Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

Cromer, M. Kyle; Vaidyanathan, Sriram; Ryan, Daniel E.; Curry, Bo; Lucas, Anne Bergstrom; Camarena, Joab; Kaushik, Milan P.; Hay, Sarah R.; Martin, Renata; Steinfeld, Israel; Bak, Rasmus O.; Dever, Daniel P.; Hendel, Ayal; Bruhn, Laurakay; Porteus, Matthew H.

doi:10.1016/j.ymthe.2018.06.002

Cited by 105 publications

(112 citation statements)

References 58 publications

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Section: Relationship Between Transcriptional Responses To Exogenousmentioning

confidence: 99%

“…As a proxy to cellular response to the delivery of exogenous RNA, we considered recently published transcriptional responses of a human cell type to delivery of non-viral RNA sequences encoding cas9 protein and CRISPR gRNA to hemoglobin B locus (HBB), 4 hereafter referred to as 'cas9 mRNA' (3). Specifically, the delivery of cas9 mRNA into hematopoietic stem and progenitor cells (HSPCs) was found to induce a strong antiviral transcriptional response (3). Several genes involved in innate antiviral response such as interferon stimulatory genes (ISGs), interferon regulatory factors (IRF1, IRF7, IRF9) and the cytosolic foreign RNA sensor RIG-1 were upregulated by cas9 mRNA delivery (3).…”

Section: Relationship Between Transcriptional Responses To Exogenousmentioning

confidence: 99%

“…Specifically, the delivery of cas9 mRNA into hematopoietic stem and progenitor cells (HSPCs) was found to induce a strong antiviral transcriptional response (3). Several genes involved in innate antiviral response such as interferon stimulatory genes (ISGs), interferon regulatory factors (IRF1, IRF7, IRF9) and the cytosolic foreign RNA sensor RIG-1 were upregulated by cas9 mRNA delivery (3).…”

Section: Relationship Between Transcriptional Responses To Exogenousmentioning

confidence: 99%

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Predicting Broad-Spectrum Antiviral Drugs against RNA Viruses Using Transcriptional Responses to Exogenous RNA

Nordor¹,

Siwo²

2020

Preprint

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All RNA viruses deliver their genomes into target host cells through processes distinct from normal trafficking of cellular RNA transcripts. The delivery of viral RNA into most cells hence triggers innate antiviral defenses that recognize viral RNA as foreign. In turn, viruses have evolved mechanisms to subvert these defenses, allowing them to thrive in target cells. Therefore, drugs activating defense to foreign or exogenous RNA could serve as broad-spectrum antiviral drugs. Here we show that transcriptional signatures associated with cellular responses to the delivery of a non-viral exogenous RNA sequence into human cells predicts small molecules with broad-spectrum antiviral activity. In particular, transcriptional responses to the delivery of cas9 mRNA into human hematopoietic stem and progenitor cells (HSPCs) highly matches those triggered by small molecules with broad-spectrum antiviral activity such as emetine, homoharringtonine, pyrvinium pamoate and anisomycin, indicating that these drugs are potentially active against other RNA viruses. Furthermore, these drugs have been approved for other indications and could thereby be repurposed to novel viruses. We propose that the antiviral activity of these drugs to SARS-CoV-2 should therefore be determined as they have been shown as active against other coronaviruses including SARS-CoV and MERS-CoV. These drugs could also be explored as potential adjuvants to COVID-19 vaccines in development due to their potential effect on the innate antiviral defenses that could bolster adaptive immunity when delivered alongside vaccine antigens.

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Section: Relationship Between Transcriptional Responses To Exogenousmentioning

confidence: 99%

Section: Relationship Between Transcriptional Responses To Exogenousmentioning

confidence: 99%

Section: Relationship Between Transcriptional Responses To Exogenousmentioning

confidence: 99%

See 1 more Smart Citation

Predicting Broad-Spectrum Antiviral Drugs against RNA Viruses Using Transcriptional Responses to Exogenous RNA

Nordor¹,

Siwo²

2020

Preprint

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“… Cromer et al [1] recently reported global transcriptional changes occuring in cells in response to CRISPR/Cas9 gene editing. Using a CD34 + hematopoietic and progenitor stem cell model, they observed differentially expressed genes enriched for immune, stress and apoptotic processes following treatment with a CRISPR/Cas9-AAV6 genome-editing system.…”

mentioning

confidence: 99%

“…The sgRNA in each case was designed to target the hemoglobin B ( HBB ) locus which is a clinically relevant model for sickle cell disease (in the scope of several translational research programs leveraging CRISPR/Cas9 technology). All experiments in Cromer et al [1] were performed in CD34 + hematopoietic stem cells obtained from four individual donors to account for potential individual variability to genome editing machinery [4]. The query signature lists were restricted to genes belonging to 10,174 best inferred genes (BING) in the L1000 assay.…”

mentioning

confidence: 99%

“Global Transcriptional Response to CRISPR/CAS9-AAV6 Based Genome Editing” Matches Transcriptional Response to Specific Small Molecule Perturbations

Nordor

Aryee

Siwo

2018

Preprint

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Cromer et al. [1] recently reported global transcriptional changes occuring in cells in response to CRISPR/Cas9 gene editing. Using a CD34+ hematopoietic and progenitor stem cell model, they observed differentially expressed genes enriched for immune, stress and apoptotic processes following treatment with a CRISPR/Cas9-AAV6 genome-editing system. Following treatment with Cas9’s mRNA they observed transcriptional changes enriched for viral response as well as a downregulation of metabolic and cell cycle processes. Similarly, they observed a downregulation of metabolic processes in response to electroporation. Surprisingly, no enrichment for viral response genes was observed following treatment with AAV6 while minor transcriptional changes enriched for DNA damage signature occurred in response to Cas9/sgRNA ribonucleoprotein.

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Materials for Improving Immune Cell Transfection

et al. 2021

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recognize and bind to tumor-associated antigens such as CD19 and epidermal growth factor receptor (EGFR) before releasing cytotoxic granules and effector cytokines to destroy cancer cells. [3] CAR-T therapy has shown remarkable success against CD19 expressing B cell hematological malignancies and, thus far, five products (Kymriah from Novartis; Yescarta and Tecartus from Kite Pharma/Gilead Sciences; and Breyanzi and Abecma from Bristol-Myers Squibb) have been approved by the US Food and Drug Administration (FDA) for clinical use. [4,5] Currently, there are at least 500 clinical trials registered on ClinicalTrials.gov database using engineered immune cells to treat various cancers including that of the brain, lungs, and skin.Despite the clinical success of CAR-T cells against B cell leukemia, CAR-T therapy still face tremendous challenges in manufacturing, safety, and affordability before it can become a viable clinical option. [6] One of the biggest technical difficulties is to transfect sensitive, primary T cells whereby biomolecules like oligonucleotides and proteins have to be delivered intracellularly and into the nuclei of cells. FDA-approved gold standard viruses and bulk electroporation suffer from low transfection efficiency while also perturbing the critical biological attributes of cells such as proliferation, metabolism, and gene expression. [7] This increases the time and costs for cell expansion, and without an efficient and cost-friendly transfection technology, the price (between USD 0.4-0.5 million per patient) for FDA-approved CAR treatments (Kymriah and Yescarta) will remain unaffordable and untimely. [8] The limitations in conventional transfection techniques have motivated the development of micro-and nanoplatforms such as microfluidics, nanoparticles, and high-aspect-ratio nanostructures to improve immune cell viability and throughput during transfection.Herein, we first provide an overview of CAR-T cell manufacturing, with emphasis on the science of transfection and limitations of traditional technology using viruses and bulk electroporation. There will also be discussion of other cell-based cancer immunotherapy using other types of promising immune cell types. Next, we describe emerging transfection platforms and companies that have been established to overcome gaps in CAR-T transfection. We then provide a list of assays constituting the polyfunctionalities of immune cells that we believe will help the field better assess the robustness and suitability of their transfection methods. Finally, we end off with existing challenges in CAR-T transfection and how overcoming these challenges can significantly enhance the clinical impact of CAR-T therapy.Chimeric antigen receptor T cell (CAR-T) therapy holds great promise for preventing and treating deadly diseases such as cancer. However, it remains challenging to transfect and engineer primary immune cells for clinical cell manufacturing. Conventional tools using viral vectors and bulk electroporation suffer from low efficiency while posing risks ...

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Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

Cited by 105 publications

References 58 publications

Predicting Broad-Spectrum Antiviral Drugs against RNA Viruses Using Transcriptional Responses to Exogenous RNA

Predicting Broad-Spectrum Antiviral Drugs against RNA Viruses Using Transcriptional Responses to Exogenous RNA

“Global Transcriptional Response to CRISPR/CAS9-AAV6 Based Genome Editing” Matches Transcriptional Response to Specific Small Molecule Perturbations

Materials for Improving Immune Cell Transfection

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