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.
Lentiviral vectors have revolutionized gene therapy by efficient and stable transduction of dividing and non-dividing cells, their large packaging capacity, and their compatibility with pseudotyping to alter viral tropism. The vesicular stomatitis virus glycoprotein (VSV-G) is widely used as a viral envelope protein of choice to pseudotype lentiviral vector particles as it confers exceptional particle stability and a broad tropism, due to the ubiquitous nature of the low-density lipoprotein receptor (LDLR). While this broad tropism facilitates transduction of diverse cell types, it precludes accurate in vivo targeting of specific cell populations. Structural insights into VSV-G have made receptor-blinding possible and revealed sites amenable to mutation while preserving fusion capacity. Coupled with targeting moieties, VSV-G pseudotyped lentiviral particles are redirected towards cells expressing target antigens. Such targeted vectors open new possibilities for in vivo gene therapy across oncology, infectious diseases, transplantation medicine, and other diseases. Use of targeted vectors will make in vivo gene therapy more accessible than cost-intensive ex vivo gene therapies. Since targeted vectors will be available as 'off-the-shelf' drugs, they will also drastically reduce time-to-treatment. This review highlights advances in bioengineering to exploit the versatility of VSV-G-pseudotyped lentiviral vectors and explores their vast potential for targeted gene delivery.
Anjali Shrivastava, Felix L. Warnecke, J. Schott et al.· Molecular Therapy· 0 citations
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
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
Efficient and reproducible lentiviral vector production and T-cell transduction remain important technical challenges in CAR-T (Chimeric Antigen Receptor T-cell) cell manufacturing. In this study, we optimized HEK293T transfection and primary T-cell transduction parameters for lentiviral CAR constructs targeting BCMA (B-cell maturation antigen) and GPRC5D (G-protein coupled receptor family C group 5 member D). Lipofectamine 3000 and TurboFectin 8.0 were compared across different seeding densities and reagent-to-DNA ratios, with vector yields quantified by qPCR (Quantitative Polymerase Chain Reaction) and p24 ELISA (Enzyme-linked Immunosorbent Assay). Lipofectamine 3000 consistently generated higher viral titers and transduction efficiencies, as reflected by a greater proportion of GFP-positive (Green Fluorescent Protein) cells than TurboFectin 8.0, reaching peak titers of 9.65 × 108 copies/mL for the anti-GPRC5D and 5.33 × 108 copies/mL for the anti-BCMA vectors. Under optimized conditions, transduction efficiencies reached 43.8% GFP+ cells for BCMA-CAR and approximately 13–14% GFP-positive transduced cells for the GPRC5D construct within the tested TU/mL range. Co-transduction experiments yielded approximately 62–66% GFP+ cells with detectable BCMA-binding and presumptive GPRC5D-CAR-expressing subpopulations identified based on GFP reporter expression. Immunophenotypic analysis demonstrated a relatively stable CD4/CD8 distribution (~65/35), enrichment of effector memory CD8+ cells, and expression of activation-associated markers. Collectively, these findings describe an optimized lentiviral transfection and transduction workflow that may support the further development of dual-targeting BCMA/GPRC5D CAR-T manufacturing strategies in research and early translational settings.
Ewa Nowak, E. Morawiec, A. Pudełko et al.· Current Issues in Molecular...· 0 citations
A detailed protocol is described for a small-scale production of AsCas12a-VLPs using three distinct transfection methods and a large-scale production of VLPs using calcium-phosphate transfection, showing that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies.
N. Kruglova, S. E. Borovikova, M. Shepelev· Frontiers in Genome Editing· 0 citations
Viral vectors are widely used for immune cell engineering but pose challenges including limited cargo capacity and high production costs. TcBuster-M™, a transposase-based editing platform, offers a non-viral cell engineering alternative with broader cargo capacity and commercial availability.
Peripheral blood-derived T and NK cells were edited via electroporation with the TcBuster-M transposase and a multicistronic CD19-CAR transposon. Cells were expanded and assessed for cell growth and viability in addition to CAR expression and cell phenotype by flow cytometry. Genomic integration of the CD19-CAR was assessed by dPCR. Cytotoxicity was evaluated using a luciferase-based CD19+ target cell assay and cytokine secretion profile by Simple Plex (Ella).
TcBuster-M-mediated cell engineering achieved high CD19-CAR expression in both T and NK cells while preserving cell viability and growth. Edited cells demonstrated potent, target-specific cytotoxicity and favorable cytokine secretion. Genomic analysis revealed stable integration with copy number variations below eight.
The TcBuster transposon system supports rapid, cost-effective cell manufacturing and enables delivery of complex therapeutic cargos, positioning it as a robust alternative to virus-mediated editing systems for immunotherapy development.
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Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Jessica K. Fiege, R. Haugen, Ellie A. Mews et al.· Journal of Immunology· 0 citations