A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.
Hyuncheol Jung, Pascal Devant, Carter Ching et al.· Nature Biotechnology· 0 citations
Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand break induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knock in (KI), we expand the use of base editor nickase activity to stimulate homology-directed repair (HDR) and insert clinically-relevant chimeric antigen receptors (CARs) into specific loci. Through a novel sgRNA design strategy and a recombinant adeno-associated virus (rAAV) delivered DNA template, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19, CD33, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3ε/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines, and a suppressive 3D spheroid culture model. This efficient engineering process of Iterative Nicking for Synchronous Engineered Reprogramming of T cells (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.
Joseph G. Skeate, Nicholas J. Slipek, Walker S. Lahr et al.· Molecular Therapy· 0 citations
Background
CRISPR-Cas genome editing offers curative potential for monogenic disorders and persistent infections, but its
clinical translation is hindered by delivery inefficiency, off-target effects, and immunogenicity. Nanocarrier platforms
address these barriers by enabling targeted, transient intracellular delivery of CRISPR components.
Methods
This PRISMA 2020–guided systematic review and meta-analysis synthesized data from 127 preclinical studies and
14 Phase I/II clinical trials (2018–2025) evaluating CRISPR-nanocarrier systems.
Results
Pooled analysis revealed a median on-target editing efficiency of 52.4% (95% CI: 48.1–56.7). Ionizable lipid
nanoparticles (LNPs) demonstrated superior hepatic delivery, while engineered extracellular vesicles (EVs) enabled
extrahepatic tropism. Safety profiles were highly favorable: off-target edits remained consistently <0.1%,
chromosomal aberrations were negligible (98.4% compliance), and pathogen loads decreased by 3.12 log₁₀. Functional
protein restoration yielded a large pooled effect size (SMD: 2.84). Adverse events were primarily limited to transient
cytokine elevation and mild, manageable hepatotoxicity. Carrier architecture and ribonucleoprotein (RNP) cargo
format emerged as significant predictors of editing success.
Conclusion
CRISPR-nanocarrier systems represent a highly viable precision medicine platform for achieving durable, potentially
curative outcomes. Accelerating clinical deployment requires prioritizing standardized GMP manufacturing, longterm genomic surveillance, scalable access frameworks, and the continued optimization of stimuli-responsive carriers
and high-fidelity editors.
A. Aslam, F. Naz, Syeda Fatima Nadeem et al.· International Journal of Dru...· 0 citations
Chimeric antigen receptor T cell immunotherapies are transforming therapies for hematological malignancies and solid tumors and can be enhanced by targeted gene knockout. Here, we report lentiviral-based virus-like particles that package and deliver Cas9 ribonucleoproteins to primary human T cells. Using distinct pseudotyping strategies for virus-like particles and for lentiviral or γ-retroviral vectors, we achieved chimeric antigen receptor expression and targeted gene disruption. Under optimized transduction conditions, more than 50% of T cells expressed a chimeric antigen receptor by flow cytometry, with vector copy numbers exceeding two. Editing efficiencies were above 70% at three different target loci tested: T cell receptor α constant chain, β2-microglobulin, and DNA methyltransferase 3α. When the editing efficiency of virus-like particles was directly compared to electroporation, electroporation achieved a higher editing efficiency (99% versus 70%–90%). However, virus-like particle treatment resulted in twice as many cells being recovered compared with electroporation with a 10% increase in cell viability. Furthermore, off-target editing in virus-like particle-treated cells was reduced compared to ribonucleoprotein electroporated cells. These results support the feasibility of using virus-like particle-mediated delivery of Cas9 ribonucleoprotein to disrupt genes of interest, enabling a more scalable and cost-effective process for generating T cell immunotherapies.
Francesca Ferrara, Matthew M Wielgosz, Jeoungeun J. Park et al.· Molecular therapy. Advances· 0 citations
Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4⁺ and CD8⁺ T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.
Miti A. Patel, Michael Singh, Hugo Sinha et al.· Journal of Visualized Experi...· 0 citations
Introduction CRISPR–Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches. Methods We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules—Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope—into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days. Results Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci. Discussion It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.
Wei Lin, Jiaru Shi, Hanyi Chen et al.· Frontiers in Immunology· 0 citations