Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing

Renauer

et al. 2023

Immunological Reviews

SummaryCRISPR technology has transformed multiple fields, including cancer and immunology. CRISPR‐based gene editing and screening empowers direct genomic manipulation of immune cells, opening doors to unbiased functional genetic screens. These screens aid in the discovery of novel factors that regulate and reprogram immune responses, offering novel drug targets. The engineering of immune cells using CRISPR has sparked a transformation in the cellular immunotherapy field, resulting in a multitude of ongoing clinical trials. In this review, we discuss the development and applications of CRISPR and related gene editing technologies in immune cells, focusing on functional genomics screening, gene editing‐based cell therapies, as well as future directions in this rapidly advancing field.

Section: Advancements Of Crispr Technology In T‐cell‐based Therapysupporting

confidence: 64%

Section: T-cell Editing With Novel Crispr Editorsmentioning

confidence: 71%

Applications of CRISPR technology in cellular immunotherapy

Renauer

et al. 2023

Immunological Reviews

“…34,41 In addition, CBE allows editing in both dividing and non-dividing cells such as brain neurons. 43 CRISPR base editing technology, including CBE 32,44,45 and other CRISPR modalities 32,44 are being used to edit genes involved in a wide range of human diseases, including AD. 32,35,44,[46][47][48][49][50][51] The first CRISPR based therapy, CASGEVY TM , was recently approved by the U.S. Food and Drug Administration (FDA) for sickle cell disease, 52 and clinical trials of gene editing for many diseases are underway, 53,54,56, with promising phase 1 results for the liver disease transthyretin amyloidosis.…”

Section: Introductionmentioning

confidence: 99%

Successful Gene Editing of Apolipoprotein E4 to E3 in Brain of Alzheimer Model Mice After a Single IV Dose of Synthetic Exosome-Delivered CRISPR

Teter,

Campagna,

Zhu

et al. 2024

Preprint

Self Cite

Background. The gene for apolipoprotein E4 (ApoE4 E4) confers an increased risk for development and lowers the age of onset of Alzheimers disease (AD). It is a highly suitable target for CRISPR-based editing because ApoE4 differs from ApoE3 by a single nucleotide polymorphism in the codon for residue 112 that codes for arginine (CGC) in E4 and cysteine (TGC) in E3. Editing of E4 to E3 could lower the risk of AD or ameliorate E4-related AD phenotypes. For AD, in order to deliver CRISPR components across the blood-brain barrier to the brain, we have developed a delivery platform termed Synthetic Exosomes (SEs) microfluidically-synthesized deformable nanovesicles approximately the size of natural exosomes that have the ability to cross the BBB and deliver cargo to the brain. Here, we describe our use of SEs carrying CRISPR to successfully edit E4 to E3 in brain tissue of an E4-expressing mouse model. Methods. Several CRISPR guide RNAs (gRNA) and Cytosine Base Editor (CBE) mRNAs were synthesized by chemical and in vitro transcription syntheses, respectively. Four combinations of gRNA and CBE mRNA were tested in vitro for their relative activity to edit the E4 (cytosine) to E3 (thymine) in E4-expressing neuroblastoma (E4-N2A) and human Kelly-neuroblastoma cells, to assess which combination produced the highest E4 to E3 base editing efficiency. The CRISPR RNA combination with the highest efficiency was encapsulated in SEs and injected intravenously (IV) via the tail vein into an AD model E4-expressing (E4-5XFAD) transgenic mouse; as a negative control, an E4-5XFAD mouse was injected with empty SEs. Five days after injection, mice were euthanized and brain, liver, and buffy coat (white blood cells (WBC)) collected to determine the editing of E4 to E3 measured by Next Generation Sequencing. In addition, E3 mRNA was measured in the brain and liver and compared to the %E3 gene editing. Results. The highest gRNA+CBE mRNA editing efficiency was ~50% in E4-N2A cells and the same gRNA+CBE combination delivered in SEs to Kelly neuroblastoma cells showed 6.5% editing efficiency. In the E4-5XFAD mouse in vivo, five days after IV delivery of a single dose of the highest-activity SE-CRISPR gRNA+CBE mRNA, the percent of E4 edited to E3 was 0.14% in brain, 0.8% in liver, and 0.36 % in WBCs. As evidence of functional editing, SE-CRISPR-treated mice had 0.03% E3 mRNA in brain and 0.09% E3 mRNA in liver. Conclusions. While this level of ApoE4 to E3 editing achieved five days after a single IV injection of SE-CRISPR is small, it provides initial in vivo proof-of-concept that the ApoE4 gene can be successfully edited and editing results in functional expression of ApoE3 mRNA. The findings presented herein supports further optimization of the SE-CRISPR approach to increase the level of editing in brain as part of clinical development of SE-CRISPR as a powerful novel therapeutic approach for AD.

ACS Appl. Mater. Interfaces

“…6,7 Additionally, issues with potential insertional mutagenesis due to semirandom gene insertion mediated by viral carriers have driven the gene-editing field away from utilizing viral vectors and toward more targeted strategies such as those employing zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced palindromic repeats (CRISPR)-Cas, and, more recently, prime and base editors. 1,8,9 However, these important gene-modifying biomolecules are often large proteins that need to be delivered to cells using non-cytotoxic and effective intracellular delivery strategies, because the latest favored viral vectors suffer from size limitations and are thus unable to carry the large DNA constructs encoding these proteins. 1,10−12 Additionally, gene manipulation through targeted knockouts is an important research tool to elucidate functional gene roles and pathways that may inform clinical targets and outcomes.…”

Section: ■ Introductionmentioning

confidence: 99%

“…To date, most clinical progress has been made in the field of viral vector-mediated gene modification, , which harnesses viruses’ natural ability to enter cells and to modify DNA. The manufacturing of these viral-based therapies has been burdened with extremely high costs, while populations that are frequently affected by prevalent hematological disorders are often located in medically underserved and/or low-resource regions of the world, underscoring the need for intracellular delivery technologies that are accessible and easy to use and require little training to operate. , Additionally, issues with potential insertional mutagenesis due to semirandom gene insertion mediated by viral carriers have driven the gene-editing field away from utilizing viral vectors and toward more targeted strategies such as those employing zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced palindromic repeats (CRISPR)-Cas, and, more recently, prime and base editors. ,, However, these important gene-modifying biomolecules are often large proteins that need to be delivered to cells using non-cytotoxic and effective intracellular delivery strategies, because the latest favored viral vectors suffer from size limitations and are thus unable to carry the large DNA constructs encoding these proteins. ,− Additionally, gene manipulation through targeted knockouts is an important research tool to elucidate functional gene roles and pathways that may inform clinical targets and outcomes …”

Section: Introductionmentioning

confidence: 99%

Fluorinated Silane-Modified Filtroporation Devices Enable Gene Knockout in Human Hematopoietic Stem and Progenitor Cells

Frost,

Mendoza,

Chiou

et al. 2023

Self Cite

Intracellular delivery technologies that are costeffective, non-cytotoxic, efficient, and cargo-agnostic are needed to enable the manufacturing of cell-based therapies as well as gene manipulation for research applications. Current technologies capable of delivering large cargoes, such as plasmids and CRISPR-Cas9 ribonucleoproteins (RNPs), are plagued with high costs and/or cytotoxicity and often require substantial specialized equipment and reagents, which may not be available in resourcelimited settings. Here, we report an intracellular delivery technology that can be assembled from materials available in most research laboratories, thus democratizing access to intracellular delivery for researchers and clinicians in low-resource areas of the world. These filtroporation devices permeabilize cells by pulling them through the pores of a cell culture insert by the application of vacuum available in biosafety cabinets. In a format that costs less than $10 in materials per experiment, we demonstrate the delivery of fluorescently labeled dextran, expression plasmids, and RNPs for gene knockout to Jurkat cells and human CD34 + hematopoietic stem and progenitor cell populations with delivery efficiencies of up to 40% for RNP knockout and viabilities of >80%. We show that functionalizing the surfaces of the filters with fluorinated silane moieties further enhances the delivery efficiency. These devices are capable of processing 500,000 to 4 million cells per experiment, and when combined with a 3D-printed vacuum application chamber, this throughput can be straightforwardly increased 6−12-fold in parallel experiments.