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