About proof of principle in genetically modulated substrate reduction therapy for Sanfilippo syndrome.FCT SFRH/BPD/101965/2014; SFRH/BD/124372/2016N/

Alves, Sandra

Coutinho, Maria Francisca

Gaspar, Paulo

Santos, Juliana Inês

English

Introduction: Mucopolysaccharidosis type III (MPS III) refers to a group of five autosomal recessive neurodegenerative lysosomal storage disorders caused by the incomplete lysosomal degradation of the heparan sulphate (HS) that accumulates in patient cells and triggers disease. The main characteristic of this disease is the degeneration of the central nervous system, resulting in mental retardation and hyperactivity. Currently, there is no effective therapy available, with treatment limited to clinical management of neurological symptoms. 
Methods: Taking advantage of the RNA interference (RNAi) technology potential, we have designed and assayed a specific siRNA targeting an early stage of the HS biosynthetic cascade (XYLT1) in order to promote an effective reduction of the accumulating substrate. Fibroblasts from MPS III patients were transfected with the designed siRNA. Total RNA was extracted and target mRNA levels evaluated through real-time PCR. The effect on GAGs accumulation was quantified over time using a modified 1,9-dimethylmethylene blue assay. 
Results: Proof of principle on the effect of siRNA targeting XYLT1 was achieved for two independent control cell lines, with 8-12 fold decreases on the target mRNA levels, after 24h of incubation with concentrations of 20nM of each siRNA. Subsequent analysis on the effect of the same siRNA on MPS III cell lines resulted in significant lower expression of XYLT1 in types A, B and C, after 24-48h of siRNA incubation. Studies on type B are also ongoing. For types C and D, we have already assessed the treatment effect on storage and observed a significant reduction (50-70%) on the total GAGs levels. 
Conclusions: The effect of siRNA targeting XYLT1 was achieved, resulting in significant lower levels of XYLT1 mRNA. Studies on MPS IIIB are ongoing.  Moreover, a significant reduction on GAGs’ accumulation was observed, and we are currently addressing this storage in the remaining MPS III cell lines.This work was supported by Fundação Millennium bcp (bcp/LIM/DGH/2014). JIS and MFC are grantees from the FCT (SFRH/BD/124372/2016 and SFRH/BPD/101965/2014).N/

Santos, J.I.

Gaspar, P.

Alves, S.

Repositório Científico do Instituto Nacional de Saúde

Genetically Modulated Substrate Reduction Therapy for Sanfilippo syndrome – proof of principle

GENETICALLY MODULATEDSUBSTRATE REDUCTION THERAPYfor SANFILIPPO SYNDROME – proof of principleJuliana Inês Santos*, Maria Francisca Coutinho*, Paulo Gaspar and Sandra AlvesLysosomal Storage Disorders GroupResearch & Development Unit,Department of Human Genetics,INSA14th International Symposium15th-17th March, 2018MUCOPOLYSACCHARIDOSIS (MPS) TYPE III Autosomal recessive  Lysosomal Storage Disorders Sub-type of MPSs;glycosaminoglycans (GAGs) Accumulated substrate: heparan sulphate  4 different diseases: III A III B III C III Ddepending on the defective enzyme Chronic Progressive Large spectrum of severity & symptomsMPS III (= Sanfilippo Syndrome)MUCOPOLYSACCHARIDOSES (MPS)AVAILABLE THERAPIES None!…only symptomatic!ameliorate symptomssupport disabled patientsERT for neurodegenerative MPS would require theintroduction of active enzyme into the CNSextra difficulties!Still, it’s being attempted withsome promissing resultsAVAILABLE THERAPIES None!…only symptomatic!ameliorate symptomssupport disabled patientsERT for neurodegenerative MPS would require theintroduction of active enzyme into the CNSStill, it’s being attempted withsome promissing resultsPerfect Target forSubstrate ReductionApproaches! gSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsubstrate reduction geneticgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIearly stage of the HS biosynthetic cascadenaturally occurring post-ranscriptionalgene silencing processDesigned to induce RNAigSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsiRNAMPS III fibroblasts siRNAsRISCControl fibroblasts gSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsiRNA24/48h incubationharvest cellsRNA extractioncDNA synthesisqRT-PCR (target gene expression assessment)  Livak methodNTmRNAMPS III A MPS III C MPS III DgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsiRNAMPS III fibroblasts GAGsgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsiRNAMPS III fibroblasts GAGsgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsiRNAMPS III fibroblasts Further validation:   nr of experiments; immunocytochemistry(anti-HS antibody) + tests in MPS IIIBgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIIsiRNAMPS III fibroblasts Promising results!Reasons to keep studying...A LOOK FORWARD… Vector design & siRNA encapsulation into liposomes  bioavailability of siRNAs; protection from degradation control of  circulation time release rate  Coupling of specific ligands to siRNA-carrying liposomeso Transferrin (Tf)o Rabies virus peptide derivative (RGV-2r) Efficiency assessment+ Targeting of brain cellsA LOOK FORWARD…in vivo studiesSUMMARY“early stages”  GAGsbiosynthesis gene GAG storagegSRT FOR MUCOPOLYSACCHARIDOSIS TYPE IIISUMMARYgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE III“early stages”  GAGsbiosynthesis geneTherapeutic use SUMMARYgSRT FOR MUCOPOLYSACCHARIDOSIS TYPE III“early stages”  GAGsbiosynthesis geneHolds potential to benefit virtually all MPS!ACKNOWLEDGMENTSDr. Sandra AlvesProf. Mª João PrataJuliana Inês SantosPaulo GasparSFRH/BPD/101965/2014SFRH/BD/124372/2016bcp/LIM/DGH/Dz2015António Reis THANK YOU!

Genetically Modulated Substrate Reduction Therapy for Sanfilippo Syndrome: Proof of Principle

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

http://repositorio.insa.pt/bitstream/10400.18/7454/1/Santos%20JI%20%28Barcelona%202018%29.pdf

Genetically Modulated Substrate Reduction Therapy for Sanfilippo Syndrome: Proof of Principle

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Repositório Científico do Instituto Nacional de Saúde

Repositório Científico do Instituto Nacional de Saúde

Scientific Repository of the National Health Institute Doutor Ricardo Jorge