scholarly journals Highly efficient and versatile plasmid-based gene editing in primary T cells

2018 ◽  
Author(s):  
Mara Kornete ◽  
Romina Marone ◽  
Lukas T. Jeker
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Genome Engineering ◽  
Genetic Manipulation ◽  
Cell Epitope ◽  
Basic Research ◽  
Foxp3 Expression ◽  
Adoptive Cell Transfer ◽  
Nucleotide Polymorphisms ◽  
Highly Efficient

AbstractAdoptive cell transfer (ACT) is an important approach for basic research and emerges as an effective treatment for various diseases including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. Here, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms (SNP) in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology directed repair (HDR), systematically optimize experimental parameters and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Both repairing the cause of the scurfy syndrome, a 2bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

scholarly journals CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Seyed Mohammad Miri ◽  
Elham Tafsiri ◽  
William Chi Shing Cho ◽  
Amir Ghaemi
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Gene Editing ◽  
Genome Engineering ◽  
Building Blocks ◽  
Cell Receptor ◽  
Adoptive Cell Transfer ◽  
Promising Strategy ◽  
Pre Treatment ◽  
Cas A

Abstract Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called “adoptive cell transfer”, or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.

scholarly journals AChlamydia trachomatisStrain Expressing Ovalbumin Stimulates an Antigen-Specific CD4+T Cell Response in Mice

2019 ◽  
Vol 87 (7) ◽  
Author(s):  
Jennifer D. Helble ◽  
Michael N. Starnbach
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Cell Epitope ◽  
T Cell Response ◽  
Developmental Cycle ◽  
Dependent Manner ◽  
Content Type

ABSTRACTAntigen-specific CD4+T cells againstChlamydiaare crucial for driving bacterial clearance and mediating protection against reinfection. Although theChlamydia trachomatisprotein Cta1 has been identified to be a dominant murine CD4+T cell antigen, its level of expression during the bacterial developmental cycle and precise localization within the host cell are unknown. Newly developed tools forChlamydiagenetic manipulation have allowed us to generate aC. trachomatisstrain expressing a heterologous CD4+T cell epitope from ovalbumin (OVA) consisting of OVA residues 323 to 339 (OVA323–339). By tagging proteins expressed inC. trachomatiswith OVA323–339, we can begin to understand how protein expression, developmental regulation, and subcellular compartmentalization affect the potential of those proteins to serve as antigens. When OVA323–339was expressed as a fusion with green fluorescent protein, we found that we were able to elicit an OT-II T cell response in an antigen-dependent manner, but surprisingly, these T cells were unable to reduce bacterial burden in mice. These data suggest that the subcellular localization of antigen, the level of antigen expression, or the timing of expression within the developmental cycle ofChlamydiamay play a crucial role in eliciting a protective CD4+T cell response.

scholarly journals Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Beau R. Webber ◽  
Cara-lin Lonetree ◽  
Mitchell G. Kluesner ◽  
Matthew J. Johnson ◽  
Emily J. Pomeroy ◽  
...  
Keyword(s):  
T Cells ◽  
Genome Engineering ◽  
Cellular Therapy ◽  
Cell Types ◽  
Strand Break ◽  
Double Strand Break ◽  
Adoptive Cellular Therapy ◽  
Highly Efficient ◽  
Human T Cell ◽  
T Cell Engineering

AbstractThe 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.

IMMU-08. NOVEL NANOPARTICLE-BASED DELIVERY OF H3.3K27M PEPTIDE TO TUMOR-ASSOCIATED MACROPHAGES ENHANCES THE TUMOR HOMING OF H3.3K27M-TCR TRANSDUCED T-CELLS IN HLA-A2/DR1 TRANSGENIC MICE WITH H3.3K27M+

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi93-vi93
Author(s):  
Akane Yamamichi ◽  
Polly Chuntova ◽  
Bunta Kakihara ◽  
Tiffany Chen ◽  
David Diebold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cell Epitope ◽  
Antigen Presenting Cells ◽  
Adoptive Cell Transfer ◽  
Adoptive T Cell Therapy ◽  
Combination Regimen ◽  
Tumor Associated Macrophages ◽  
Antigen Presenting

Abstract We have identified a novel HLA-A*02:01-restricted CD8 T-cell epitope encompassing the H3.3K27M mutation and a corresponding high-affinity T-cell receptor (TCR) that recognizes the epitope. While the development of adoptive cell transfer therapy using TCR-transduced T-cells holds a promise, we still need to overcome multiple challenges, such as suboptimal T-cell trafficking and the immunosuppressive environment of malignant glioma. For example, tumor-associated macrophages (TAMs) mediate immunosuppression but do not function as effective antigen-presenting cells. We have developed a novel cholesteryl pullulan (CHP) nanogel as a highly biocompatible and efficient vaccine delivery system targeting TAMs. In this study, we investigated whether the CHP nanogel loaded with the H3.3K27M peptide would deliver the peptide to TAMs and convert TAMs to better antigen-presenting cells that enhance the anti- H3.3K27M+ glioma activity of the TCR-transduced T-cells. As a clinically relevant mouse model, we used HLA-A2/HLA-DR1-transgenic mice and generated a syngeneic glioma cell line that expresses H3.3K27M from their astrocytes. We also generated a retroviral vector encoding the H3.3K27M-specific TCR for transduction of mouse T cells. HLA-A2/HLA-DR1-transgenic mice bearing day 16 intracerebral H3.3K27M+ glioma received an intravenous administration of the CHP nanogel along with poly-ICLC, a Toll-like receptor 3 agonist. The mice then received an intravenous infusion of TCR-transduced or control, non-transduced T-cells on the following day. The triple combination regimen with the CHP, poly-ICLC and TCR-transduced T-cells significantly suppressed the tumor growth, associated with increased levels of T-cell infiltration into the tumors compared with the dual-therapy with poly-ICLC and TCR-T-cells without the CHP. Furthermore, TAMs isolated from CHP-treated mice showed evidence of CHP-uptake, abilities to stimulate proliferation of TCR-transduced T-cells, and higher levels of HLA.A2 expression. These results suggest that the antigen-loaded CHP nanogel can promote the local antigen-presentation to T-cells and represent a promising approach for improving the efficacy of adoptive T-cell therapy for gliomas.

scholarly journals Improved Cas9 activity by specific modifications of the tracrRNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tristan Scott ◽  
Ryan Urak ◽  
Citradewi Soemardy ◽  
Kevin V. Morris
Keyword(s):  
T Cells ◽  
Streptococcus Pyogenes ◽  
Gene Editing ◽  
Basic Research ◽  
Clinical Applications ◽  
Receptor Expression ◽  
Guide Rna ◽  
Surface Receptor ◽  
Endogenous Genes ◽  
Mediated Reduction

Abstract CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.

scholarly journals CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research

2015 ◽  
Author(s):  
Fillip Port ◽  
Nadine Muschalik ◽  
Simon L Bullock
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Basic Research ◽  
Evidence Based ◽  
Gene Drive ◽  
Highly Active ◽  
Genome Modification ◽  
Target Sites ◽  
Gene Drives

CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. CRISPR components produced by integrated transgenes have been shown to mutagenise some genomic target sites in Drosophila melanogaster with high efficiency, but whether this is a general feature of this system remains unknown. Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.

scholarly journals Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

2020 ◽  
Author(s):  
Ezgi Akidil ◽  
Manuel Albanese ◽  
Alexander Buschle ◽  
Adrian Ruhle ◽  
Oliver T. Keppler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
B Cells ◽  
Epstein Barr Virus ◽  
Gene Editing ◽  
Barr Virus ◽  
Highly Efficient ◽  
Ribonucleoprotein Complexes ◽  
Human B Cells ◽  
Epstein Barr

AbstractGene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.Author summaryHuman hematopoietic stem cells and their derivatives of the myeloid and lymphoid lineages are important targets for gene correction or modifications using the CRISPR-Cas9 technology. Among others, this approach can support site-specific insertion of chimeric antigen receptors (CARs) or T cell receptors (TCRs) into primary T cells. Their subsequent adoptive transfer to patient donors is a promising immunotherapeutic concept that may control chronic infection or certain types of cancer. Human B cells have a similar potential but, in contrast to T cells, they are very sensitive, difficult to handle, and short-lived ex vivo precluding their genetic modification. Here, we provide means to manipulate primary human B cells genetically using in vitro assembled Cas9 ribonucleoprotein complexes and electroporation for their delivery. Our study demonstrates near-to-complete loss of a model target gene and provides examples to evaluate a cellular gene with a critical role during infection with Epstein-Barr virus (EBV).

scholarly journals Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

2018 ◽  
Vol 200 (7) ◽  
pp. 2489-2501 ◽  
Author(s):  
Mara Kornete ◽  
Romina Marone ◽  
Lukas T. Jeker
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Highly Efficient

scholarly journals Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

2015 ◽  
Vol 112 (33) ◽  
pp. 10437-10442 ◽  
Author(s):  
Kathrin Schumann ◽  
Steven Lin ◽  
Eric Boyer ◽  
Dimitre R. Simeonov ◽  
Meena Subramaniam ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Genome Engineering ◽  
Cell Function ◽  
Genetic Manipulation ◽  
Cell Surface Expression ◽  
Great Promise ◽  
Knock Out ◽  
Human T Cells ◽  
Genome Modifications

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved inCXCR4andPD-1(PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

scholarly journals Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

2021 ◽  
Author(s):  
Manuel Albanese ◽  
Adrian Ruhle ◽  
Jennifer Mittermaier ◽  
Ernesto Mejías-Pérez ◽  
Madeleine Gapp ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Immune Functions ◽  
Hiv Replication ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Neutral Gene ◽  
Endogenous Loci

AbstractCD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

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