614. Microfluidic Manufacture of RNA-Lipid Nanoparticles Leads to Highly Efficient Delivery of Potent Nucleic Acid Therapeutics for Controlling Gene Expression
Authors: Grace Tharmarajah, Eric Ouellet, Oscar Seira, Jie Liu, Anitha Thomas, Timothy Leaver, Andre Wild, Yuping Li, Yu Tian Wang, Wolfram Tetzlaff, Carl Hansen, Pieter Cullis, James R. Taylor, Euan Ramsay
Journal: Molecular Therapy (2016)

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 (< 4 hours) and sustained gene expression (>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.