The Potency Chains of Events for mRNA/LNP Therapeutics

Steve Wolk, September 1, 2025

There are two key chains of events that can impact therapeutic potency. A break in the chain at any stage can cause reduction or catastrophic failure of therapeutic potency.

The first is the manufacturing chain of events, or the cold chain path, which may include any or all of the following steps:

Manufacturer --> Distributor --> Wholesaler --> Pharmacist --> Patient

Factors affecting potency include:

• Temperature

• Light exposure

• Air (oxidation) exposure

• Moisture (for lyo’d product)

• Contamination exposure

Understanding the impact of each of these factors will inform the development of a good process, and monitoring will help ensure that integrity is maintained.  Important steps where monitoring/characterization should be considered include:

• Manufacturing Steps

  • cargo prep (gRNA, mRNA, possibly template)

  • assembly of LNP

• Storage

  • storage stability studies to inform

  • monitoring

• Transportation

  • shipping stability studies

  • monitoring

• Inventory management

The second potency chain is the in vivo chain of events, which is exquisitely complex, and includes each of the following steps:

• Circulation Steps

  • protein corona formation

  • biodistribution

• Cell Penetration/Uptake

  • endocytosis

  • endosomal escape

    • internal organization of LNP cargo components

  • intracellular sequestration

  • degradation (inside the endosome, in the cytoplasm, and in sequestered states)

• Translation Efficiency

  • transport to the ribosome

  • endogenous protein expression regulation mechanisms

  • expression of full-length, correctly folded protein

• Immune Activation/Impact on Each Step

  • opsonization in circulation?

  • reduction/shutdown of translation machinery resulting from immune response

For a CRISPR-based mRNA/LNP therapeutic, the following additional steps come into play:

• Formation of the RNP in cytoplasm

• Transport of the RNP to the nucleus via NLS

• Unwinding chromatin

• Cleavage at locus

Considering how many opportunities there are for failure, it’s truly amazing that this in vivo pathway works, and the current data makes it clear that it does.

It would be ideal to have analytics at each step of the chains in order to correctly identify and engineer past failure points.  For the in vivo chain, analytics at each step are not yet available, but the science continues to advance every day.