Adeno-associated viruses (AAVs) induce dose-dependent neonatal ventriculomegaly following intracerebroventricular administration

Cell-type-specific expression of synthetic/endogenous proteins or genetic sequences has significantly advanced our understanding of the central nervous system (CNS). Adeno-associated virus (AAV)-delivery to cerebrospinal fluid (CSF) mediates transfection of target cells to enable sustained delivery of secretory proteins into the CSF, offering promising avenues for both CNS therapy and mechanistic studies. However, despite the advantages afforded by AAV tropism-based cellular selectivity and transgene delivery, both preclinical studies and clinical trials report short- and long-term adverse effects, particularly immune activation. Especially relevant for CSF biology, CNS immune insults raise the risk of CSF dysregulation, including hydrocephalus. These risks may be exacerbated in pediatric populations, where ongoing CNS development, including immature meninges, choroid plexus (ChP), and skull structures, may further impair any ability to compensate for CSF dysregulation. To systematically address these risks and provide guidelines for minimizing CNS immune insults by AAVs, we test the dose-dependent effects of intracerebroventricular (ICV) injections of 3 AAV serotypes (AAV2/5, AAV2/4, and AAV.PHP.eB) to neonatal (P0.5) CD1 mouse pups. Histological analysis verified AAV2/5 tropism limited to ChP epithelial cells (CPECs), whereas AAV.PHP.eB transfected both CPECs and ependymal regions. By contrast, AAV2/4 shows limited transfection in the brain. Further, we find that ICV injections of all 3 AAV serotypes at the high dose (4x10 9 genome copy GC/pup) induced ventriculomegaly by P7.5, while the low dose (1x10 9 GC/pup) was well tolerated. Additionally, high-dose AAV2/5 tropism became more permissive, transfecting the ChP and also ependymal cells and some neurons. Longitudinal MRI of P0.5 pups with high-dose AAV2/5 ICV injections highlighted quick progression of ventriculomegaly. CSF ELISA analysis detected elevated pro-inflammatory cytokine CCL2 in AAV2/5 and AAV2/4 high-dose groups, indicating CNS inflammation. Moreover, decreased CSF TTR concentrations in high-dose groups suggest ChP dysfunction. We further revealed that earlier in utero high-dose ICV AAV2/5 injections at E13.5 induced even more severe ventriculomegaly and that adult animals were also susceptible to ventriculomegaly after high-dose AAV ICV delivery. Taken together, our data emphasize critical safety considerations of CSF-based AAV delivery, particularly during brain development. Further, these results call for an optimized dosage for perinatal ICV AAV applications. Highlights CSF-administration of AAVs results in unique cellular tropism in neonatal brains. Intracerebroventricular AAV delivery induces dose-dependent neonatal ventriculomegaly. Neonatal ventriculomegaly by AAV overdose develops quickly and progressively. AAV overdose ventriculomegaly correlates with ChP/CSF dyshomeostasis and inflammation. Adult and embryonic brains are also susceptible to dose-dependent AAV-induced ventriculomegaly.