Hyaluronidase-enhanced delivery in gene therapy and regenerative medicine for improved vector, cell, and cargo access

Efficient delivery of gene therapy vectors and regenerative cargo to the central nervous system (CNS) is limited by the hyaluronic acid (HA)-rich extracellular matrix (ECM). Adeno-associated virus (AAV) particles, mesenchymal stem cells (MSCs), multilineage-differentiating stress-enduring (MUSE) cells, and small extracellular vesicles (sEVs) must traverse the HA-rich ECM of the nasal submucosa, the perineural and perivascular compartments, and brain parenchyma to reach CNS targets. Dense ECM imposes viscosity, steric, and hydrodynamic barriers that limit tissue penetration, and perineuronal nets impose an additional HA-based barrier around subsets of neurons. Hyaluronidase enzymes transiently depolymerize HA, reducing tissue viscosity and increasing interstitial permeability through a well-defined, reversible mechanism. Recombinant human hyaluronidase PH20 (rHuPH20) is FDA-approved as a subcutaneous delivery adjunct, demonstrating that controlled ECM modulation is clinically feasible and safe. Preclinical studies demonstrate that hyaluronidase pretreatment enhances intranasal CNS delivery of AAV vectors and sEVs, with quantified improvements in olfactory bulb and cortical distribution. Intranasal MSC and MUSE cell delivery is similarly enhanced: 100 USP units of hyaluronidase applied 30 min before cell administration significantly increased MSC delivery to the olfactory bulb and total brain area in rodent models (p = 0.02). MUSE cells, which express sphingosine-1-phosphate receptor 2 (S1PR2) and home actively to sites of neuroinflammation via the S1P-S1PR2 axis, achieve selective CNS distribution after intranasal administration in Parkinson's disease models, outperforming non-MUSE MSCs and restoring dopaminergic neuron markers and motor function. This narrative review synthesizes the mechanistic basis, evidence tiers, practical intranasal dosing and technique protocols, safety considerations, and translational knowledge gaps for hyaluronidase as a CNS delivery adjunct across viral gene therapy, non-viral platforms, gene-modified cell products, and sEV-based therapeutics. We distinguish established, off-label, and investigational applications, and provide practical guidance for clinicians developing hyaluronidase-augmented CNS programs.