Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency

Abstract

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1+) and human (CD34+) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.

A Microfluidic Device to Enhance Viral Transduction Efficiency During Manufacture of Engineered Cellular Therapies

“…Tran et al . also achieved 2.5 to 4-fold improvements in transduction efficiencies using microfluidic channels 24 . In their work, Tran and colleagues used geometric constraints related to the microfluidic channels to shorten the diffusion path length for the initial binding of cells with vector.…”

“…While these methods yielded a high rate of transduction, a significant fraction of virus flows past target cells and through the membrane without interaction, and therefore the efficiency of vector usage (described as the ratio of cells transduced to number of virus particles used) is low, reducing the utility of this method for clinical-scale manufacturing. Alternatively, microfluidic channels have been used to colocalize target cells and concentrated virus in microliter volumes resulting in >4 fold increases in transduction efficiency relative to static controls 24 . Such microchannels work most efficiently at volumes where cells are present at multi-fold higher concentration above typical culture conditions leading to rapid depletion of nutrients and oxygen and limiting the time in which cells can reside in the device.…”

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis

“…Microfluidic channels were fabricated as previously described (Tran et al, 2017). Briefly, double sided adhesive sheets (2 in wide, 9 cm long, 125 mm thick) were backed with non-silicone release liner into which a U-shaped channel consisting of two parallel 4 mm wide by 8 cm long channels connected by a 4 mm wide by 1.5 cm long perpendicular channel ( Figures 4A and 4B) was cut.…”

High-Throughput Platform for Efficient Chemical Transfection, Virus Packaging, and Transduction

“…Microfluidic devices for gene delivery have been numerously reported, which offer the advantages of high throughput, low reagent consumption, and independently controlled liquid conditions, however most of the studies focused on chemical transfection [13,14] or electroporation [15,16]. Recently, microfluidic systems were employed to increase virus production [17] and improve the target cell transduction efficiencies [18,19] through mass transport-based approaches that overcome the diffusion limitation of the viral particles. However, a high throughput platform integrating virus packaging and transduction has not been reported.…”