Optimal Window of Opportunity for Granulosa Cell Gene Therapy in Galactosemia

PROJECT SUMMARY / ABSTRACT Hereditary deficiency of galactose-1-phosphate uridylyltransferase (GALT, E.C. 2.7.7.12) activity in humans can lead to a potentially lethal disease called Classic Galactosemia (OMIM 230400). Despite the life-saving consequences of newborn screening, early diagnosis, and a galactose-restricted diet, affected women

invariably show ovarian damage and fertility impairment representing the greatest burden of their disease. There are currently no satisfactory treatments available to prevent this complication. Recently, insights into the underlying pathophysiology and innovative technological advancements have become available, and

options for curative treatment have emerged. Using this momentum, we aim to develop a treatment to prevent fertility impairments. We aim to 1) develop a non-viral nucleic acid therapy approach that targets the ovary and 2) determine the optimal timing to rescue the ovarian function. Folliculogenesis is driven by the

cross-talk between oocytes and granulosa cells. Restoration of GALT activity in these cells could possibly rescue the ovarian damage. In this pilot grant application, we will explore a new modality of gene therapy using nanoparticles encapsulating episomal GALT cDNA within a non-viral expression vector. This will include determination of

the optimal cationic polymer layer for delivery of episomal DNA plasmids expressing GFP or GALT to granulosa cell lines and the addition of ligands to encourage uptake and promote specificity. In addition, we will determine the optimal window of opportunity for this treatment. A prenatal origin has been

considered for decades whereas the data currently available, albeit scarce, favor a postnatal origin. There is undoubtedly early damage, but whether treatments aimed to prevent ovarian damage needs to be started in the neonatal period, or treating in infancy or adolescence would suffice, is not clear. This will be studied using

the zebrafish model for classic galactosemia that mimics the fertility impairments and is suitable for studies through development. Zebrafish reproduction is regulated by the brain-pituitary-gonadal axis, comparable to the human situation. For this purpose, RNA-seq, histology/immunohistochemistry of follicole

development and single cell RNA-seq of oocytes and granulosa cells at 4 developmental stages will be studied. These studies will uncover developmental stage and cell-specific alterations in the affected ovaries. Distinctive patterns at the different stages are expected to reveal at which stage(s) damage

can be prevented/halted using ovary targeted gene therapy.