scholarly journals Electro-mechanical transfection for non-viral primary immune cell engineering

2021 ◽  
Author(s):  
Jessica Margaret Sido ◽  
James B Hemphill ◽  
Rameech N McCormack ◽  
Ross D Beighley ◽  
Bethany F Grant ◽  
...  
Keyword(s):  
T Cells ◽  
Electric Fields ◽  
Immune Cell ◽  
Scale Up ◽  
Genetic Material ◽  
Multiple Use ◽  
Delivery Methods ◽  
Human T Cells ◽  
High Fluid ◽  
Cell And Gene Therapy

Non-viral approaches to transfection have emerged a viable option for gene transfer. Electro-mechanical transfection involving use of electric fields coupled with high fluid flow rates is a scalable strategy for cell therapy development and manufacturing. Unlike purely electric field-based or mechanical-based delivery methods, the combined effects result in delivery of genetic material at high efficiencies and low toxicity. This study focuses on delivery of reporter mRNA to show electro-mechanical transfection can be used successfully in human T cells. Rapid optimization of delivery to T cells was observed with efficiency over 90% and viability over 80%. Confirmation of optimized electro-mechanical transfection parameters was assessed in multiple use cases including a 50-fold scale up demonstration. Transcriptome and ontology analysis show that delivery, via electro-mechanical transfection, does not result in gene dysregulation. This study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for cell and gene therapy engineering and development.

scholarly journals CTS™ immune cell SR for serum free culture and expansion of human T cells

2015 ◽  
Vol 3 (Suppl 2) ◽  
pp. P1
Author(s):  
Grethe Okern ◽  
Angel Varela-Rohena ◽  
Sandra Kuligowski ◽  
Brian Newsom ◽  
Tanja Aarvak
Keyword(s):  
T Cells ◽  
Immune Cell ◽  
Serum Free ◽  
Human T Cells

scholarly journals A tissue-like platform for studying engineered quiescent human T-cells’ interactions with dendritic cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Enas Abu-Shah ◽  
Philippos Demetriou ◽  
Štefan Bálint ◽  
Viveka Mayya ◽  
Mikhail A Kutuzov ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Immune Cell ◽  
Population Based ◽  
Functional Responses ◽  
Human Immunology ◽  
Human T Cells ◽  
Activation Dynamics ◽  
Effect Of Pd

Research in the field of human immunology is restricted by the lack of a system that reconstitutes the in-situactivation dynamics of quiescent human antigen-specific T-cells interacting with dendritic cells. Here we report a tissue-like system that recapitulates the dynamics of engineered primary human immune cell. Our approach facilitates real-time single-cell manipulations, tracking of interactions and functional responses complemented by population-based measurements of cytokines, activation status and proliferation. As a proof of concept, we recapitulate immunological phenomenon such as CD4 T-cells' help to CD8 T-cells through enhanced maturation of DCs and the effect of PD-1 checkpoint blockades. In addition, we characterise unique dynamics of T-cell/DC interactions as a function of antigen affinity.

CTS™ immune cell SR for serum free culture and expansion of human T cells

Cytotherapy ◽  
2015 ◽  
Vol 17 (6) ◽  
pp. S77
Author(s):  
Karoline Schjetne ◽  
Grethe Økern ◽  
Sandra Kuligowski ◽  
Angel Varela-Rohena ◽  
Tanja Aarvak
Keyword(s):  
T Cells ◽  
Immune Cell ◽  
Serum Free ◽  
Human T Cells

scholarly journals Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement

2015 ◽  
Vol 4 (1) ◽  
pp. e31 ◽  
Author(s):  
Corey Smith ◽  
Grethe Økern ◽  
Sweera Rehan ◽  
Leone Beagley ◽  
Sau K Lee ◽  
...  
Keyword(s):  
T Cells ◽  
Adoptive Immunotherapy ◽  
Immune Cell ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
The Novel ◽  
Serum Replacement ◽  
Human T Cells

scholarly journals 685 A highly selective and potent HPK1 inhibitor enhances immune cell activation and induces robust tumor growth inhibition in a murine syngeneic tumor model

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A724-A724
Author(s):  
David Ciccone ◽  
Vad Lazari ◽  
Ian Linney ◽  
Michael Briggs ◽  
Samantha Carreiro ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Activation ◽  
Immune Cell ◽  
Tumor Model ◽  
Tumor Growth Inhibition ◽  
Immune Cell Activation ◽  
Syngeneic Tumor ◽  
Human T Cells

BackgroundHPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in regulating signal transduction cascades in cells of hematopoietic origin. Recent data from HPK1 knockout animals and kinase-inactive knock-in animals underscores the role of HPK1 in negatively regulating immune cell activation. This negative-feedback role of HPK1 combined with its restricted expression in cells of hematopoietic origin, make it a compelling drug target for enhancing anti-tumor immunity.MethodsA structure-based drug design approach was used to identify potent and selective inhibitors of HPK1. Biochemical assays, as well as primary human and mouse immune cell-based activation assays, were utilized for multiple iterations of structure-activity relationship (SAR) studies. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models.ResultsA highly potent, HPK1 inhibitor was identified, that showed high selectivity against T cell-specific kinases and kinases in the MAP4K family. In vitro, HPK1 small molecule inhibition resulted in enhanced IL-2 production in primary mouse and human T cells, enhanced IL-6 and IgG production in primary human B cells, and enhanced mouse dendritic cell activation and antigen presentation capacity. Furthermore, HPK1 inhibition alleviated the immuno-suppressive effects of PGE2 on naïve human T cells and restored the proliferative capacity of exhausted human T cells. In vivo, HPK1 inhibitionHPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model.ConclusionsPharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity.

scholarly journals A tissue-like platform for studying engineered quiescent human T-cells’ interactions with dendritic cells

2019 ◽  
Author(s):  
Enas Abu-Shah ◽  
Philippos Demetriou ◽  
Stefan Balint ◽  
Viveka Mayya ◽  
Mikhail A. Kutuzov ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Immune Cell ◽  
Population Based ◽  
Functional Responses ◽  
Human Immunology ◽  
Human T Cells ◽  
Activation Dynamics ◽  
Cd4 Help ◽  
Effect Of Pd

AbstractResearch in the field of human immunology is restricted by the lack of a system that reconstitutes the in-situ activation dynamics of quiescent human antigen-specific T-cells interacting with dendritic cells. Here we report a tissue-like system that recapitulates the dynamics of engineered primary human immune cell. Our approach facilitates real-time single cell manipulations, tracking of interactions and functional responses complemented by population-based measurements of cytokines, activation status and proliferation. As a proof of concept, we recapitulate immunological phenomenon such as CD4 help to CD8 T-cells through enhanced maturation of DCs and effect of PD-1 checkpoint blockades. In addition, we characterise unique dynamics of T-cell/DC interactions as a function of antigen affinity.

scholarly journals Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

2021 ◽  
Author(s):  
Beau R Webber ◽  
Matthew J Johnson ◽  
Nicholas S Slipek ◽  
Walker S Lahr ◽  
Anthony P DeFeo ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Viral Vectors ◽  
Cellular Therapy ◽  
Viral Vector ◽  
Scale Up ◽  
Genetically Engineered ◽  
Adoptive Cellular Therapy ◽  
Human T Cells ◽  
Targeted Integration

Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a significant bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to cGMP compliant manufacturing and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.

scholarly journals CRISPR activation and interference screens in primary human T cells decode cytokine regulation

2021 ◽  
Author(s):  
Ralf Schmidt ◽  
Zachary Steinhart ◽  
Madeline Layeghi ◽  
Jacob W Freimer ◽  
Vinh Q Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Networks ◽  
Immune Cell ◽  
Interleukin 2 ◽  
Interferon Γ ◽  
Loss Of Function ◽  
Cell Functions ◽  
Cytokine Responses ◽  
Human T Cells ◽  
Crispr Activation

The pathways that regulate cytokine responses in T cells are disrupted in autoimmunity, immune deficiencies, and cancer, and include immunotherapy targets. Systematic discovery of cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now have accomplished genome-wide pooled CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens in primary human T cells to map gene networks controlling Interleukin-2 and Interferon-γ production. Arrayed CRISPRa confirmed key hits and enabled multiplexed T cell secretome characterization, revealing reshaped cytokine responses driven by individual regulators. CRISPRa uncovered genes not canonically expressed in T cells, including the transcription factor FOXQ1, whose overexpression promoted the expression of most cytokines, while selectively dampening T helper 2 (Th2) cytokines. Paired CRISPRa and CRISPRi screens reveal signaling components that tune critical immune cell functions, which could inform design of future immunotherapies.

scholarly journals Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

2019 ◽  
Author(s):  
Theodore L. Roth ◽  
P. Jonathan Li ◽  
Jasper F. Nies ◽  
Ruby Yu ◽  
Michelle L.T. Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dna Sequences ◽  
Immune Cell ◽  
Genetically Engineered ◽  
Regulatory Control ◽  
Cell Therapies ◽  
Synthetic Dna ◽  
Cell Functions ◽  
Human T Cells

ABSTRACTGenetically-engineered immune cell therapies have been in development for decades1–3 and recently have proven effective to treat some types of cancer4. CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies5,6. Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo. Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

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