Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.
Giridhar Murlidharan, Andrew Crowther, Rebecca A. Reardon, Juan Song, Aravind Asokan
Comparison of AAV vector accumulation within juvenile and aging mouse brains.
Three-month-old and eighteen-month-old mice were injected with equal viral titers (1.75 × 109 vg per animal) of Alexa 647–tagged fluorescent AAV9 vectors mixed with 0.5% Alexa 488–tagged fluorescent 10-kDa dextran tracer (5 mg/ml) into the left lateral ventricle. 45 minutes after injections, the mice were anesthetized and sacrificed for organ harvests. Paraformaldehyde-fixed brains were sectioned on a vibratome, and a Zeiss 700 confocal microscopy was used to generate fluorescence images of the mouse brains. (