EXTL2 and EXTL3 inhibition with siRNAs as a promising substrate reduction therapy for Sanfilippo C syndrome

Abstract

Sanfilippo syndrome is a rare lysosomal storage disorder caused by an impaired degradation of heparan sulfate (HS). It presents severe and progressive neurodegeneration and currently there is no effective treatment. Substrate reduction therapy (SRT) may be a useful option for neurological disorders of this kind, and several approaches have been tested to date. Here we use different siRNAs targeting EXTL2 and EXTL3 genes, which are important for HS synthesis, as SRT in Sanfilippo C patients' fibroblasts in order to decrease glycosaminoglycan (GAG) storage inside the lysosomes. The results show a high inhibition of the EXTL gene mRNAs (around 90%), a decrease in GAG synthesis after three days (30-60%) and a decrease in GAG storage after 14 days (up to 24%). Moreover, immunocytochemistry analyses showed a clear reversion of the phenotype after treatment. The in vitro inhibition of HS synthesis genes using siRNAs shown here is a first step in the development of a future therapeutic option for Sanfilippo C syndrome.

Figure 1. Inhibition of GAG synthesis.

SFC6 and SFC7 fibroblasts were transfected with all four siRNAs and a negative control siRNA and after 3 days incorporation of ³⁵S sodium sulfate was analysed. Results for both patients are the mean ± standard error of three experiments performed in quadruplicate and are expressed as disintegrations per minute per μg of DNA. Differences between EXTL siRNAs with respect to negative control siRNA were evaluated using the non-parametric Mann-Whitney U test, and statistical significance was set at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***).

Figure 2. Decrease in GAG storage.

SFC6 and SFC7 fibroblasts were transfected with all four siRNAs and a negative control siRNA and after 3, 7 and 14 days, GAG storage was analysed. Results for both patients are the mean ± standard error of three experiments performed in duplicate and are expressed in μg GAGs/μg DNA at 3 days, 7 days and 14 days after transfection. Differences between EXTL siRNAs respect to negative control siRNA were evaluated using the non-parametric Mann-Whitney U test, and statistical significance was set at p < 0.05 (*) or p < 0.01 (**).

Figure 3. Heparan sulfate storage in WT and patients’ fibroblasts.

(A) Immunocytochemistry analysis of HS accumulation (green) in untreated WT and SFC6 and SFC7 cells, using a specific anti-HS antibody. The same images are shown using the white channel to highlight HS staining. Fibroblasts from patient SFC6 (B) and patient SFC7 (C) were transfected with si4899 and with a negative control siRNA (siC-) for three days. HS was detected as in A, and shown in the green and white versions. Bar = 20 μm.