(T1530-07-46) Mechanistic Differences of NaCl vs. MgCl₂ in Preventing AAV8 Capsid Aggregation

Purpose: Adeno-associated virus (AAV) vectors are widely utilized in gene therapy, yet their large-scale manufacturing is hindered by capsid aggregation, which compromises stability and efficacy. Ionic excipients such as monovalent (NaCl) and divalent (MgCl₂) salts are commonly used to mitigate aggregation in many FDA-approved AAV products, but their distinct mechanisms remain inadequately understood. This study aims to elucidate the differential effects of NaCl and MgCl₂ on AAV8 capsid aggregation and stability, a process that is challenging to investigate experimentally due to the complexity of nanoscale interactions. To overcome these limitations, we employ coarse-grained molecular dynamics (CG-MD) simulations, which allow us to capture detailed molecular interactions that are difficult to isolate in laboratory settings.

Methods: Coarse-grained molecular dynamics (CG-MD) simulations were conducted to assess the aggregation behavior of two AAV8 capsids loaded with single-stranded DNA (ssDNA) under varying NaCl concentrations (0.05–0.3 M) and a fixed MgCl₂ concentration (1 mM). Key parameters, including center-of-mass (COM) distance, electrostatic potential distribution, and van der Waals interaction energy, were analyzed to determine aggregation propensity and stabilization mechanisms.

Results: NaCl influenced capsid aggregation in a concentration-dependent manner. At low concentrations (0.05 M), the insufficient electrostatic screening led to an increased likelihood of capsid aggregation due to enhanced attractive interactions between surface-exposed charged residues. In contrast, higher NaCl concentrations (0.3 M) provided stronger charge shielding, diminishing attractive forces and promoting repulsive electrostatic interactions, thereby mitigating aggregation. MgCl₂, however, stabilized capsids through a dual mechanism: electrostatic charge screening, similar to NaCl, and direct binding to ssDNA, which led to its compaction and further redistribution of surface charge on the capsid, thereby minimizing aggregation. Electrostatic potential analyses confirmed that Mg²⁺ ions provided localized charge stabilization, distinguishing their role from Na⁺ ions.

Conclusion: This study provides mechanistic insights into the roles of monovalent and divalent ions in AAV8 capsid aggregation. While NaCl modulates aggregation through electrostatic charge screening, MgCl₂ stabilizes capsids through both electrostatic charge screening and ssDNA compaction, leading to a more pronounced reduction in aggregation. These findings offer valuable insight to developing AAV formulations, improving vector stability during manufacturing, and enhancing gene therapy efficacy.