Sequential Delivery of Genetic Coding Molecules Using An Acoustic-Electric Microfluidic Platform

Introduction: Intracellular delivery of multiple genetic coding molecules is an essential step in cellular and gene therapies. One major bottleneck is that a majority of currently existing technologies utilize co-transfection methods, commonly by mixing target molecules and then delivering them simultaneously. Consequently, co-transfection has fallen short in terms of transfection efficiency due to interactions and/or competition between cargos during intracellular delivery. Here we present a new modality of our previously reported a microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP), for sequential intracellular transfection of genetic coding cargos with high throughput. AESOP utilizes acoustic microstreaming to trap millions of cells in a series of microvortices, allowing for the sequential pumping of target molecules. The cells are then transfected using a combination of mechanical shearing and electric field. As a proof of concept, we demonstrate that the sequential transfection of mRNA or plasmid with Cas9 ribonucleoprotein (RNP) via AESOP overcomes the co-transfection challenge of obtaining efficient multi cargo delivery. The utilization of AESOP for sequential delivery facilitates the development of cellular/gene therapies based on multiplex intracellular delivery such as cancer immunotherapy, mRNA therapies, and CRISPR-Cas9 gene editing.

Materials and

Methods: Sequential and co-transfection was evaluated with Cas9 RNP in conjunction with CleanCap mCherry mRNA or pmaxGFP plasmid in K562 and HeLa cells, respectively. Cas9 RNP expression was assessed through transmembrane protein CD47 knockout (KO). The sgRNA sequence used to target CD47 was ATCGAGCTAAAATATCGTGT. KO efficiency was evaluated using flow cytometry 7 days after transfection upon staining with a PE anti-human CD47 antibody. mRNA expression was evaluated by fluorescence using flow cytometry one day after transfection. Plasmid expression was evaluated by fluorescence using flow cytometry two days after transfection. Sequential transfection was performed using AESOP (Figure 1A). Acoustic microstreaming vortices was formed by placing the chip on top of a piezoelectric transducer (PZT) run by a square wave with 6 Vpp and 50.2 kHz frequency. Cell membrane pores were modulated by applying a sinusoidal wave with 70 Vpp amplitude and 30 kHz frequency to the interdigitated electrodes (IDE) for 6 ms (Figure 1B). This was applied two times at each delivery step. Co-transfection was performed using the Lonza 4D Nucleofector X Kit S.

Results, Conclusions, and Discussions: In K562 cells, we looked mRNA expression at two concentrations (0.5ug, 1ug) with 60pmol Cas9 at two different molar ratios of Cas9:sgRNA (1:1, 1:3). At a 1:1 molar ratio of Cas9:sgRNA, mRNA expression was ~20 and ~15 fold higher using sequential delivery compared to co-transfection at 0.5ug and 1ug mRNA, respectively (Figure 2A). At a 1:3 molar ratio of Cas9:sgRNA, there was no significant difference in mRNA expression between sequential delivery and co-transfection (Figure 2B). Next, we evaluated the CD47 KO efficiency with 60pmol Cas9 with a Cas9:sgRNA molar ratio of 1:3 with two mRNA concentrations (0.5ug, 1ug). There was no significant difference in CD47 KO efficiency between sequential delivery and co-transfection, both of which are comparable to the KO efficiency when there is no mRNA present (Figure 2C). We then looked at plasmid expression at three concentrations (0.5ug, 1ug, 1.5ug) with 60pmol Cas9 at two different molar ratios of Cas9:sgRNA (1:1, 1:3) in HeLa cells. At a 1:1 molar ratio of Cas9:sgRNA, plasmid expression was ~23, ~6, and ~3 fold higher using sequential delivery compared to co-transfection at 0.5ug, 1, and 1.5ug plasmid, respectively (Figure 2D). At a 1:3 molar ratio of Cas9:sgRNA, plasmid expression was ~6, ~1, and ~2 fold higher using sequential delivery compared to co-transfection (Figure 2E). Based on the results, sequential delivery significantly increases both mRNA and plasmid expression compared to co-transfection in conjunction with a 1:1 molar ratio of Cas9:sgRNA. Interestingly, when the Cas9:sgRNA molar ratio was increased to 1:3, the difference between sequential and co-transfection became significantly less prominent. We suspect that Cas9 protein not bound to sgRNA interacts with mRNA. Thus, by increasing the sgRNA concentration, the chances of mRNA interacting with unbound Cas9 protein is decreased. The proof of concept using AESOP to sequentially deliver mRNA and plasmid in conjunction with Cas9 RNP demonstrates the ability to sufficiently express multiple cargos simultaneously, overcoming the challenges present with co-transfection. We aim to ultimately use this system in hopes of developing universal CAR T cells where mRNA or plasmid encoding CSPG4-CARs will be delivered while simultaneously knocking out endogenous TCR, β-2 microglobulin (B2M).

Acknowledgements (Optional):