Liquids in Dishes in a Lab
Messenger RNA

mRNA Therapeutics GraphicManipulating gene expression with mRNA has several important advantages over viral DNA delivery.

 

• Lower risk of immune reactions
• Avoids the risk of permanent integration into the host genome
• Transient expression

 

These advantages make mRNA a desirable solution both in vitro and in vivo to express:

 

• Therapeutic proteins and peptides
• Vaccine antigens
• Gene editing components such as Cas9, transposons, and other nucleases

 

However, mRNA requires a delivery vehicle to protect against nucleases and facilitate cellular uptake and release into the cytoplasm. Lipid nanoparticles (LNPs) are the leading non-viral delivery system for mRNA that effectively solves several challenges facing mRNA delivery:

 

• Protection from nucleases
• Delivery and release into the cytoplasm
• Laborious and time-consuming viral packaging
• Safety concerns with viral vectors
• Limitations in nucleic acid length with viral vectors.


Overcome Challenges in mRNA Delivery

Nanoparticle formulations offer a desirable alternative to viral delivery and with NanoAssemblr® technology, these formulations can be prepared on demand in seconds. Several nanoparticle formulations are being explored to package and deliver mRNA including:

Liposome
Liposomes
Polymeric Nanoparticle and Micelles
Polymer NPs and Micelles
Nucleic Acid Lipid Nanoparticle LNP
LNPs

Among these, nucleic acid-LNPs are the most clinically advanced. There are however challenges to producing mRNA-loaded nanoparticles that NanoAssemblr® technology addresses:


Challenges with Conventional Methods Benefits of NanoAssemblr® Technology
Limited control over particle sizeorangeRightArrowFine-tune particle size by changing formulation parameters
Significant batch-to-batch variabilityorangeRightArrowLaminar conditions lead to highly reproducible mRNA lipid nanoparticles
Substantial material loss from low encapsulation efficiencyorangeRightArrowAchieve high mRNA encapsulation efficacy and potency
A labor-intensive production process that is difficult to scale-uporangeRightArrowRapidly produce formulations for screening and optimization with a straightforward path to scale-up for clinical applications

Key Benefits


Fine-Tuning Particle Size by Changing Formulation Parameters


mRNA Size Tuning NanoAssemblr SparkControl particle size by changing lipid:RNA ratio (top) and composition of lipids (bottom) among others. Size and PDI measured by dynamic light scattering from triplicate formulations produced using the NanoAssemblr® Spark.


Highly Reproducible mRNA LNP Formulations


mRNA Consistency BenchtopEncapsulation efficiency (left) and particle size & PDI (right) were consistent between formulations with different mRNA lengths. Formulations were produced in triplicate on the NanoAssemblr® Benchtop.


High Encapsulation Efficiency and Potency


Encapsulation Efficiency & PotencyFlow cytometry showing expression of reporter protein GFP on primary rat cortical neurons 48h after in vitro treatment with mRNA-LNP.  GFP expression was dose-dependent with over 90% of cells expressing GFP at doses as low as 0.5 µg RNA per mL media. mRNA LNPs were produced on the NanoAssemblr® Spark.


Straightforward to Scale Production


Consistent Results on NanoAssemblrConsistent results for size, encapsulation efficiency, composition and morphology achieved across the NanoAssemblr® Platform.

How It Works

mRNA Production and Delivery



1) An organic solvent containing dissolved lipids and an aqueous solution containing nucleic acids are injected into the two inlet channels of the NanoAssemblr® cartridge.
2) Under laminar flow, the two solutions do not immediately mix, but microscopic features engineered into the channel cause the two fluids to intermingle in a controlled and reproducible way.
3) Within a millisecond, the two fluids are completely mixed, causing a change in solvent polarity that triggers the self-assembly of lipid nanoparticles loaded with nucleic acids.


4) Changing the speed and ratio of fluid injection controls the size of the lipid nanoparticles.
5) Lipid nanoparticles mimic low-density lipoproteins, which allows them to be taken up by an endogenous cellular transport pathway to deliver nucleic acids to cells.
6) Using pH-sensitive lipids allow lipid nanoparticles to release encapsulated nucleic acids into the cytoplasm when vesicle pH decreases.

mRNA Resources

Poster

July 01, 2018

Robust low-volume production of nanoparticles for genetic manipulation of cells

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Publication - Abstract

April 26, 2018

Small Methods

State‐of‐the‐Art Design and Rapid‐Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

Evers, M. J. W., Kulkarni, J. A., van der Meel, R., Cullis, P. R., Vader, P., & Schiffelers, R. M.

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Publication - Abstract

February 27, 2018

Cell Reports

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

J. Finn, A. Smith, M. Patel, L. Shaw, M. Youniss, J. Heteren, T. Dirstine, C. Ciullo, R. Lescarbeau, J. Seitzer, R. Shah...

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Publication - Abstract

June 15, 2018

Molecular therapy

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis

E Robinson, KD MacDonald, K Slaughter, M McKinney

Read More

Publication - Abstract

June 13, 2018

Journal of nucleic acids

SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency

B Connolly, C Isaacs, L Cheng, KH Asrani, R R Subramanian

Read More

Poster

June 11, 2018

Rapid development and seamless scale-up of genetic nanomedicines

Read More PDF
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