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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

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 (***).
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f1: 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 (***).

Mentions: After three days of transfection, fibroblasts showed a decrease in the incorporation of 35S sulfate of about 30% to 60% (depending on the siRNA used) compared to control samples (Fig. 1). Both patients showed similar results for each siRNA, indicating a more efficient inhibition of GAG synthesis for siRNAs designed to target the EXTL2 gene (more than 50%) than in those targeting the EXTL3 gene (less than 50%).


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)

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

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

f1: 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 (***).
Mentions: After three days of transfection, fibroblasts showed a decrease in the incorporation of 35S sulfate of about 30% to 60% (depending on the siRNA used) compared to control samples (Fig. 1). Both patients showed similar results for each siRNA, indicating a more efficient inhibition of GAG synthesis for siRNAs designed to target the EXTL2 gene (more than 50%) than in those targeting the EXTL3 gene (less than 50%).

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