Timothy Leaver

Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities.

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by “flow-controlled wetting,” a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

614. Microfluidic Manufacture of RNA-Lipid Nanoparticles Leads to Highly Efficient Delivery of Potent Nucleic Acid Therapeutics for Controlling Gene Expression

Lipid nanoparticles (LNPs) have demonstrated efficient nucleic acid delivery in vitro and in vivo, as well as in clinical development. They exploit endogenous delivery pathways, by co-opting apolipoprotein E (apoE), to mediate effective delivery of the encapsulated nucleic acids into cells via the low-density lipoprotein receptor (LDLR). However, use of LNPs from the bench to the clinic has been considerably limited by challenges in manufacturing at both small and large scales. Here, we bridge that gap by describing the robust manufacture and use of clinical-grade lipid-based nanoparticles for highly efficient delivery of nucleic acids at scales suitable for both in vitro screening and in vivo applications.RNA-LNPs manufactured using an optimized microfluidic platform enables efficient encapsulation of nucleic acids (e.g. siRNA, mRNA, pDNA) into biocompatible “solid-core” nanoparticles (~50 nm). The resultant nanoparticles can then be applied to cell cultures in vitro or administered in vivo. The following reports a comprehensive set of studies conducted to evaluate the merits of the technology and further provide insights for delivering short interfering RNA (siRNA) and mRNA in difficult-to-transfect cells both in vitro and in vivo.RNA-LNPs were formulated to encapsulate a potent siRNA directed against PTEN - a clinically relevant gene associated with neural regeneration. Exceptional cellular uptake (>98%) with minimal toxicity was observed in both primary rat hippocampal and mixed cortical cell cultures. High transfection efficency (>95%) of the encapsulated material resulted in concomitant high-level (>85%) PTEN knockdown within the first 4 hours of a low dose (100 ng/ml) treatment; that level of knockdown was further sustained for 21 days. Similarly, RNA-LNPs encapsulating mRNA were also found to mediate early ( 75% for 7 days) following a single (500 ng/ml) treatment in primary rat mixed cortical cultures.Strategies for locally administering RNA-LNPs into the brain and spinal cord of adult Sprague Dawley rats were also investigated. Controlled localized injections of PTEN-encaspulated siRNA into the motorcortex resulted in significant and sustained (7 days) knockdown. Similarly, local administration at the site of a cervical spinal cord injury significantly reduced target PTEN expression, 10 days later. Visible uptake of RNA-LNPs characterized by their presence in the soma of neurons found in the red nucleus provides further insights into a regtrograde transport mechanism involving the axons.Collectively, these studies reflect the simplicity and efficacy of this commercially available technology in presenting a cost-effective and advantageous avenue for screening and validating new targeted nucleic acid therapies.