Evaluating the Effect of an Antisense Oligonucleotide Treatment Targeting RAN Translation in FMR1 Premutation Cell-line Model

Introduction:
Antisense Oligonucleotide (ASO) technology has broadened the spectrum of possible therapeutic targets in a variety of genetic diseases. An ASO is a single stranded oligonucleotide that upon binding sterically blocks specific portions of the targeted RNA and modulate RNA splicing or proteins translation. Fragile X (FMR1) premutation carriers may suffer from Fragile X-Associated Premature Ovarian Insufficiency. One of the foremost proposed causative mechanism in FMR1 premutation male carriers is Repeat associated non-AUG initiated (RAN) translation. RAN translation’s most abundant product is a cryptic poly (glycine)-containing protein, named FMRpolyG, which accumulates in large ubiquitin-positive inclusion bodies and was shown to be toxic. A recent study used a targeted RNA based ASO to selectively block the formation of FMRpolyG. In human and rodent neurons, RAN-blocking ASO suppressed toxicity and prolonged neural survival in-vitro. This CGG RAN-ASO blockade also enhanced endogenous FMR1 protein (FMRP) expression in neurons in-vitro. To treat fertility related genetic disorders it is necessary to explore the effect of CGG RAN-ASO on cells involved in folliculogenesis (e.g. granulosa cells).

Aim:
To assess the activity of CGG RAN-ASO in FMR1 premutation transfected human granulosa tumor cells (COV434).

Methods:
FMR1 premutation transfected COV434 cells were treated with consecutive concentrations of CGG RAN-ASO. After 48 hours FMRP expression was measured using western blot analysis.

Results:
Elevated FMRP expression was demonstrated in FMR1 premutation transfected COV434 cells following ASO treatments.

Conclusions:
CGG RAN-ASO targeting RAN translation may have a potential therapeutic effect and assist FMR1 premutation carriers following more investigation.