Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different ...CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted “off-the-shelf” lymphocytes for anti-cancer immunotherapy.
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•Human iPSC-derived NK cells provide an “off-the-shelf” resource for cancer therapy•New CAR constructs optimize targeted anti-tumor activity of iPSC-derived NK cells•Optimized NK-CAR-expressing iPSC-NK cells activate specific intracellular signals•NK-CAR iPSC-NK cells demonstrate increased in vivo expansion and improved activity
Natural killer (NK) cells are a key part of the immune system’s ability to mediate anti-cancer activity. Li et al. utilize human iPSCs to produce NK cells with novel chimeric antigen receptors that specifically target cancer cells in an antigen-specific manner to improve survival in an ovarian cancer xenograft model.
The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used ...to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.
Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing ...the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.
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Enhancing natural killer (NK) cell cytotoxicity could lead to improved cancer immunotherapy. In this issue of Molecular Therapy, Pomeroy et al. describe an efficient method for editing the genome of primary human NK cells using CRISPR/Cas9. The authors target genes using this method to enhance NK cell anti-tumor function.
CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss ...of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms. Negative selection is capable of detecting dead or slow-growing cells efficiently, and it can identify survival-essential genes, which can be promising candidates for molecularly targeted drugs. In addition, negative selection can be applied for synthetic lethality interactions, where the perturbation of both genes simultaneously results in the loss of viability, but that of either gene alone does not affect viability. This mechanism is highly important to identifying the optimal combination of molecularly targeted drugs. Survival-co-essential genes in cancer cells can be identified using new methods, such as the paired guide RNA system and in combination with single-cell RNA sequencing techniques. These efficient methods can clarify interesting biological mechanisms and suggest candidates for molecularly targeted drugs. This review identifies what types of screenings were performed and suggests ideas for the next CRISPR screenings to develop new drugs.
The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in ...research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CRISPR-Cas9-Cytidine deaminase fusion enzymes—termed “base editors”—allow targeted editing of genomic deoxycytidine to deoxythymidine (C:G→T:A) without the need for double-stranded break induction. ...Base editors represent a paradigm shift in gene editing technology due to their unprecedented efficiency to mediate targeted, single-base conversion. However, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time-consuming. To overcome these limitations, we developed a simple, cost-effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed “EditR.” We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence. EditR is more accurate than enzymatic assays, and provides added insight to the position, type, and efficiency of base editing. Furthermore, EditR is likely amenable to quantify base editing from the recently developed adenosine deaminase base editors that act on either DNA (adenosine deaminase base editors ABEs) or RNA (REPAIRs) (catalyzes A:T→G:C). Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.
'Gain' of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) ...oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent 'gene desert' of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop ...(pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.
B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. ...Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19
B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+ IgM+ CD27- naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modified sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specific integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics.
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