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
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) and 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
An optimized lentiviral transfection and transduction workflow is described 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
TcBuster-M-mediated cell engineering achieved high CD19-CAR expression in both T and NK cells while preserving cell viability and growth, and demonstrated potent, target-specific cytotoxicity and favorable cytokine secretion.
Jessica K. Fiege, R. Haugen, Ellie A. Mews et al.· Journal of Immunology· 0 citations