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.
Abstract
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)
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
CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.
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The potential of circRNA-based CD19-targeted CAR-NK therapy as an effective approach for enhancing the safety and efficacy of cancer immunotherapy is supported.
Qisheng Dong, Ying Liu, Na Chen et al.· International Immunopharmaco...· 0 citations
EF-1α preserved the antitumor potency of nanobody-based CD19-redirected CAR-T cells, supporting its use as a clinically relevant promoter for further development of this VHH-based CAR-T cell platform.
Marzieh Mazinani, Pooria Safarzadeh Kozani, F. Rahbarizadeh· Molecular and Cellular Bioch...· 0 citations
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.
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