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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

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 (**).
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f2: 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 (**).

Mentions: Sulfated GAG storage was quantified in patients’ fibroblasts after treatment (Fig. 2). After three days, SFC6 fibroblasts showed 1.346 ± 0.085 μg GAGs/μg DNA, while treated fibroblasts presented between 1.075 and 1.235 μg GAGs/μg DNA. SFC7 fibroblasts had 0.953 ± 0.049 μg GAGs/μg DNA without treatment, and between 0.812 and 1.168 μg GAGs/μg DNA after treatment. After seven days, SFC6 fibroblasts showed 1.824 ± 0.136 μg GAGs/μg DNA, while treated fibroblasts presented between 1.538 and 1.701 μg GAGs/μg DNA. SFC7 fibroblasts had 1.521 ± 0.081 μg GAGs/μg DNA without treatment and between 1.354 and 1.677 μg GAGs/μg DNA after treatment. After 14 days, SFC6 fibroblasts showed 3.259 ± 0.085 μg GAGs/μg DNA, while treated fibroblasts presented between 2.618 and 2.945 μg GAGs/μg DNA. SFC7 fibroblasts had 2.569 ± 0.049 μg GAGs/μg DNA without treatment and between 1.973 and 2.66 μg GAGs/μg DNA after treatment. In general, patients’ storage decreased at each time point, reaching a maximum reduction of 24% for siRNA si4901 at 14 days in the fibroblasts of patient SFC7. GAG levels in WT fibroblasts were 0.796 ± 0.105 throughout the experiment.


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)

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

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

f2: 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 (**).
Mentions: Sulfated GAG storage was quantified in patients’ fibroblasts after treatment (Fig. 2). After three days, SFC6 fibroblasts showed 1.346 ± 0.085 μg GAGs/μg DNA, while treated fibroblasts presented between 1.075 and 1.235 μg GAGs/μg DNA. SFC7 fibroblasts had 0.953 ± 0.049 μg GAGs/μg DNA without treatment, and between 0.812 and 1.168 μg GAGs/μg DNA after treatment. After seven days, SFC6 fibroblasts showed 1.824 ± 0.136 μg GAGs/μg DNA, while treated fibroblasts presented between 1.538 and 1.701 μg GAGs/μg DNA. SFC7 fibroblasts had 1.521 ± 0.081 μg GAGs/μg DNA without treatment and between 1.354 and 1.677 μg GAGs/μg DNA after treatment. After 14 days, SFC6 fibroblasts showed 3.259 ± 0.085 μg GAGs/μg DNA, while treated fibroblasts presented between 2.618 and 2.945 μg GAGs/μg DNA. SFC7 fibroblasts had 2.569 ± 0.049 μg GAGs/μg DNA without treatment and between 1.973 and 2.66 μg GAGs/μg DNA after treatment. In general, patients’ storage decreased at each time point, reaching a maximum reduction of 24% for siRNA si4901 at 14 days in the fibroblasts of patient SFC7. GAG levels in WT fibroblasts were 0.796 ± 0.105 throughout the experiment.

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