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- Antisense oligonucleotide exon-skipping as a therapeutic approach for a rare diseasePublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana I.; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Omidi, Maryam; Pohl, Sandra; Alves, SandraMucolipidosis II (MLII) is a Lysosomal Storage Disorder caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase, which is responsible for the Mannose- 6-Phosphate marker addition to lysosomal enzymes. Of all MLII mutations, the c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) for MLII. Previously, on MLII patients’ fibroblasts, ASOs were used to skip exon 19 of the GNPTAB pre-mRNA, successfully resulting in the production of an in-frame mRNA[1]. Now, our aim is to analyze if these results are translated to the enzymatic and cellular phenotype level.
- Exploring an antisense oligonucleotide exon-skipping therapeutic strategy for Mucolipidosis IIPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Alves, SandraIntroduction: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase, which is responsible for the Mannose-6-Phosphate marker addition to lysosomal enzymes. Of all ML II mutations, the c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) for ML II. Previously, on ML II patients’ fibroblasts, ASOs were used to skip exon 19 of the GNPTAB pre-mRNA, successfully resulting in the production of an in-frame mRNA. Now, our aim is to analyze if these results are translated to the enzymatic and cellular phenotype level.
- Is Antisense Oligonucleotide-Mediated Exon Skipping a Potential Therapeutic Approach for Mucolipidosis II?Publication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Duarte, Ana Joana; Gaspar, Paulo; Santos, Juliana Inês; Coutinho Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntridution: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs lysosomal hydrolases trafficking. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) to promote targeted skipping of GNPTAB exon 19, which harbors c.3503_3504del, the most frequent disease-causing variant. Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping, successfully generating an in-frame mRNA1. Now, our aim is to determine if this in-frame transcript leads to increased GlcNAc-PT levels. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts, but activity levels were similar in ML II and control fibroblasts (treated/non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without exon 19) and a mutant (with the mutation c.3503_3504del) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Also, we measured the activity of several hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment. To further validate this therapy we also generated a novel GlcNAc-PT antibody in rabbits. Results: Our results showed that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β-subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, in the delTC construct WB no α/β precursor band was detected. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts after treatment. However, only the α-Gal values were statistically significant, but the WB analysis for this enzyme did not reveal any band in ASO-treated ML II cells. We also developed a novel antibody for GlcNAc-PT. Preliminary results showed a β-subunit band both in control and patient fibroblasts (unexpected), but in overexpression both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess its specificity. Conclusion: Our ASO-based approach effectively promotes exon 19 skipping. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.
- Is exon skipping the key to correct N-acetylglucosamine-1-phosphotransferase deficiency? An antisense oligonucleotide therapeutic approachPublication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Gaspar, Paulo; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntroduction: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs the trafficking of lysosomal hydrolases. Of all ML II mutations, c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs). Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping in pre-mRNA, successfully generating an in-frame mRNA. Aims: Now, our aim is to determine whether this in-frame transcript leads to increased GlcNAc-PT levels improving ML II cellular phenotype. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts by a radioactive assay, but activity levels were similar in ML II and control fibroblasts (treated and non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without the exon 19) and a mutant (with the mutation c.3503_3504delTC) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Additionally, we have measured the activity of several lysosomal hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment of control and patient cells. To further help in the validation of this therapy we are also generating a novel GlcNAc-PT antibody in rabbits. Results: Our results demonstrated that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, the c.3503_3504delTC construct showed no expression, with no detectable α/β precursor band. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts treated with the ASO, particularly 24h and 48h post-treatment. However, only the values relatively to the α-Gal were statistically significant, but the WB analysis using an antibody against this enzyme did not detect any band in ASO-treated ML II fibroblasts. We also developed a novel antibody for GlcNAc-PT. Preliminary results revealed a band corresponding to the β-subunit in both control and ML II patient fibroblasts (unexpected), but in overexpression assays both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess their specificity. Conclusion: Our ASO-based approach effectively promotes the skipping of exon 19. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.
- Mucolipidosis II-Related Mutations Inhibit the Exit from the Endoplasmic Reticulum and Proteolytic Cleavage of GlcNAc-1-Phosphotransferase Precursor Protein (GNPTAB)Publication . De Pace, Raffaella; Coutinho, Maria Francisca; Koch-Nolte, Friedrich; Haag, Friedrich; Prata, Maria João; Alves, Sandra; Braulke, Thomas; Pohl, SandraMucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β-subunit precursor protein of GlcNAc-1-phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α-subunit-specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild-type and mutant α/β-subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β-subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc-1-phosphotransferase that may help to explain the clinical phenotype of MLII patients
- The lysosomal storage disorders mucolipidosis type II, type III alpha/beta, and type III gamma: update on GNPTAB and GNPTG mutationsPublication . Velho, Renata Voltolini; Harms, Frederike L.; Danyukova, Tatyana; Ludwig, Nataniel F.; Friez, Michael J.; Cathey, Sara S.; Filocamo, Mirella; Tappino, Barbara; Güneş, Nilay; Tüysüz, Beyhan; Tylee, Karen L.; Brammeier, Kathryn L.; Heptinstall, Lesley; Oussoren, Esmee; Ploeg, Ans T.; Petersen, Christine; Alves, Sandra; Saavedra, Gloria Durán; Schwartz, Ida V.; Muschol, Nicole; Kutsche, Kerstin; Pohl, SandraMutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/β-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.
- The lysosomal storage disorders mucolipidosis type II, type III alpha/beta, and type III gamma: Update on GNPTAB and GNPTG mutationsPublication . Velho, Renata Voltolini; Harms, Frederike L.; Danyukova, Tatyana; Ludwig, Nataniel F.; Friez, Michael J.; Cathey, Sara S.; Filocamo, Mirella; Tappino, Barbara; Güneş, Nilay; Tüysüz, Beyhan; Tylee, Karen L.; Brammeier, Kathryn L.; Heptinstall, Lesley; Oussoren, Esmee; van der Ploeg, Ans T.; Petersen, Christine; Alves, Sandra; Saavedra, Gloria Durán; Schwartz, Ida V.; Muschol, Nicole; Kutsche, Kerstin; Pohl, SandraMutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/β-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.