Less is more  – substrate reduction therapy for mucopolysaccharidoses through RNAi

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Assessing the potential of RNA-based therapeutics for a group of Lysosomal Storage Diseases with neurological involvement

Publication . Santos, Juliana Inês; Gonçalves, Mariana; Matos, Liliana; Gaspar, Paulo; Pires, Maria João; Oliveira, Paula; Prata, Maria João

During the first two decades of the 21st century, remarkable progresses have been achieved in the field of RNA-based therapeutics. From antisense RNA to RNA modification, the therapeutic potential of RNA-based technologies has nothing but increased. In our lab, we have been addressing the potential of different RNA-based drugs to either correct or ameliorate the sub-cellular phenotype of a number of severe, life-threatening diseases: the so-called Lysosomal Storage Disorders (LSDs). Among them, we are focusing our efforts on those which present with a predominant neurological phenotype, since there are virtually no approved treatments for any of them. Briefly, two major research lines are being pursued: the first relies on the design of mutation-specific approaches to correct abnormal splicing processes in LSD-related genes, whenever they underlie pathology. The second depends upon selective downregulation of genes involved in the biosynthethic cascades that give origin to the substrates that accumulate in each pathology. Here we present an overview on our results with both approaches on Sanfilippo syndrome, a sub-group of severe neurodegenerative LSDs. For the mutation-specific, splicing correction approach, we are using U1snRNA vectors to restore the splicing defect caused by the HGSNAT mutation c.234+1G>A, that leads to Sanfilippo C disease. We started by demonstrating in vitro that a modified U1snRNA vector designed to improve the definition of HGSNAT exon 2 could partially restore its normal splicing process. Now, we are evaluating its therapeutic potential in vivo, in mice expressing the human splicing defect. For the substrate reduction approach, we are using siRNAs. By acting over a specific biosynthethic cascade, siRNAs promote an overall decrease of the accumulating substrate. So far, we have already tested this approach in patients’ fibroblasts and observed a high inhibition of the target mRNAs and a decrease in storage. Overall, there are substantial differences between these two approaches but they also face common challenges and show equally promising results.

2021-11Conference object

Open access

NPC1 silent variant induces skipping of exon 11 (p.V562V) and unfolded protein response was found in a specific Niemann-Pick type C patient

Publication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo Min; Cardoso, Maria Teresa; Ribeiro, Isaura; Chaves, Paulo; Santos, Juliana Inês; Quelhas, Dulce; Lacerda, Lúcia; Leão Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, Sandra

Background: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis. Methods: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels. Results: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER. Conclusion: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.

2020-11Journal article

Open access

When less is actually more: in vitro assessment of the potential of anti-XYLT1 siRNAs to promote substrate reduction in Mucopolysaccharidosis type III

Publication . Santos, Juliana Inês; Coutinho, Maria Francisca; Gaspar, Paulo; Prata, Maria João; Alves, Sandra

Mucopolysaccharidosis type III (MPS III) refers to a group of four autosomal recessive neurodegenerative lysosomal storage disorders (LSD) caused by the incomplete lysosomal degradation of the heparan sulphate (HS) that accumulates in patient cells and triggers disease. Degeneration of the central nervous system is the major hallmark of these disorders, resulting in mental retardation and hyperactivity. By their mid-teenage years most affected patients are dependent on their caregivers for all needs and death occurs at the end of the second or early in the third decade of life. The classical therapeutic approach for LSDs, enzyme replacement therapy, would hardly rise as a potentially successful tool to reduce the disease burden in MPS III patients due to the inability of the recombinant enzymes to cross the blood-brain barrier (BBB), having no impact in neuropathology. Thus, there is no effective therapy available, with treatment limited to clinical management of neurological symptoms. A tempting alternative, however, would be to block substrate accumulation upstream, by decreasing its synthesis. That concept is known as substrate reduction therapy (SRT). In order to decrease HS storage inside the lysosomes, we designed and assayed in MPS III patients’ fibroblasts a specific siRNA pool targeting XYLT1, a gene that encodes an enzyme involved in an early stage of the HS biosynthetic cascade. Fibroblasts from MPS IIIA, B, C and D patients were transfected with the designed siRNAs pool to inhibit XYLT1. Cell pellets were collected 24/48/72 hours and 7 days post- transfection and total RNA extracted. Target mRNA levels were evaluated through qRT-PCR using the 2-∆∆Cq method. Additionally, the effect on HS accumulation was quantified 24 and 48h after transfection using a modified 1,9-dimethylmethylene blue assay. The results showed a high inhibition of the XYLT1 gene mRNAs (around 80%) and a decrease in GAGs storage (only assessed for types C and D until now). Currently, we are evaluating the effect of that decrease on the overall GAGs storage 7 days post-transfection, also with promising results. Here we present an overview on the current results of this project, while discussing its next steps, namely the development and evaluation of vectors for in vivo delivery. Our goal is to develop targeted stable nucleic acid lipid particles (t-SNALPs) coupled with different ligands, to promote cell uptake of the ‘anti-GAG’ siRNAs in a variety of cells, including neurons.

2021-06Conference object

Open access

Genetic Substrate Reduction Therapy for Mucopolysaccharidoses type III: toward a siRNA-containing nanoparticle targeted to brain cells

Publication . Coutinho, Maria Francisca; Santos, Juliana Inês; Gaspar, Paulo; Prata, Maria João; Jurado, Amália Silva; Pedroso de Lima, Maria da Conceição; Alves, Sandra

The classical therapeutic approach for LSDs, enzyme replacement therapy, would hardly rise as a potentially successful tool to reduce the disease burden in MPS III patients, as it is long known to have no impact on neuropathology. A tempting alternative, however, would be to block substrate accumulation upstream, by decreasing its synthesis. That concept is known as substrate reduction therapy (SRT). Having this in mind, we designed an RNA-based strategy based upon the selective downregulation of one gene involved in the very early stages of the glycosaminoglycans’ (GAG) biosynthethic cascade. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPS’ symptoms. As tools to achieve substrate reduction, we are evaluating a specific type of antisense oligonucleotides, able to trigger a naturally-occurring post-transcriptional gene silencing process called RNA interference: the small interfering RNAs (siRNAs). So far, the obtained results are quite promising with marked decreases of the target mRNA levels in fibroblast cell lines for all the different MPS III disease sub-types. Initial studies addressing the overall storage of sulphated GAGs used either the routine alcian blue or a modified, more sensitive 1,9-dimethylmethylene blue assay at different time points. Nevertheless, the low confluency levels required for siRNA transfection did not allow detection of GAGs excreted to the culture media. Similar problems have been noted by other authors, including over- and under-estimation of sulphated GAGs. This is particularly relevant in small samples, like the ones we have been using. In fact, even the direct assessment of the intralysosomal suphated GAGs on those samples, while more reliable, does show some limitations. That is why we are currently implementing a novel, more sensitive method for GAG detection by liquid chromatography and quantification with electrospray ionization–tandem mass spectrometry (Saville et al., 2018). Thus, additional data on the effect of the designed siRNAs on substrate accumulation will be collected over the next months and other methods will be used to further address this issue. Here we present an overview on the current results of this project, while discussing its’ next steps, namely the development and evaluation of vectors for in vivo delivery. Our goal is to develop targeted stable nucleic acid lipid particles (t-SNALPs) coupled with different ligands, which promote cell uptake of the ‘anti-GAG’ siRNAs in a variety of cells, including neurons.

2020-11Conference object

Open access

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

Publication . Santos, J.I.; Coutinho, Maria Francisca; Gaspar, P.; Alves, S.

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.

2018-09Conference object

Open access