Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

in Molecular therapy : the journal of the American Society of Gene Therapy by Joseph G Skeate, Emily J Pomeroy, Nicholas J Slipek, Bryan J Jones, Bryce J Wick, Jae-Woong Chang, Walker S Lahr, Erin M Stelljes, Xiaobai Patrinostro, Blake Barnes, Trevor Zarecki, Joshua B Krueger, Jacob E Bridge, Gabrielle M Robbins, Madeline D McCormick, John R Leerar, Kari T Wenzel, Kathlyn M Hornberger, Kirsti Walker, Dalton Smedley, David A Largaespada, Neil Otto, Beau R Webber, Branden S Moriarity

TLDR

  • The study is about making a new way to put genetic material into cells that can help fight diseases. The new way is called TcB-M and it's made by changing the structure of a protein called a transposon. The transposon is used to put the genetic material into the cells. The study shows that TcB-M is better at putting the genetic material into the cells than the current way, which uses viruses. The study also shows that the cells made with TcB-M work well in the lab and in mice. TcB-M is a safe and efficient way to make cells that can fight diseases.

Abstract

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and NK cells have shown remarkable clinical efficacy in treating hematological malignancies, however current methods mainly utilize viral vectors which are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof of principle TcB-M engineering of CAR-NK and CAR-T cells show low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Overview

  • The study focuses on developing a novel hyperactive transposase engineered through structure guided and in vitro evolution approaches for efficient integration of large, multicistronic CAR-expression cassettes in primary human cells. The methodology used includes the engineering of TcB-M transposase, the integration of CAR-NK and CAR-T cells, and the evaluation of the efficiency and safety of the engineered cells in vitro and in vivo. The primary objective of the study is to demonstrate the feasibility and potential of TcB-M as a versatile, safe, efficient, and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Comparative Analysis & Findings

  • The study compares the efficiency and safety of TcB-M transposition with current viral vector methods for delivering genetic cargo in primary human cells. The results show that TcB-M achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells with low integrated vector copy number and a safe insertion site profile. The engineered CAR-NK and CAR-T cells show robust in vitro function and improve survival in a Burkitt lymphoma xenograft model in vivo. These findings suggest that TcB-M is a more efficient and safe option for delivering large, multicistronic genetic cargo in primary human cells compared to current viral vector methods.

Implications and Future Directions

  • The study's findings have significant implications for the development of more efficient and safe cell therapies for human diseases. TcB-M is a versatile, open-source option that can be rapidly manufactured and preclinically tested for various primary human immune cell therapies. Future research directions could focus on further optimization of TcB-M transposition efficiency, expanding its applicability to other cell types, and evaluating its potential for clinical translation.
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