Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system
Harjeet Singh1, Pallavi R Manuri, Simon Olivares
1Division of Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Abstract:
Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR(+) T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19(+) artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4(+) and CD8(+) T cells expressing CAR to redirect specificity for CD19(+) tumor cells.
Insights
Researchers developed a novel method using the Sleeping Beauty transposon system to genetically modify T cells for cancer therapy. This approach efficiently generates CD19-specific CAR T cells without drug selection, overcoming key manufacturing barriers.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) T cell therapy shows promise for cancer treatment by redirecting T cell specificity.
- Current CAR T cell manufacturing faces challenges with viral vector production and ex vivo drug selection for efficient gene transfer.
Purpose of the Study:
- To develop a cost-effective and efficient method for generating clinical-grade CAR T cells.
- To overcome limitations associated with viral transduction and drug selection in CAR T cell therapy.
Main Methods:
- Utilized the Sleeping Beauty (SB) transposon/transposase system for DNA plasmid-based gene transfer into T cells.
- Engineered T cells to express a CD19-specific CAR without the need for drug selection.
- Employed numerical expansion on artificial antigen-presenting cells for CAR T cell amplification.
Main Results:
- Achieved efficient direct expression of a CD19-specific CAR in both memory and effector T cells.
- Demonstrated successful generation of CAR T cells without requiring cytocidal drug selection.
- Observed rapid outgrowth of CD4+ and CD8+ T cells with redirected specificity for CD19+ tumor cells.
Conclusions:
- The Sleeping Beauty transposon system offers a viable, non-viral alternative for CAR T cell engineering.
- This method facilitates the efficient generation of CAR T cells, potentially reducing manufacturing costs and complexity.
- The approach enables the production of functional CAR T cells for targeting CD19-expressing malignancies.
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