Role of Microglia in Vanishing White Matter Disease Leukodystrophy

PROJECT SUMMARY
Leukoencephalopathy with Vanishing White Matter (VWM) is a rare neurodegenerative leukodystrophy that
presents with ataxia, seizures, and progression to death. There are no clinicaltreatments for VWM, and our ultimate
goal is the development of a therapy for VWM. VWM is caused by autosomal recessive mutations in the five
subunit genes of the Eukaryotic Initiation Factor 2B (EIF2B) complex, which is necessary for regulation of the
integrated stress response (ISR). VWM has abnormal, persistent activation of the ISR. Current dogma in the field
has settled on astrocytes as responsible for VWM pathophysiology. We developed adeno-associated virus (AAV)-
mediated EIF2B5 gene (the most commonly mutated subunit) replacement therapy for VWM with targeted
astrocyte expression. In both the classic R191H mouse model and a more severe I98M mouse model, we
showed rescue of VWM-disease related phenotypes (Herstine et al., 2024). However, we observed waning
efficacy in treated VWM mice. Interestingly, our preliminary data showed recrudescent ISR activation that
mirrored presence of activated microglia. Further, we found that untreated mice have extensive infiltration of
Iba1+/CD68+ microglia into the CNS. Activated microglia secrete cytokines which induce reactive astrocytes in
vitro and in vivo. Our hypothesis is that activated microglia contribute to VWM pathophysiology, and that
transplant of wild-type microglia is necessary for full gene therapy rescue of VWM by interrupting the
cycle of microglia-driven reactive astrocyte activation.To test our hypothesis, we propose first, to
Characterize microglial phenotypes in wild-type, mutant, and AAV9-treated mutant VWM mice. We will
characterize the timing and extent of activated microglia involvement in the CNS, in R191H and I98M mice, at
defined ages; in comparison to wild-type and AAV9-treated VWM affected mice. Histological, molecular, and
biochemical targets include CD68 and Iba1 (for activated and stable microglia); and CNS tissue markers (GFAP,
astrocytes; NeuN and neurofilament light, neurons; and Olig2, fluoromyelin, oligodendrocytes and myelin).
Second, we will Perform microglia replacement using circulation-derived myeloid cells (CDMCs) in VWM
mice in conjunction with or without AAV9 gene therapy. Since dysregulated microglia may interfere with
gene therapy rescue, we will test a combination approach of microglia transplantation and gene therapy. We will
use a published protocol, which we have demonstrated in preliminary data is effective for VWM mice at our lab
(busulfan ablation of endogenous hematopoietic cells, followed by bone marrow transplant with donor circulation
derived myeloid cells (CDMC)). With use of PLX5622 (a CSF1R inhibitor required for native microglia), the CDMC
cross the blood-brain barrier and replace endogenous microglia. Our experiments will include 3 mouse groups:
VWM mice; VWM mice receiving CDMC replacement and AAV9-EIF2B5 gene therapy; and VWM mice with
CDMC only (no gene therapy).

Grant Number: 1R21HD119649-01
NIH Institute/Center: NIH
Principal Investigator: Joshua Bonkowsky