Abstract In recent years, the field of gene therapy has taken significant strides towards clear cut demonstrations of safety, effectiveness, and superiority to current therapies for a variety of human diseases. Two approaches in particular, lentiviral vector (LV)-based hematopoietic stem cell (HSC) and T cell gene therapies, are especially promising. However, two major limitations, and definitive commercialization barriers, remain, which are the low efficiency of current clinical LV transduction protocols and the need inefficient and scale-limited manufacturing methods. As clinical gene therapy necessitates the transduction of high numbers of HSCs or T-cells, usually in the range of 107-8 cells, the low efficiency of clinical transduction protocols involving even maximal LV concentrations requires the production of logistically non-feasible quantities of LV for treating patient populations at large. While various methods and technologies have been developed with some achieving a degree of success, transformative strategies to improve LV-based gene transfer will be critical to bring HSC and T cell gene therapies closer to mainstream clinical utilization. To that end, the Lam bioengineering laboratory has recently reported the development of microfluidic devices that demonstrates significantly improved LV transduction efficiency (Tran R et al, Molecular Therapy, in press). The microfluidic platform leverages high surface area to volume ratios of microchannels and fluid mechanics and mass transport principles to increase the probability of interaction between LV particles in suspension and typically non-adherent cells bound to the microfluidic surface. For this Bioengineering Research Grant (BRG), the Lam bioengineering laboratory and Doering and Spencer gene therapy labs will continue to partner to further research, develop/optimize and translate to scale and clinical application our platform technology. We will test the general hypothesis that microfluidic systems improve LV transduction efficiency of CD34+ HSCs and human T cells while minimizing LV product waste. Collectively, these advances will shift the current paradigm in clinical LV gene therapy by removing the two major barriers to widespread clinical success. Furthermore, our studies will provide a more comprehensive understanding of the general guiding biophysical principles of microfluidic devices and the LV transduction process. This, in turn, will enable us to optimize our microfluidic devices further and to develop scaled-up systems capable of transducing >108 CD34+ cells and >107 T cells. We will then test the full scale microfluidic device to facilitate LV transduction of both human CD34+ cells and T cells using a GFP-reporter LV as well as two clinically-relevant LV-based products currently under preclinical development in the Aflac Gene Therapy Program under the direction of Drs. Doering and Spencer. Overall, these studies will apply microfluidic technology to introduce a new para...