Short term scientific mission about therapeutic strategies to modulate splicing in lysosomal storage disorders.COST ACTION BM1207N/

Matos, Liliana

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

Development of antisense U1 snRNA-mediated therapeutic strategies to modulate splicing in lysosomal storage disorders

Repositório Científico do Instituto Nacional de Saúde

Short term scientific mission Development of antisense U1 snRNA-mediated therapeutic strategies to modulate splicing in lysosomal storage disordersLiliana MatosCOST Action BM1207Porto Meeting22nd March 2017Lysosomal Storage Disorders (LSDs) Large group of inherited metabolic disorders (>50) The majority are caused by deficiencies in specificlysosomal enzymes Individually rare Autosomal recessive (majority) Prevalence as a group is estimated in 1:4000 – 1:8000live birthsProgressive lysosomal accumulation of undegraded metabolites results:- generalised cell and tissue dysfunction- multi-systemic pathologyearly and severe phenotype  Clinical presentation:can vary late-onset mild diseaseReviewed in Filocamo and Morrone, 2011 Study of 2 different splice donor site mutations2 patients diagnosed with different LSDs foundPatient Allele 1 Allele 2 LocationPatient 1 (MPS I) c.1650+5G>A c.1205G>A (p.W402X) Intron 11/Exon 9Patient 2 (ML III α/β) c.3335+6T>G c.2864C>T (p.A955V) Intron 17/Exon 14Project SPLICETHER from FCT (PTDC /BBB-BMD/6301/2014)Work in progress :  Splicing mutations are frequent in these pathologies (10 to ~16% of the mutations described) Good targets for mutation specific U1 snRNA therapeutic approaches5’ donor site mutations~64% MPS I~47% ML III α/β To correct the pathogenic effect of both splice donor site mutations throughU1 snRNA-mediated therapeutic strategiesMain aim: Mucopolysaccharidosis I(Hurler; Hurler-Scheie; Scheie syndromes) Autossomal recessive disorder Caused by mutations in IDUA gene (14 exons)α-L-IduronidaseResponsible for the degradation of Therapy: Enzyme replacement therapy – laronidase (Aldurazyme®) Hematopoietic stem cell transplantation Still with some limitationsDevelopment of alternative or adjuncttherapies would be importantdermatan and heparan sulfateMucopolysaccharidosis  I IDUA gene mutation analysis (RT-PCR)c.1650+5G>A/p.W402XPrimers for exon 9 and 12:- Normal length transcript- One with exon 11 skipping due to the donor site mutationPatient 1: two transcripts- One with normal length arising fromthe nonsense alleleNC WT P1500 bpHeteroduplex200 bpM9 10 11 12Exon109 121. Analysis of IDUA exon 11 splice donor sites (SDS)Loss of  Splicing therapeutic approach for the +5 IDUA gene mutation with modified U1 vectorsU1 WTGUCcauucaua3’ 3’ U1 WTGUCcauucauaMucopolysaccharidosis  Ibase pairing (+5)IDUA exon 11 WT SDSCAGgcaagugg…Exon 11Splice donor site5’2. Design and construction of U1 modified vectors (pG3U1)Increased complementarity to the SDSU1 IDUA mut 2 (+2G) 3’…GUCcguucaua…5’…CAGgcaaaugg…Exon 11 mutant SDSb) U1 IDUA mut 3 (+2G;+5T;+7C;+8C)3’…GUCcguuuacc…5’…CAGgcaaaugg…Exon 11 mutant SDSc) a) U1 IDUA mut 1 (+5T)5’…CAGgcaaaugg…3’…GUCcauuuaua…Exon 11 mutant SDSIDUA exon 11 mutant SDSCAGgcaaaugg…Exon 11Splice donor site5’X Transfection of wild-type and patient 1 fibroblasts with 2.5 µg of modified U1 vectors for 24 hPrimers for exon 9 and 12: RT-PCR analysisWild-type control Patient 1Mucopolysaccharidosis  IM WTNC P1500 bp300 bp 109 129 10 11 12ExonHeteroduplexc.1205G>ANo heterozygous peak Not even a partial correction observed→ No correction with 3.5 or 4.5 µg of each U1 vector or even in a 48 h treatment Mucolipidosis  III alpha/beta(pseudoHurler polydystrophy) Autossomal recessive disorder Caused by mutations in GNPTAB gene (21 exons)α/β-subunit of the N-acetylglucosamine (GlcNAc)-1-phosphotransferaseTherapy: No causal therapies available for treatment Disease management only symptomatic  Development of therapeutic strategies is essentialFormation of the mannose-6-phosphate residues on lysosomal enzymes GNPTAB gene mutation analysis (RT-PCR)c.3335+6T>G/p.A955VPrimers for exon 13 and 18:Mucolipidosis III alpha/beta- Normal length transcript- One with exon 17 skipping due to the donor site mutationPatient 2: two transcripts- One with normal length arising from the missense alleleM NC WT P2500 bp800 bp Exon13 14 15 16 17 1813 14 15 16 181. Analysis of GNPTAB exon 17 splice donor sites (SDS)Loss of  Splicing therapeutic approach for the +6 GNPTAB gene mutation with modified U1 vectors U1 WTGUCcauucaua3’ 3’ U1 WTGUCcauucauaMucolipidosis III alpha/betabase pairing (+6)2. Design and construction of U1 modified vectors (pG3U1)GNPTAB exon 17 WT SDSUAGguaaguag…Exon 17Splice donor site5’a) U1 GNPTAB mut 1 (+6C)5’…UAGguaaggag…3’…GUCcauuccua…Exon 17 mutant SDSb) U1 GNPTAB mut 2(-3A;+6C;+8C)5’…UAGguaaggag…3’…AUCcauuccuc…Exon 17 mutant SDSGNPTAB exon 17mutant SDSUAGguaaggag…Exon 17Splice donor site5’XIncreased complementarity to the SDS Transfection of wild-type and patient 2 fibroblasts with 2.5 µg of modified U1 vectors for 24 hPrimers for exon 13 and 18: RT-PCR analysisWild-type control Patient 2Mucolipidosis III alpha/beta500 bpM NC WT P2800 bp13 14 15 16 18Exon13 14 15 16 17 18c.2864C>TNo heterozygous peak Not even a partial correction observed→ No correction with 3.5 or 4.5 µg of each U1 vector or even in a 48 h treatment Transfection results conclusions Absence of correction for both 5’ splice site mutations - High level of cell death after transfection- Presence of different modified U1’s (?)- Lipo-mediated transfection toxicity (?)Low transfection eficiency?  Co-transfection of minigene constructs with the U1’s (Hep 3B cell line) Lentiviral transduction of U1’s in patients fibroblastsNext steps:Minigenes expression (Hep 3B)• MPS I (IDUA minigenes)Intron 11Exon 11 Exon 12Splice Donor Splice AcceptorpSPL3 vectorIntron 10 Intron 12SA2SD6EcoRIEcoRI EcoRI• ML III α/β (GNPTAB minigenes)Splice Donor Splice AcceptorpSPL3 vectorSA2SD6EcoRIExon 17Intron 16 Intron 17EcoRI EcoRI• RT-PCR analysisExon 11/Intron 11Wild-type: …CCGGGCAGgcaagtgg…Mutant: …CCGGGCAGgcaaatgg…c.1650+5G>AExon 17/Intron 17Wild-type: … AAATATAGgtaagtag…Mutant: … AAATATAGgtaaggag…c.3335+6T>GM NCMinigene500 bp200 bpV VExon 12V VExon 17V VMinigenes:- WT intron 11- c.1650+5G>A- c.3335+6T>G- WT intron 17XMinigenes co-transfection with different U1’s (Hep 3B)• MPS I – IDUA gene (RT-PCR analysis)M NC500 bp200 bpc.1650+5G>A minigeneExon 12V VV VExon 12V VExon 11X New constructs!• ML III α/β – GNPTAB gene (RT-PCR analysis)M NC500 bp200 bpc.3335+6T>G minigeneWT intron 17 minigeneV VExon 17V VInsertion of exon 17Short term scientific missionLearn and perform the lentiviral transduction technique of the differentmodified U1’s into patients’ fibroblasts to try the therapeutic rescue of the 5’splice site mutations, c.1650+5G>A (IDUA gene) and c.3335+6T>G (GNPTAB gene)Main aim: Some different splice donor site mutations have been partially or totally rescued through viral transduction of U1 snRNAs (Glaus et al., 2011; Schmid et al., 2011; Dal Mas et al., 2015) Lentiviral transduction of U1 constructs in fibroblasts More efficient technique to the acquisition of external vectors   Less toxic for cells than lipo-mediated transfection ( ↓ mortality )Workflow1. Cloning of the different U1 snRNAs in the lentiviral vector U1 WTU1 IDUA mut 1 U1 IDUA mut 2 U1 IDUA mut 3pG3U1U1 GNPTAB mut 1 U1 GNPTAB mut 2 GFPp.RRLSIN.cPPT.SFFV/GFP.WPRE2. HEK293T cells infection for lentivirus productionGFPp.RRLSIN.cPPT.SFFV/GFP.WPREpSPAX2 + +pMD2.G (VSV-G) TransfectionHEK293THarvest:- 24 h- 48 hLentiviral vector supernatant3. Lentivirus transduction in fibroblast cells MPS I patient (c.1650+5G>A) ML III α/β patient (c.3335+6T>G) Wild-type control Positive control (BBS-1 gene)  Schmid et al., 20115 mlCells harvest after 4 days  RT-PCR analysis- Splicing pattern qPCR analysis- Nr of lentivirus copiesLentiviral transduction results: MPS I RT-PCR analysisM NC1000 bp500 bpWild-type control Patient 1 (c.1650+5G>A)9 10 11 12Exon109 12 qPCR analysis0,00100,00200,00300,00400,00500,00600,00700,00Nr of lentivirus copiesNr of lentivirus copies = 2(1+∆Cq)Albumin (n=2) vs GFP (n=?)Reference gene vs Target gene:Lentiviral transduction results: ML III α/β RT-PCR analysisM NC1000 bp13 14 15 16 18Exon13 14 15 16 17 18Wild-type control Patient 2 (c.3335+6T>G)500 bp qPCR analysis0,0050,00100,00150,00200,00250,00300,00350,00400,00450,00Nr of lentivirus copiesNr of lentivirus copies = 2(1+∆Cq)Albumin (n=2) vs GFP (n=?)Reference gene vs Target gene:Lentiviral transduction results: BBS (+ control)c.479G>A Last nt exon 51 3 6 9lentiviral supernatant (ml) ML III α/β parallel experimentM500 bp100 bpBBS patient5 ml lentiviral supernatantFully-adapted MPS I (c.1650+5G>A) ML III α/β (c.3335+6T>G)- ↑ lentivirus volume- Optimization !!!Our cases:Main STSM achievements Cloning of different modified U1’s in a lentiviral vector (p.RRLSIN.cPPT.SFFV/GFP.WPRE)  Learning and performing the lentiviral transduction technique inhuman fibroblasts Obtaining the preliminary results of the lentiviral transduction ofdifferent modified U1’s into MPS I and ML III α/β patients’ fibroblasts totry the therapeutic rescue of the 5’ ss mutations c.1650+5G>A andc.3335+6T>G Acquisition of the lentiviral transduction technique allowing, in a nearfuture, its implementation in our laboratory to be applied not only tothe 5’ ss mutations in study as to other donor site mutations reported inLSDs patient’sLysosomal storage disorders research groupDr. Sandra AlvesDr. Olga AmaralFrancisca CoutinhoJuliana SantosAcknowledgmentsProf. John NeidhardtDr. Christoph JüschkeHuman Genetics groupRegina VilelaAna Joana DuarteProf. Belén PérezProf. Lourdes Ruiz-DesviatFunding:Dr. Mirella FilocamoSPLICETHER (PTDC /BBB-BMD/6301/2014)BM1207Prof. Mª  João Prata Thank you for your attention

http://repositorio.insa.pt/bitstream/10400.18/5488/1/STSM%20COST%20presentation%20Matos%20L%2c%202017.pdf

Development of antisense U1 snRNA-mediated therapeutic strategies to modulate splicing in lysosomal storage disorders

Abstract

Similar works

Full text

Available Versions

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

Repositório Científico do Instituto Nacional de Saúde