Andre Wild

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.

Microfluidic-Based Manufacture of siRNA-Lipid Nanoparticles for Therapeutic Applications

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.

A Scalable Microfluidic Platform for the Development of LipidNanoparticles for Gene Delivery

Extended Abstract Microfluidic devices have been broadly used to produce nucleic acid-delivery nanoparticles for genetic medicine because they offer control, reproducibility and scalability of the nanoparticle precipitation process to overcome a significant challenge in the translation of these therapeutics [1-5]. Control over process parameters afforded by microfluidics, allows optimization of nanoparticle quality and encapsulation efficiency [2]. Automation improves the reproducibility and optimization of formulations. The continuous nature of the microfluidic process is inherently scalable, allowing optimization at low volumes to conserve scarce or costly materials, and seamless scale-up of optimized formulations by employing multiple microfluidic mixers performing identical unit operations in parallel. In this study, we present a scalable microfluidic platform for producing nanomedicines. The platform includes a system designed for production under cGMP conditions employing 8 parallel microfluidic mixers capable of producing a 25 L formulation of RNA lipid nanoparticles (LNP) in ~4 h. Seamless scale up of production was demonstrated by producing test batches of siRNA-LNPs against the blood clotting protein Factor VII (FVII) on each of 3 systems designed for different stages of nanomedicine development. The physico-chemical characteristics were determined by DLS, and HPLC, and in vivo efficacy was measured by assaying serum FVII levels in murine models. With a system designed for bench-scale formulation development we produced 10 mL batches of siRNA LNPs of avg. diameter ~60 nm (PDI <0.1) with encapsulation efficiency >95 %. No differences were observed in physicochemical properties of these particles when batch sizes were scaled-up by 10x on a pre-clinical scale-up system or by 100x with a system employing 8 microfluidic chips arrayed in parallel. The particles exhibited consistent lipid composition and N/P ratio within the target specifications. In addition, nanoparticles manufactured across the microfluidic platform showed a similar dose-dependent gene knockdown achieving >90 % reduction in protein levels at a dose of 1 mg/kg. These studies demonstrated the seamless scale-up of nanoparticle formulations across the platform with the potential for producing large scale, clinically relevant volumes, of lipid nanoparticles. The system employing 8 parallel mixers can prepare up to 25 L of product under 4.5 hours at 12 mL/min per mixer and incorporates a disposable fluid path that eliminates the need for costly and time consuming cleaning validation.

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.