| Resumo: | Mucopolysaccharidosis type III (MPS III) refers to a group of four autosomal recessive neurodegenerative lysosomal storage disorders (LSD) caused by the incomplete lysosomal degradation of the heparan sulphate (HS) that accumulates in patient cells and triggers disease. Degeneration of the central nervous system is the major hallmark of these disorders, resulting in mental retardation and hyperactivity. By their mid-teenage years most affected patients are dependent on their caregivers for all needs and death occurs at the end of the second or early in the third decade of life. The classical therapeutic approach for LSDs, enzyme replacement therapy, would hardly rise as a potentially successful tool to reduce the disease burden in MPS III patients due to the inability of the recombinant enzymes to cross the blood-brain barrier (BBB), having no impact in neuropathology. Thus, there is no effective therapy available, with treatment limited to clinical management of neurological symptoms. A tempting alternative, however, would be to block substrate accumulation upstream, by decreasing its synthesis. That concept is known as substrate reduction therapy (SRT). In order to decrease HS storage inside the lysosomes, we designed and assayed in MPS III patients’ fibroblasts a specific siRNA pool targeting XYLT1, a gene that encodes an enzyme involved in an early stage of the HS biosynthetic cascade. Fibroblasts from MPS IIIA, B, C and D patients were transfected with the designed siRNAs pool to inhibit XYLT1. Cell pellets were collected 24/48/72 hours and 7 days post- transfection and total RNA extracted. Target mRNA levels were evaluated through qRT-PCR using the 2-∆∆Cq method. Additionally, the effect on HS accumulation was quantified 24 and 48h after transfection using a modified 1,9-dimethylmethylene blue assay. The results showed a high inhibition of the XYLT1 gene mRNAs (around 80%) and a decrease in GAGs storage (only assessed for types C and D until now). Currently, we are evaluating the effect of that decrease on the overall GAGs storage 7 days post-transfection, also with promising results. Here we present an overview on the current results of this project, while discussing its next steps, namely the development and evaluation of vectors for in vivo delivery. Our goal is to develop targeted stable nucleic acid lipid particles (t-SNALPs) coupled with different ligands, to promote cell uptake of the ‘anti-GAG’ siRNAs in a variety of cells, including neurons.
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