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

Canals I, Benetó N, Cozar M, Vilageliu L, Grinberg D - Sci Rep (2015)

Bottom Line: It presents severe and progressive neurodegeneration and currently there is no effective treatment.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%).

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: Heparan sulfate storage was evaluated through immunocytochemistry. Clear differences between WT and patients’ fibroblasts were observed after cells were fixed and stained with anti-HS antibodies (Fig. 3A). After a 3-day treatment with siRNA 4899 (as described above), patients’ fibroblasts showed a clear reduction in HS accumulation (Fig. 3B,C). siRNA 4899 was chosen for this experiment because it was the one that gave the best results in the reduction of GAG synthesis (see Fig. 1).


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

Canals I, Benetó N, Cozar M, Vilageliu L, Grinberg D - Sci Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561882&req=5

f3: 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.
Mentions: Heparan sulfate storage was evaluated through immunocytochemistry. Clear differences between WT and patients’ fibroblasts were observed after cells were fixed and stained with anti-HS antibodies (Fig. 3A). After a 3-day treatment with siRNA 4899 (as described above), patients’ fibroblasts showed a clear reduction in HS accumulation (Fig. 3B,C). siRNA 4899 was chosen for this experiment because it was the one that gave the best results in the reduction of GAG synthesis (see Fig. 1).

Bottom Line: It presents severe and progressive neurodegeneration and currently there is no effective treatment.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%).

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

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.

No MeSH data available.


Related in: MedlinePlus