LNP Formulation

LNP Service Options

Manufacturing Workflow

Our integrated IVT RNA manufacturing workflow streamlines production from gene synthesis to LNP packaging.

Codon/ Ready-to-use UTR designs

• Codon optimization for protein expression
• Codon optimization with >200 factors screened (patent)
• Screened and validated UTR sequences

Gene
Synthesis

• 100 bp – 200 kb gene inserts
• TAT as fast as 7 BD to 14 BD
• Gene synthesis with Poly(A)-built-in vectors

Cloning/ Plasmid Preparation

• Ready-to-use linearized plasmid DNA for IVT
• Vectors encoded stable Poly(A) tracts with variance of only +/- 5 nt

mRNA
Synthesis

• RUO to GMP-like grade
• Advanced purification (Oligo dT, dsRNA removal) and variety of QC options

Lipid Nanoparticles Delivery

• Generic LNP, Targeted LNP  and ReadyEdit LNP (for gene editing)
• Load  0.1 mg ~ Gram Level mRNA/circRNA
  /saRNA/siRNA/ASO
  /DNA
• Load up to 20 kb mRNA

LNP for Different Payloads

• mRNA

• siRNA

• DNA

• Peptide

GenScript’s LNP platform demonstrates robust performance with mRNA payloads, showing uniform morphology, low empty-particle rate, high transfection efficiency, and potent in vivo delivery.

GenScript LNPs showed uniform morphology under cryo-EM characterization

GenScript LNPs exhibit a low percentage of empty LNPs (<2%).

GenScript LNPs demonstrate high transfection efficiency.GenScript LNP showed better transfection efficiency than Lipofectamine

GenScript LNPs showed strong in vivo signal 8 hours after IV (left) or IM (right) injection in mice

GenScript siRNA-LNP Demonstrates Effective Gene Silencing

LNP 1: EGFP mRNA-LNP (SM102)

LNP 2: EGFP siRNA-LNP (MC3)

LNP 3: Scramble siRNA-LNP (MC3)

When the EGFP-mRNA-LNP (LNP 1) were co-transfected with EGFP-siRNA-LNP (LNP 2), there is no EGFP expression observed (LNP 1 + LNP 2), indicating the excellent gene silencing effects of siRNA LNP; Scramble-siRNA-LNP (LNP 3) was served as positive control (LNP 1 + LNP 3)

LNPs for Efficient Plasmid DNA Delivery

SM102-NLS formulation

Incorporation of the NLS peptide into the general SM102 LNP formulation significantly enhances plasmid DNA delivery efficiency.

C12-200 formulation

The C12-200 LNP formulation exhibits significantly higher plasmid DNA delivery efficiency compared with the general SM102 formulation.

Peptide encapsulation with LNP

FITC-labeled peptide was either directly incubated with A549 cells or encapsulated in LNPs for delivery. Fluorescence intensity and cell viability were analyzed 24 hours post-transfection. LNP encapsulation markedly enhanced peptide delivery efficiency with minimal cytotoxicity.

Novel LNPs

GenScript is introducing novel LNP formulations featuring unique lipid compositions that deliver strong in vitro and in vivo performance, comparable to or exceeding benchmark systems like SM102 and ALC0315. These next-generation LNPs also avoid dependence on conventional benchmark lipids and show promising tissue/cell targeting potential, offering powerful new options for mRNA delivery, vaccines, gene editing, and cell therapy.

• CXM-2

• CX108

• CX175

• SM-CX712

mRNA-LNP for hCCR9 antibody production. We designed hCCR9 mRNA sequence and encapsulated the mRNA into different LNP formulations, and the results showed the mRNA-LNPs could successfully induce corresponding antibody of hCCR9 protein in mice. 1WP3: 1 week post 3^rd serum; 7WP3: 7 weeks post 3^rd serum; 11WP3: 11 weeks post 3^rd serum.

For in vitro test, HEK293T Cells were plated in 6-well plate at 300,000 cells per well. Cells were transfected with 2.5ug of mRNA-LNP per well. 48h after transfection, the expression of hCCR9 protein was detected using flow cytometry analysis with primary antibody against CCR9 and fluorescent secondary antibody.

For in vivo test, mice were intramuscularly injected with hCCR9 mRNA encapsulated in SM102, CXM-2, or ALC-0315 LNPs (0.5 mg/kg), without adjuvants. Mice received multiple immunizations, and serum samples were collected at weeks 1, 7, and 11 after the third immunization. To assess the CCR9-specific antibody response, a hCCR9 overexpressing CHO-K1 stable cell line was used as the screening material. Cells were incubated with diluted mouse serum, followed by staining with a fluorescently labeled secondary antibody, and analyzed by flow cytometry.

CXM-2 is also a promising candidate for gene editing. See ReadyEdit LNPs for prime editing application

CX108 LNP exhibited comparable in vivo performance to the benchmark SM102 LNP, and demonstrated enhanced expression in primary T cells after CD3 antibody conjugation, highlighting its potential for in vivo CAR-T applications.

CX175 LNP exhibited comparable in vivo performance to the benchmark SM102 LNP, and demonstrated enhanced expression in primary T cells after CD3 antibody conjugation, highlighting its potential for in vivo CAR-T applications.

SM-CX712 LNP can enhance RNA’s distribution to muscle, and reduce its expression in liver by IM injection

Quality Control and Specification

Case Studies

• GenScript's LP01-LNP Effectively Delivers Cas9 mRNA/sgRNA to HEK293T Cells
  • Espcas9 mRNA/sgRNA targeting TARC (GenScript) were formulated in LP01-LNP formulation at 1:1 mass ratio achieved 97% editing efficiency, comparing to 61% by lipofectamine.
  • 2.4 ug of RNA/LNP formulation were incubated with HEK293T cells, cell were lysed at day 3 and day 5 for PCR and sanger sequencing.

  
• GenScript’s SM102-LNP Outperforms Other Delivery Methods in Immune Cells
  

  

  

  

  

  

  Transfection efficiency was compared by delivering eGFP mRNA (100% N1-methyl-pseU modified, GenScript) into Jurkat and THP-1 immune cells using SM102-LNP (GenScript), Lipofectamine™ MessengerMAX™ (ThermoFisher Scientific) and TransIT^® (Mirus). 100ng of mRNA were transfected into cells in 24 well plates. Starvation medium (OptiMEM) was used for lipofectamine and TransIT per the manufacturer’s instruction, and complete medium was used for LNP. The expression of eGFP was measured by flow cytometry after 48 hours.

• GenScript’s MC3-LNP Outperforms Other LNP Formulations in Mouse via IM Injection
  

  

  

  

  • GS-MC3 LNP formulation has strong mRNA expression
  • GS-MC3 LNP as generic formulation for customers’ mRNA screening project

  In vivo delivery efficiency of four different LNP formulations was compared by delivering Fluc mRNA (100% N1-methyl-pseU modified, GenScript) into Balb-C mice via IM injection at a dose of 0.25mg/kg. The expression of Fluc mRNA was measured by whole-body bioluminescence imaging.  

  Fluc mRNA delivered by MC3-LNP showed the highest expression level after 8 and 48 hours by imaging at both Dorsal and Ventral pose of mice. To evaluate the biodistribution profile of different formulations, the heart, liver, lung, spleen, kidney, and brain of the mice were collected and imaged after 48 hours, MC3 -LNP formulation showed the strongest liver accumulation compared to others after 48 hours.

• GenScript’s SM102-LNP Outperforms Other LNP Formulations in Mouse via IV Injection
  

  

  

  

  • Both LNP formulations are safe for IV injection
  • SM102 has stronger liver accumulation among the two tested formulations

  In vivo delivery efficiency of two different LNP formulations was compared by delivering Fluc mRNA (100% N1-methyl-pseU modified, GenScript) into Balb-C mice via IV injection at a dose of 0.3mg/kg. The expression of Fluc mRNA was measured by whole-body bioluminescence imaging.  

  In imaging analysis, Fluc-mRNA delivered by SM102-LNP showed higher expression efficiency than MC3 after 8 and 48 hours. To evaluate the biodistribution profile of different formulations, heart, liver, lung, spleen, kidney, and brain of the mice were collected and imaged after 48 hours, SM102 -LNP showed stronger liver accumulation compared to MC3-LNP after 48 hours. MC3-LNP showed stronger spleen accumulation after 48 hrs. Neither formulation caused significant body weight loss during the experiment period for 3 days.

Resources

LNP Case Study Report

Case studies demonstrating encapsulation and delivery efficiency of GenScript’s new LNP formulations in cell models, and mouse models via both IM and IV.

Free Download

Questions On Storage And Usage Guidelines_mRNA, saRNA and LNP

Free Download

mRNA LNP in vitro Cell Transfection

Free Download

FAQs

• What is an LNP?

  Lipid nanoparticles (LNPs) are natural or synthetic molecules that can self-assemble into a spherical structure. LNPs are typically composed of lipid layer, which forms a shell around an inner compartment that can be used to deliver drugs or other therapeutic agents. LNPs have become increasingly popular as a drug delivery system due to their ability to protect therapeutic agents from degradation and improve their bioavailability. They can also be designed to target specific cells or tissues, allowing for more precise delivery of drugs. LNPs have been used in various applications, including the delivery of RNA-based therapeutics such as mRNA vaccines. The lipid layer of LNPs can fuse with the cell membrane, allowing the encapsulated RNA to enter the cell and exert its therapeutic effect. LNPs have also been investigated as a potential delivery system for other types of drugs, including small molecules and proteins.

• What’s the mRNA length limit that we can load in LNP?

  Our LNP service encapsulates 20nt - 10knt RNA.

• What are the differences between LNP formulations?
  1. MC3 LNP is formulated with the main ionizable lipid MC3, which was used for siRNA LNP formulation, it is less effective in mRNA delivery compared to SM102 and ALC0315 formulation.
  2. SM102 LNP was used in COVID vaccine, it has good delivery efficiency for mRNA, and accumulates in the liver.
  3. ALC0315 LNP was used in COVID vaccine, it has good delivery efficiency for mRNA and CRISPR (mRNA +sgRNA), and accumulates in the liver.
  4. LP01 LNP was tested for its efficienct delivery of CRISPR (mRNA +sgRNA).
• Are GenScript LNP formulations cell or organ targeted?

  All of these LNP formulation (MC3 / SM102/ ALC0315 or LP01) are not active targeting formulations, so they tend to accumulate in the liver instead of targeting to specific tissues. However, if you need organ-specific LNP, we can offer customized formulation per the customer's request. Please reach out to our technical support team for details.

• What is the mRNA LNP transfection protocol?
  

  mRNA LNP In Vitro CellTransfection Protocol GenScrip

  Free Download
• Which lipid do we recommend for mRNA-LNP?

  We recommend SM102 and ALC0315 LNPs for mRNA, these two has shown great efficiency in delivery mRNA in vivo. You can acquire off-the-shelf LNP loaded with mRNA coding for marker gene to perform initial studies for their project needs.

• Which lipid do we recommend for oligo-LNP?

  MC3-LNP has been successfully used in siRNA delivery previously.

• Can GenScript encapsulate sgRNA and Cas9 mRNA in LNP?

  Yes, We can formulate multiple guide RNA and Cas9 mRNA into one LNP formulation. GenScript also provides sgRNA synthesis service up to 160nt long and customized Cas9 virants mRNA production.

• Can GenScript encapsulate with customer-provided lipid and mRNA?

  Yes, we do. Please reach out to our technical support team for details.

• Does GenScript offer LNP packaging without mRNA synthesis?

  Yes, we do. Please reach out to our technical support team for details.

• How does GenScript prepare LNP?

  We use microfluidic device and chip to prepare LNP.

• What’s the encapsulation/recovery efficiency of LNP formulation?

  The final encapsulation efficiency is >90%. However, we expect ~50% payload loss during the encapsulation process. For example, 2mg payload input could expect 1mg encapsulated, with a variance of ±10% in final amount encapsulated.

• How much mRNA is needed if I want to want to have 100ug LNP to use after encapsulation?

  We suggest to order 200ug of mRNA for encapsulation.

• What’s the highest concentration GenScript can make for LNP?

  Typical concentration we provide is 0.1-0.4 mg/ml. The highest concentration we can provide is 2mg/mL.

• What if the final LNP buffer solution?

  For the LNP final buffer solution, we use Tris buffer which will enable longer-term stability compared with PBS, according to the research results from Moderna. We also offer the option of using PBS with sucrose or Tris with sucrose as the final buffer for LNP. Sucrose is used as a cryoprotectant. Default buffer is PBS with sucrose.

• How to dilute LNP?

  We suggest to thaw LNP and keep at 4°C after thaw for up to 1 month. Dilute with PBS or tris based on your final buffer.

• What is your recommendations for LNP storage and transport temperatures?

  If you plan to store LNP for longer than 3 months, it is recommended to add a cryoprotectant. GenScript will ship LNP in default buffer PBS with sucrose on dry ice. We recommend to store LNP at -80°C upon receipt until use. Avoid freeze-thaw cycles. Suggest to thaw on the day of use, otherwise keep your thawed LNP at 4°C for up to 1 month.

• How long is our LNP stable in the freezer and can it undergo freeze thaw cycles?

  The LNP should be stable for at least 3 months if stored properly at -80°C upon receival and avoiding repeated freezing and thawing cycles. The amount of freeze/thaw cycles depends on the mRNA length. For 1-2kb, we suggest not to freeze/thaw more than 2 times. For 2-5kb, we suggest no more than once freeze/thaw. If you need to thaw the LNP, it is suggested to thaw them on ice and keep the thawed formulation at 4°C.

• Do you have a LNP cell transfection protocol?
  1. Plate cells and incubate overnight to 24 hours. Cells grown and transfected in complete media (DMEM +10% FBS) has higher expression than reduced serum medium.
  2. Calculate mRNA and LNP dose needed. Add LNP directly to cells incubated in complete media.
  3. Incubate for expression and analysis. For eGFP expression and analysis on plate reader, we recommend using low fourescent background media such as Flourobrite® with 10% FBS, 1X GlutaMax® and 1X sodium pyruvate.

  Same protocol can be used for adherent and immune cell lines as well.

Additional mRNA Offerings