Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors

Area of Science:

• Cellular and Gene Therapy
• Immunology
• Molecular Biology

Background:

• Chimeric antigen receptor (CAR) T cell immunotherapy shows promise for hematologic malignancies.
• Viral vectors (gamma-retroviral, lentiviral) used for CAR T cell engineering pose risks of insertional mutagenesis and genotoxicity.
• High cost and regulatory hurdles limit widespread clinical translation of viral vector-based CAR T cell therapies.

Purpose of the Study:

• To develop a non-viral method for CAR T cell engineering using Sleeping Beauty (SB) transposition and minicircles (MCs).
• To compare the genotoxicity and integration profiles of SB-engineered CAR T cells with those engineered by lentiviral (LV) vectors.
• To assess the efficacy and safety of SB-engineered CAR T cells in preclinical models.

Main Methods:

• Engineering CAR T cells using Sleeping Beauty (SB) transposition of CAR genes from minicircles (MCs).
• Analyzing genomic integration sites of SB transposons and LV integrants.
• Evaluating CD19-CAR T cell reactivity in vitro and efficacy in a lymphoma xenograft model in vivo.
• Comparing electroporation efficiency and toxicity of SB MCs versus conventional plasmids.

Main Results:

• SB transposition resulted in a significantly higher proportion of CAR transposons integrating into safe harbor loci compared to LV integrants.
• SB-engineered CAR T cells demonstrated potent in vitro reactivity and eradicated lymphoma in vivo.
• Electroporation of SB MCs was more effective and less toxic than conventional plasmids, enabling rapid, cost-effective CAR T cell preparation.

Conclusions:

• Non-viral SB transposition using MCs provides a safer and potentially less mutagenic alternative to viral vectors for CAR T cell engineering.
• This enhanced SB approach offers a cost-effective and rapid method for producing therapeutic CAR T cells.
• The findings support SB MCs as a new standard for advanced cellular and gene therapy, potentially increasing accessibility of CAR T cell therapy for hematologic malignancies.