Lipid Nanoparticle Formulation for mRNA Delivery

LNPs were formulated as previously described^{25 (link)}, with the lipid components (SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-2000 at molar ratios of 50:10:38.5:1.5) being rapidly mixed with an aqueous buffer solution containing ABE8.8 mRNA and either PAH1 gRNA or PAH2 gRNA in a 1:1 ratio by weight in 25 mM sodium acetate (pH 4.0), with an N:P ratio of 5.6. The resulting LNP formulations were subsequently dialyzed against sucrose-containing buffer, concentrated using Amicon Ultra-15 mL Centrifugal Filter Units (Millipore Sigma), sterile-filtered using 0.2-µm filters, and frozen until use. The LNPs had particle sizes of 69–89 nm (Z-Ave, hydrodynamic diameter), with a polydispersity index of <0.21 as determined by dynamic light scattering (Malvern NanoZS Zetasizer) and 90–100% total RNA encapsulation as measured by the Quant-iT Ribogreen Assay (Thermo Fisher Scientific).

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Brooks D.L., Carrasco M.J., Qu P., Peranteau W.H., Ahrens-Nicklas R.C., Musunuru K., Alameh M.G, & Wang X. (2023). Rapid and definitive treatment of phenylketonuria in variant-humanized mice with corrective editing. Nature Communications, 14, 3451.

Publication 2023

Amicon Ultra-15 mL Centrifugal Filter Units NanoZS Quant-iT Ribogreen Assay

Assay Buffer Cholesterol Distearoyl sn glycero 3 phosphocholine Frozen Hydrodynamic Lipid Molar Mrna Sm 102 Sodium acetate Sterile Sucrose

Corresponding Organization :

Other organizations : Cardiovascular Institute of the South, University of Pennsylvania, George Mason University, Children's Hospital of Philadelphia

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Protocol cited in 1 other protocol

Variable analysis

independent variables

Lipid components (SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-2000) at molar ratios of 50:10:38.5:1.5
ABE8.8 mRNA and either PAH1 gRNA or PAH2 gRNA in a 1:1 ratio by weight

dependent variables

Particle sizes of the LNPs (69–89 nm Z-Ave, hydrodynamic diameter)
Polydispersity index of the LNPs (<0.21)
Total RNA encapsulation (90–100%)

control variables

Rapid mixing of the lipid components with the aqueous buffer solution
Dialysis against sucrose-containing buffer
Concentration using Amicon Ultra-15 mL Centrifugal Filter Units
Sterile filtration using 0.2-µm filters
N:P ratio of 5.6

controls

Positive control: Not specified
Negative control: Not specified

Annotations

Based on most similar protocols

Optimize the N:P ratio for efficient encapsulation of mRNA and gRNA. The protocols used an N:P ratio of 5.6 (Protocol 1) or 6 (Protocol 2), which could be further optimized to improve RNA encapsulation efficiency.

Validate the quality of the LNP formulations by measuring particle size (Z-Ave, hydrodynamic diameter), polydispersity index, and total RNA encapsulation. These parameters should be within the desired ranges to ensure the integrity and effectiveness of the LNPs (Protocols 1, 2, 4, and 5).

Compare the lipid compositions used in the different protocols. Protocol 1 used SM-102, DSPC, cholesterol, and PEG-2000, while Protocols 2, 3, 4, and 5 used different lipid compositions. The choice of lipid components can impact the physicochemical properties and performance of the LNPs.

Ensure consistent pH conditions during the LNP formulation process. Protocols 1, 2, and 4 used sodium acetate buffer at pH 4.0-5.0, while Protocol 3 used sodium citrate buffer at pH 4.0 and Protocol 5 used citrate buffer at pH 3.0. Maintaining the appropriate pH is crucial for efficient encapsulation and stability of the LNPs.

Consider the use of positive controls, such as well-characterized LNP formulations with known performance, to validate the reproducibility and accuracy of the LNP preparation and characterization methods across different laboratories (not explicitly stated in the protocols).

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