Spencer Fox Eccles School of Medicine
90 Advancements in Gene Therapy Approaches for Treating Vanishing White Matter Disease
Sophia Peralta and Joshua Bonkowsky
Faculty Mentor: Joshua Bonkowsky (Pediatrics, University of Utah)
Background
Vanishing White Matter (VWM) disease is a rare leukodystrophy, an inherited genetic disorder affecting the white matter of the central nervous system. This disease primarily affects children and young adults, with an incidence of approximately 1 in every 80,000 births. This disease is caused by a mutation within any of the 5 subunits that comprise the eukaryotic initiation factor (EIF2B) protein complex. As there is no cure, our research team is dedicated to advancing our knowledge of VWM disease and developing a robust adeno-associated virus (AAV)-mediated gene therapy using a mouse model with an EIF2B5 R191H mutation. Given that astrocytes are central in disease pathology, the research team is investigating astrocyte-specific promoters for AAV9 gene therapy.
Methods
This study consisted of WT and mutant characterization groups, including 7 cohorts of approximately 15-20 mice each. Each of the 7 cohorts received an intracerebroventricular (ICV) injection at one day old to deliver the AAV9 construct, testing 4 promoters for driving the expression of human EIF2B5. The 4 promoters include: 1) GFAP – a large astrocyte specific promoter, 2) CAG – a ubiquitous promoter to express in many cell types, 3) gfaABC(1)D – a small astrocyte specific promoter, 4) and gfa1405 – a medium-sized astrocyte specific promoter with inhibitory regions deleted.
At endpoint, the brain and spinal cord were collected for analysis via histology, immunohistochemistry (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) to examine the efficacy of the gene therapy. Histology analysis was performed using Luxol Fast Blue (LFB) staining to visualize and quantify myelinated fibers of the corpus callosum and cervical spinal cord. Immunohistochemistry data was collected for three 3 antibodies: ATF3, an ISR marker, in the cervical spinal cord; GFAP, an astrocyte marker, in the brain; and Iba1, a marker for activated microglia, in the brain and cervical spinal cord. Additionally, qRT-PCR was conducted to analyze the expression of 7 integrated stress response (ISR) genes in the brains and cervical spinal cords.
Results
The gene therapy appears to have no adverse effects and shows early signs of efficacy through improved motor function, increased body weight, and reduced expression of ISR genes. Partial rescue of ATF3 staining in the cervical spinal cord was observed using the CAG, gfaABC(1)D, and gfa1405 promoters. Analysis of ISR gene expression levels revealed partial rescue in CAG, gfaABC(1)D, and gfa1405 brains. However, some outcomes were not improved with the gene therapy. LFB staining revealed little to no rescue of myelination in GFAP, CAG, gfaABC(1)D, and gfa1405 brains or cervical spinal cords. Similarly, none of the promoters effectively rescued Iba1 staining or ISR gene expression levels of the cervical spinal cords.
Conclusion
Gene therapy for VWM with AAV9 gives partial rescue of the disease phenotype with CAG, gfaABC(1)D, and gfa1405 promoters. However, the GFAP promoter could not package well in AAV9 due to its large size and showed poor efficacy. Improvements in motor behavior, body weight, and ATF3 IHC are promising, while the regional variation in ISR gene expression levels prompts further investigation into the therapy delivery site.
Discussion
Our lab remains committed to developing an effective gene therapy, which is paramount in offering hope and therapeutic intervention to individuals and families grappling with Vanishing White Matter disease. Due to the greater rescue of ISR genes in the brain versus the spinal cord, the team has considered possible explanations for this regional variation. To address this, a trial is underway using intra cisterna magna (ICM) injections, which can better target the spinal cord. Additionally, while targeting astrocytes has shown partial rescue, emerging research has highlighted the role of other cell types that are key in VWM disease pathology. Microglia, a cell type involved in VWM and other neurological disorders, pose a challenge due to their inability to be infected by AAV9. To address this, we are currently exploring microglial replacement through a bone marrow transplant alone or with AAV9 gene therapy to enhance treatment efficacy.
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