Proteostasis regulators as potential rescuers of PMM2 activity.

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-glycosylation disorder. To date there is no treatment. Following the identification of a number of destabilizing pathogenic variants, our group suggested PMM2-CDG to be a conformational disease. The aim of the present study was to evaluate the possible use of proteostasis network regulators to increase the stability, and subsequently the enzymatic activity, of misfolded PMM2 mutant proteins. Patient-derived fibroblasts transduced with their own PMM2 folding or oligomerization variants were treated with different concentrations of the proteostasis regulators celastrol or MG132. Celastrol treatment led to a significant increase in mutant PMM2 protein concentration and activity, while MG132 had a small effect on protein concentration only. The increase in enzymatic activity with celastrol correlated with an increase in the transcriptional and proteome levels of the heat shock proteins Hsp90 and Hsp70. The use of specific Hsp70 or Hsp90 inhibitors showed the positive effect of celastrol on PMM2 stability and activity to occur through Hsp90-driven modulation of the proteostasis network. The synergistic effect of celastrol and a previously described pharmacological chaperone was also examined, and a mutation-dependent synergistic effect on PMM2 activity was noted. These results provide proof-of-concept regarding the potential treatment of PMM2-CDG by proteostasis regulators, either alone or in combination with pharmacological chaperones.

Protein misfolding diseases: Prospects of pharmacological treatment.

Protein misfolding has been linked to numerous inherited diseases. Loss- and gain-of-function mutations (common features of genetic diseases) may cause the destabilization of proteins, leading to alterations in their properties and/or cellular location, resulting in their incorrect functioning. Misfolded proteins can, however, be rescued via the use of proteostasis regulators and/or pharmacological chaperones, suggesting that treatments with small molecules might be developed for a range of genetic diseases. This work describes the potential of these small molecules in this respect, including for the treatment of congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG).

Antisense-mediated therapeutic pseudoexon skipping in TMEM165-CDG.

Deficiencies in glycosyltransferases, glycosidases or nucleotide-sugar transporters involved in protein glycosylation lead to congenital disorders of glycosylation (CDG), a group of genetic diseases mostly showing multisystem phenotype. Despite recent advances in the biochemical and molecular knowledge of these diseases, no effective therapy exists for most. Efforts are now being directed toward therapies based on identifying new targets, which would allow to treat specific patients in a personalized way. This work presents proof-of concept for the antisense RNA rescue of the Golgi-resident protein TMEM165, a gene involved in a new type of CDG with a characteristic skeletal phenotype. Using a functional in vitro splicing assay based on minigenes, it was found that the deep intronic change c.792+182G>A is responsible for the insertion of an aberrant exon, corresponding to an intronic sequence. Antisense morpholino oligonucleotide therapy targeted toward TMEM165 mRNA recovered normal protein levels in the Golgi apparatus of patient-derived fibroblasts. This work expands the application of antisense oligonucleotide-mediated pseudoexon skipping to the treatment of a Golgi-resident protein, and opens up a promising treatment option for this specific TMEM165-CDG.