My Publications

Nanoparticle-based RNA delivery has shown great progress in recent years with the approval of two mRNA vaccines for SARS-CoV-2 and a liver-targeted siRNA therapy. Here, we discuss the preclinical and clinical advancement of new generations of RNA delivery therapies along multiple axes. Improvements in cargo design such as RNA circularization and data-driven untranslated region optimization can drive better mRNA expression. New materials discovery research has driven improved delivery to extrahepatic targets such as the lung and splenic immune cells, which could lead to pulmonary gene therapy and better cancer vaccines, respectively. Other organs and even specific cell types can be targeted for delivery via conjugation of small molecule ligands, antibodies, or peptides to RNA delivery nanoparticles. Moreover, the immune response to any RNA delivery nanoparticle plays a crucial role in determining efficacy. Targeting increased immunogenicity without induction of reactogenic side effects is crucial for vaccines, while minimization of immune response is important for gene therapies. New developments have addressed each of these priorities. Lastly, we discuss the range of RNA delivery clinical trials targeting diverse organs, cell types, and diseases, and suggest some key advances that may play a role in the next wave of therapies. 

Nanoparticles formed by electrostatic assembly of nucleic acids with cationic lipids or polymers are the most advanced non-viral vehicles for gene delivery. The nanoparticle size is a central property of these vehicles as it not only determines their physical characteristics, such as payload capacity and relative surface area, but also drives their biological performances such as biodistribution, intracellular trafficking (including cellular uptake and endosomal escape), cargo release, and clearance. Therefore, to precisely control the size of nucleic acid delivery vehicles and to optimize it for different in vitro and in vivo applications is of great importance for successful gene delivery. However, most literature regards nanoparticle size only as a quality control measure, while a diameter around 100 nm is default as target without a strong basis. We attribute this to the lack of approaches to understanding and controlling the size of nucleic acid delivery vehicles.

Our research group has been at the forefront of studying the macromolecular assembly of nucleic acid nanoparticles. We first demonstrated that due to the fast kinetics of electrostatic complexation between negatively charged nucleic acids and positively charged polymers, a faster mixing kinetics of the two materials is critical to assembly uniformity and is a prerequisite for size control. By controlling the concentration of nucleic acids, the size of polymeric DNA nanoparticles can then be controlled between 30 to 200 nm through a diffusion-dominated mechanism. With emerging evidence of the value of a vehicle size above 200 nm, we subsequently explored secondary nanoparticle assembly of sub-100 nm “building blocks” through modulation of nanoparticle surface charge as a switch for size growth and stabilization. Through controlling surface protonation degree and charge screening by solution pH and ionic strength, respectively, polymeric DNA nanoparticles were assembled with a freely tunable size between 200 and 1000 nm. Combining both studies gives rise to a generalizable strategy to control the size of nucleic acid delivery vehicles in the full nano-to-micro size range of 30 to 1000 nm. We showcased the generalizability by switching the carrier from poly(ethylenimine) (PEI) to poly(beta-amino ester) (PBAE), and the cargo from DNA to messenger RNA (mRNA), and achieved size control between 100 to 1000 nm with a neutral surface coating, suitable for in vivo delivery. With this PBAE/mRNA system, we realized optimal in vivo gene delivery to monocytes and macrophages by size-controlled 400-nm particles.

Most recently, we broadened our understanding of the size of nucleic acid delivery vehicles by characterizing cargo loading and loading-size correlations at the single-nanoparticle level through modern chromatographic and spectroscopic techniques. While we found that, on average, the loading of nucleic acids positively correlates with vehicle size, a significant degree of heterogeneity in loading density was discovered in the commonly referenced RNA lipid nanoparticle (LNP) delivery system. This means that size uniformity, which has always been a priority in optimizing nanoparticles, does not spontaneously mean loading uniformity. It inspires further developments for loading optimizations of nucleic acid-loaded LNPs to boost their biological performance.

Cost-effective and scalable production is critical for advancing the clinical translation of adeno associated virus (AAV)-mediated gene therapy. The widely used transient transfection method using plasmid DNA (pDNA)-loaded transfection particles for AAV production faces technical challenges due to instability of the particles and the concentration limits for particle preparation, hindering reproducibility and scalability. Here, we report a streamlined and scalable strategy to generate shelf-stable, highly concentrated pDNA/poly(ethylenimine) (PEI) transfection particles. By incorporating trivalent citrate ions in the dilution buffers, we kinetically modulate electrostatic complexation to achieve uniform nanoparticle assembly and prevent aggregation at high concentrations. This enables a tenfold increase in pDNA concentration in stabilized transfection particles from a typical range of 10–20 μg/mL to 200 μg/mL, while reducing the required dosing volume from 5–10% to 0.5% of the cell culture medium. The particle assembly process is robust to changes in mixing scale and timing and is compatible with standard workflows. We demonstrate equivalent AAV production efficiencies to standard methods and consistent performance in various production scales, which confirms the practical utility of this assembly method in developing robust, scalable, and cost-effective AAV manufacturing processes. 

Size-dependent phagocytosis is a well characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate inter-nanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester) (PBAE)/mRNA nanoparticles into particles with a size that is kinetically tunable between 200 to 1000 nm with high colloidal stability in physiological media. We discovered that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anti-cancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables new therapeutic paradigms via monocyte programming.

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multi-component LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.

The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400 to 500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a 2-inlet confined impinging jet (CIJ) mixer with a dual syringe pump setup, we produced a 1-L batch at a flow rate of 100 mL/min; and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot-size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%–80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.

Polyelectrolyte complex particles assembled from plasmid DNA (pDNA) and poly(ethylenimine) (PEI) have been widely used to produce lentiviral vectors (LVVs) for gene therapy. The current batch-mode preparation for pDNA/PEI particles presents limited reproducibility in large-scale LVV manufacturing processes, leading to challenges in tightly controlling particle stability, transfection outcomes, and LVV production yield. Here we identified the size of pDNA/PEI particles as a key determinant for a high transfection efficiency with an optimal size of 400–500 nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based approach to assemble size-controlled and shelf-stable particles using preassembled nanoparticles as building blocks and demonstrated production scalability on a scale of at least 100 mL. The preservation of colloidal stability and transfection efficiency was benchmarked against particles generated using an industry standard protocol. This particle manufacturing method effectively streamlines the viral manufacturing process and improves the production quality and consistency. 

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at −20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.