Microfluidic platforms to generate 'off-the-shelf' fratricide-resistant CAR T cells for T-cell malignancies

项目总结

Project Summary Chimeric antigen receptor (CAR) T-cell therapy has been remarkably successful in treating B-cell malignancies; however, fewer studies have evaluated CAR T-cell therapy for the treatment of T-cell malignancies. Two main manufacturing challenges exist in translating this therapy for T-cell disease. First, given the lack of a cancer-specific antigen on malignant T cells, CAR T cells targeting T-cell antigens undergo fratricide, thus making effective expansion of a CAR T-cell product difficult. Second, the difficulty in isolating healthy T cells during leukapheresis results in product contamination, wherein malignant T cells inadvertently transduced to express the CAR become treatment-resistant. Thus targeting T-cell disease ideally requires an allogeneic “off-the-shelf” fratricide-resistant CAR T-cell product. This can be achieved by multiplex genome editing of T cells prior to transduction with the CAR-expressing vector. Genome editing of the target T-cell antigen via CRISPR/Cas9 technology would prevent fratricide, while knocking down T-cell receptor (TCR) expression through T-cell receptor alpha chain (TRAC) locus editing would prevent life-threatening graft- versus-host disease. However, new delivery technologies are needed to facilitate production of T-cell therapies requiring multiple genome edits. Inefficient transfection and combinatorial stochasticity can produce a final product that contain subsets of cells that are unsafe or ineffective, decreasing yield as well as product potency. The current goal standard is to perform knockout edits using a non-viral delivery system through electroporation. Electroporation when conducted serially for multiple genome edits results in a substantial decrease in cell proliferation and low yield. Alternatively, when performed as a batch process, electroporation can result in the interference of CRISPR edits, or worse, a plethora of double strand breaks that culminate in genomic instability and low proliferation in vivo. In this collaborative multiple principle investigator (mPI) proposal, we plan to test a novel microfluidic transfection technology to generate an effective CAR T-cell product for T-cell malignancies. Our microfluidic platform, called volume exchange for convective transfer (VECT) mechanoporation, is a non-viral, biomechanical approach that enables efficient delivery of genome editing products into the cell interior. It has the potential to permit multiple CRISPR edits with high transfection efficiency and viability, while being gentle enough to avoid detrimental off-target damage to therapeutic cells. VECT mechanoporation has shown low damage to the nucleus of T cells and therefore, offers a route to produce more proliferative therapeutic T cells. In Aim 1, we will establish the microfluidic device and process parameters to optimally deliver CD5 and TRAC CRISPR-Cas9 editing molecules to T cells, in both serial and multiplexed approaches. In Aim 2, edited T cells will be transduced with CD5-CAR encoding lentiviral vector and cytotoxicity will be tested in in vitro and in vivo experiments.