Sleeping beauty system to redirect T-cell specificity for human applications

Area of Science:

• Cellular and Molecular Immunology
• Gene Therapy
• Cancer Immunotherapy

Background:

• The Sleeping Beauty (SB) transposon system facilitates genetic modification for sustained transgene expression.
• Adapting the SB system for human applications is crucial for developing novel cell therapies.

Purpose of the Study:

• To adapt the SB system for generating chimeric antigen receptor (CAR) T cells targeting CD19 for potential human application.
• To evaluate the efficacy, safety, and persistence of SB-modified CAR T cells in vitro.

Main Methods:

• Utilized the SB DNA plasmid system for electrotransfer into human peripheral blood mononuclear cells.
• Expanded T cells expressing CD19-specific CARs using artificial antigen-presenting cells.
• Assessed CAR T cell expression, cytotoxicity, phenotype, genetic stability, and integration patterns using quantitative PCR and FISH.

Main Results:

• >90% of expanded CD3 T cells expressed CAR by day 28.
• CAR T cells demonstrated specific killing of CD19 target cells and maintained memory phenotypes.
• Transposon integration occurred once per T-cell genome, with preserved telomere length and polyclonal TCR repertoire. CAR T cells were detectable at 0.01% sensitivity.

Conclusions:

• The nonviral SB system effectively generates CD19-specific CAR T cells with potent anti-tumor activity and favorable safety profiles.
• These findings support the initiation of first-in-human clinical trials for CD19 B-cell malignancies using this SB-based CAR T-cell therapy.