Method ◽  
Myeloid Leukemia ◽  
Human Peripheral Blood ◽  
Transduction Efficiency ◽  
Proof Of Concept ◽  
Activation Markers ◽  
Adeno Associated Virus ◽  
Gene Insertion ◽  
Signaling Domains

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology 30 arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3146-3151 ◽  
Author(s):  
Megan A. Cooper ◽  
Todd A. Fehniger ◽  
Sarah C. Turner ◽  
Kenneth S. Chen ◽  
Bobak A. Ghaheri ◽  
...  
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Innate Immune Response ◽  
Nk Cell ◽  
Primary Source ◽  
Interferon Γ ◽  
Innate Immune ◽  
Immunoregulatory Cytokines ◽  
Human Natural Killer Cells

Abstract During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56bright and CD56dim). In this report, it is shown that CD56bright NK cells produce significantly greater levels of interferon-γ, tumor necrosis factor-β, granulocyte macrophage–colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56dim NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56bright NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine production by CD56bright NK cells. It is proposed that human CD56bright NK cells have a unique functional role in the innate immune response as the primary source of NK cell–derived immunoregulatory cytokines, regulated in part by differential monokine production.

scholarly journals Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 841-846 ◽  
Author(s):  
MR Silva ◽  
R Hoffman ◽  
EF Srour ◽  
JL Ascensao
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Mononuclear Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Specific Lysis ◽  
Human Natural Killer Cells

Abstract Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

scholarly journals AKIRIN1: A Potential New Reference Gene in Human Natural Killer Cells and Granulocytes in Sepsis

2019 ◽  
Vol 20 (9) ◽  
pp. 2290 ◽  
Author(s):  
Anna Coulibaly ◽  
Sonia Y. Velásquez ◽  
Carsten Sticht ◽  
Ana Sofia Figueiredo ◽  
Bianca S. Himmelhan ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Reference Gene ◽  
Reference Genes ◽  
Inflammatory Diseases ◽  
Transcriptional Profiling ◽  
Lps Challenge ◽  
Human Natural Killer Cells ◽  
Suitable Reference

Timely and reliable distinction of sepsis from non-infectious systemic inflammatory response syndrome (SIRS) supports adequate antimicrobial therapy and saves lives but is clinically challenging. Blood transcriptional profiling promises to deliver insights into the pathomechanisms of SIRS and sepsis and to accelerate the discovery of urgently sought sepsis biomarkers. However, suitable reference genes for normalizing gene expression in these disease conditions are lacking. In addition, variability in blood leukocyte subtype composition complicates gene profile interpretation. Here, we aimed to identify potential reference genes in natural killer (NK) cells and granulocytes from patients with SIRS and sepsis on intensive care unit (ICU) admission. Discovery by a two-step probabilistic selection from microarray data followed by validation through branched DNA assays in independent patients revealed several candidate reference genes in NK cells including AKIRIN1, PPP6R3, TAX1BP1, and ADRBK1. Initially, no candidate genes could be validated in patient granulocytes. However, we determined highly similar AKIRIN1 expression also in SIRS and sepsis granulocytes and no change by in vitro LPS challenge in granulocytes from healthy donors. Inspection of external neutrophil transcriptome datasets further support unchanged AKIRIN1 expression in human systemic inflammation. As a potential new reference gene in NK cells and granulocytes in infectious and inflammatory diseases, AKIRIN1 may improve our pathomechanistic understanding of SIRS and sepsis and help identifying new sepsis biomarkers.

scholarly journals Classification of human natural killer cells based on migration behavior and cytotoxic response

Blood ◽  
2013 ◽  
Vol 121 (8) ◽  
pp. 1326-1334 ◽  
Author(s):  
Bruno Vanherberghen ◽  
Per E. Olofsson ◽  
Elin Forslund ◽  
Michal Sternberg-Simon ◽  
Mohammad Ali Khorshidi ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Serial Killer ◽  
Migration Behavior ◽  
Killer Cells ◽  
Key Points ◽  
Human Natural Killer Cells ◽  
Cytotoxic Responses

Key Points Activated NK cells display heterogeneity in their cytotoxic responses that justifies grouping them into 5 distinct classes of NK cells. A subpopulation of particularly active “serial killer” NK cells deliver their lytic hits faster and release more perforin in each hit.

scholarly journals A simple method for in vitro preparation of natural killer cells from cord blood

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Yong Xu Mu ◽  
Yu Xia Zhao ◽  
Bing Yao Li ◽  
Hong Jing Bao ◽  
Hui Jiang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Sorting ◽  
Interleukin 2 ◽  
Feeder Cells ◽  
Simple Method ◽  
Economical Method ◽  
Large Numbers ◽  
Promising Source

Abstract Background Cord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines. Results The large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells. Conclusion We develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

scholarly journals Human natural killer cells can inhibit clonogenic growth of fresh leukemic cells

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 596-599 ◽  
Author(s):  
M Beran ◽  
M Hansson ◽  
R Kiessling
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Colony Growth ◽  
Inhibitory Effect ◽  
Leukemic Cells ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Release Assay

Abstract The effect of allogenic human natural killer (NK) cells on fresh leukemic cells from three patients was investigated. The low levels of leukemic target cell lysis in the conventional 51Cr-release assay contrasted with a pronounced inhibitory effect on the colony growth of the clonogeneic leukemic target cells (L-CFC). The ability of allogeneic lymphocytes to inhibit L-CFC increased if they were pretreated with interferon (IFN), which also increased their NK activity, monitored in parallel cytotoxicity assay, against K562. Furthermore, cell separation procedures, based on differences in density among nonadherent lymphocytes, revealed that only NK cell containing fractions were inhibitory. We have also compared the susceptibility to NK-mediated L-CFC inhibition of IFN pretreated leukemic target cells with that of nontreated target cells. As in the case of NK lysis in general, this pretreatment of target cells abolished the presumably NK-mediated L-CFC inhibition. In conclusion, these data provide the first indication that NK cells can inhibit the in vitro growth of fresh clonogenic leukemia cells from patients with nonlymphocytic leukemia. The identity of NK cells as effector is strongly suggested by Percoll separation and responsiveness to interferon; the final proof awaits more sophisticated purification of these cells.

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