(588cj) Understanding the Role of Poloxamer 188 in Inhibiting AAV8 Capsid Aggregation: A Coarse-Grained Molecular Dynamics Study

Purpose:

Adeno-associated virus (AAV) is one of the widely used vectors in gene delivery with great features such as a high safety profile and high transduction efficacy.

However, large-scale production and long-term formulation stability are challenging due to product degradation as a result of capsid aggregations.

Aggregation mainly happens due to hydrophobic interactions between the capsid proteins and subsequently leads to loss of potency, reduced shelf life, and higher immunogenic responses.

Many FDA-approved AAV based formulations contain Poloxamer 188 (P188), a nonionic surfactant, to modify the capsid interactions, improve stability, and prevent aggregation. However, the molecular mechanisms of poloxamer 188 interaction with AAV capsids and the optimal concentration needed for aggregation inhibition have remained unknown. To fill these knowledge gaps, we employed coarse-grained molecular dynamics (CG-MD) simulations to investigate the concentration required to prevent aggregation of AAV8 capsids and how Poloxamer 188 interacts with AAV8 capsids in an aqueous environment and its mechanism of action.

Methods:

We performed CG-MD simulations using GROMACS with the MARTINI force field to model AAV8 capsids (PDB: 6V12) in solution with varying amounts of Poloxamer 188 (2, 5, 10, 24 poloxamer molecules in system equals to mol/Litr). The system underwent energy minimization using the steepest descent algorithm, followed by 10 ns equilibration and production runs in an NPT ensemble using the Nosé−Hoover thermostat and the Parrinello−Rahman barostat at 1 atm pressure with a 20-fs time step.

Results:

Our simulations revealed that the low amount of Poloxamer 188 (2 and 5 poloxamers molecules) was insufficient to prevent aggregation, as capsids still interacted via hydrophobic patches. However, higher amounts (10 and 24 poloxamers molecules) significantly reduced aggregation, as Poloxamer 188 preferentially adsorbed onto capsid surfaces, forming a steric barrier that disrupted capsid-capsid interactions.

Mechanistically, Poloxamer 188 inserted its hydrophobic block into exposed hydrophobic regions on the capsid surface, while its hydrophilic segments remained in the aqueous phase, effectively stabilizing the viral particles and reducing aggregation propensity. We determined that a minimum number of poloxamers, more than 10 poloxamers in our system, is required to effectively prevent AAV8 capsid aggregation.

Conclusion:

This study shows the interactions between AAV8 capsids in the presence of poloxamer in an aqueous environment by molecular-level insight of the CG-MD model, highlighting the minimum required amount of poloxamer for aggregation inhibition.

By understanding these interactions between hydrophobic and hydrophilic parts of the molecules, and gaining deeper knowledge, we would have a better understanding of the effect of surfactants on the AAV formulation stability and ultimately, we could improve drug formulation development for gene therapy applications

These findings are particularly relevant to FDA-approved AAV-based products, where the use of nonionic surfactants like Poloxamer 188 is critical for ensuring product stability and efficacy. Our results demonstrate that CG-MD simulations serve as a powerful predictive tool for optimizing surfactant formulations, offering valuable guidance for improving AAV-based gene therapies in biopharmaceutical manufacturing.