Poster - #UCLA-31 - Keywords: Cancer immunotherapy, CAR-T cell, gene delivery, acoustofluidics

Acoustofluidic Sonoporation Gene Delivery Utilizing DNA-encapsulated Supramolecular Nanoparticles for Cancer Immunotherapies

Yao Gong, Ruth A. Foley, Jason N. Belling, Tzu-Ting Chiou, David M. Cunningham, Micah Lim, Andras A. Heczey, Hsian-Rong Tseng, Satiro De Oliveira, Paul S. Weiss, Steven J. Jonas
1California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, United States. 2Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, United States. 3Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, United States. 4Children’s Discovery and Innovation Institute, University of California, Los Angeles, Los Angeles, CA, United States. 5Department of Pediatrics, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA, United States. 6Center for Advanced Innate Cell Therapy, Department of Pediatrics, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX, United States. 7Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, United States. 8Departments of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, CA, United States. 9Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, United States.

ABSTRACT:
Acoustofluidic Sonoporation Gene Delivery Utilizing DNA-encapsulated Supramolecular Nanoparticles for Cancer Immunotherapies Gene therapies that leverage engineered cells to produce a therapeutic effect via gene correction or modification are increasingly offering exciting treatment solutions for patients with cancer, genetic diseases, and infectious diseases. Recent decades have witnessed the advent of many remarkable trials including autologous stem cell transplantation for inherited disorders and hemoglobinopathies, and T cell-based cancer immunotherapy. Accelerating the clinical translation of these exciting clinical interventions will require the development and application of new methods for engineering target cell populations rapidly, efficiently, safely, and cost effectively. To address concerns with safety, immunogenicity, and limitations in cargo capacity of traditional viral-based gene delivery methods, we report an acoustofluidic intracellular delivery technology that enables precise sonoporation of cells as they are passed through a microfluidic channel. The acoustic pressure field generated within the microfluidic system is engineered to drive cells to the sidewall of a glass microcapillary, which transiently increases membrane permeability via shear forces to promote uptake of biomolecular cargoes. We have previously applied this acoustofluidic approach to demonstrate the delivery of model expression plasmid cargoes encoding for a non-integrating enhanced green fluorescent protein (eGFP) cassette to umbilical blood CD34+ hematopoietic stem and progenitor cells (CD34+ HSPCs) with 92% cell viability and 20% GFP expression. Here we adapt this biophysical cell therapy manufacturing platform for non-virally generating chimeric antigen receptor (CAR) T-cell populations via delivery of plasmids designed to encode for expression of a Glypican-3 (GPC3)-targeting CAR and that include a Sleeping Beauty transposon cassette, which we package into supramolecular nanoparticle (SMNP) carriers. Incorporation of therapeutic payloads into SMNPs adds new capabilities for packaging and delivering more complex and multiple cargo types during acoustofluidic processing. In preliminary tests in human primary T cells, we observed ~40% cell and stable integration of the CAR transgene with ~30% CAR expression after two weeks. We observe that the addition of a thermoelectric cooling system improves both the operation lifetime and output consistency of the acoustofluidic platform. To better understand the mechanisms of the acoustofluidic-mediated membrane disruption, we are beginning to probe the dynamics of membrane pore formation at both the plasma and nuclear membranes that occurs during acoustofluidic manipulation. Altogether, this study represents an initial step toward expanding the library of acoustofluidic-engineered cell products. Leveraging this versatile acoustofluidic platform to deliver an increasing array of biomolecular cargoes such as plasmids, mRNA, CRISPR-Cas9 cocktail ultimately will inform strategies for rapidly advancing the generation of future stem cell-based gene therapeutics.