Mucopolysaccharidoses II is an X-linked recessive disorder caused by lysosomal enzyme iduronate-2-sulfatase (I2S) deficiency. The lack of I2S causes glycosaminoglycans (GAGs) such as chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) to accumulate in all body tissues, causing organ abnormalities. In severe cases, this leads to death during the teenage years. In MPS II, enzyme replacement therapy (ERT) appears to be the most effective treatment. However, ERT is unable to cross the blood-brain barrier and shows several limitations. The isoflavone genistein has been studied as a potential therapy for the MPS because of its putative ability to inhibit GAG synthesis and subsequent accumulation. Furthermore, genistein can cross the blood-brain barrier in murine models, but previous studies showed variable outcomes. This project is focused on GAGs qualitative and quantitative characterization in a murine MPS II model (I2S knock-out) non-treated, treated with ERT or treated with genistein, analyzed at different weeks after the beginning of therapy. GAGs were extracted from plasma, urine, liver and brain samples of 225 mice, according to a standardized protocol. The crude GAGs fraction was digested with specific enzymes to isolate Hyaluronic Acid (HA), CS, DS and HS disaccharide units. Disaccharides were lyophilized and tagged with a fluorophore. Finally, derivatized disaccharides were separated and quantified by capillary electrophoresis interfaced with laser-induced fluorescence. Compared to wild type (WT), the I2S KO samples showed from the first weeks significant differences for all the analyzed parameters. In these subjects, CS-DS, HS and HA concentration, as well as total GAGs concentration, increased significantly compared to WT subjects and remains high during all the analyzed weeks. We observe, higher GAGs concentration, CS / HS imbalance towards HS, increase in charge density, and alteration in the sulfation profile. In particular, CS-DS was mainly sulfated in the 2,4s position, compared to the 6s position which was prevalent in WT subjects. These differences are particularly pronounced in liver samples, suggesting a higher GAGs accumulation in solid organs. As for the therapies, we can see how ERT can reduce many of the altered parameters, but it cannot restore the WT condition completely. Moreover, ERT did not show significant alterations in the brain samples, confirming that the enzyme cannot cross the blood-brain barrier. Genistein treatment, showed no significant alterations, either quantitatively or qualitatively, compared to non-treated I2S KO subjects. Comparing the results obtained from WT mice and I2S KO mice we observe higher values of each index considered. We can also evidence how the increase in disaccharides sulfated in position 2,4s indicates an accumulation of DS in plasma, urine, liver and brain, specific of MPSII. From these results, we can propose the analysis of 2,4s sulfation index in plasma, as a useful tool in the diagnosis of this pathology. Moreover, as in MPSII patients, ERT therapy can significantly reduce GAGs accumulation. These findings further strengthen the coherence of this animal model to MPSII pathology. However, ERT is not able to induce total normalization of the parameters. On the contrary, genistein therapy did not show significant differences in I2S KO subjects, in any of the parameters considered. Therefore, this therapy would not seem to benefit the patient. In the next stage, we will also analyze different organs. Furthermore, we will have more indications of the effectiveness of different therapies in reducing GAGs accumulation, providing the basis for future clinical trials on humans.

La mucopolisaccaridosi II è una malattia genetica recessiva legata al cromosoma X, causata dal deficit dell'enzima lisosomiale iduronato-2-solfatasi (I2S). La mancanza di I2S fa sì che i glicosaminoglicani (GAG) come il condroitin solfato (CS), il dermatan solfato (DS) e l'eparan solfato (HS) si accumulino in tutti i tessuti del corpo, causando anomalie a diversi organi. Per MPS II, la terapia enzimatica sostitutiva (ERT) sembra essere il trattamento più efficace. Tuttavia, ERT non è in grado di attraversare la barriera emato-encefalica e mostra diversi limiti. L'isoflavone della soia genisteina è stata studiato come potenziale terapia per le MPS grazie alla sua presunta capacità di inibire la sintesi dei GAG e il loro accumulo. Inoltre, Genisteina può attraversare la barriera ematoencefalica nei modelli murini, ma studi precedenti hanno mostrato esiti contrastanti. Questo progetto è focalizzato sulla caratterizzazione qualitativa e quantitativa dei GAG in un modello murino di MPS II (I2S knock-out) non trattato, trattato con ERT o trattato con Genisteina. I GAG sono stati estratti da campioni di plasma, urina, fegato e cervello di 225 topi, secondo un protocollo standardizzato. La frazione di GAG grezza è stata digerita con enzimi specifici per isolare le unità disaccaridiche di acido ialuronico (HA), CS, DS e HS. I disaccaridi sono stati poi liofilizzati e contrassegnati con un fluoroforo. Infine, i disaccaridi derivatizzati sono stati quantificati mediante elettroforesi capillare interfacciata con LIF (laser-induced fluorescence). Rispetto al Wild Type (WT), i campioni I2S KO hanno mostrato sin dalle prime settimane differenze significative per tutti i parametri analizzati. In questi soggetti, la concentrazione di CS-DS, HS e HA, così come la concentrazione totale di GAG, è aumentata in modo significativo rispetto al WT e rimane elevata durante tutte le settimane analizzate. Si evidenzia una elevata concentrazione di GAG, uno squilibrio del rapporto CS / HS verso HS, un aumento della densità di carica e un’alterazione del profilo di solfatazione. In particolare, CS-DS è principalmente solfatato nella posizione 2,4s, rispetto alla posizione 6s che è prevalente nei soggetti WT. Queste differenze sono particolarmente pronunciate nei campioni di fegato, suggerendo un maggiore accumulo di GAG negli organi solidi. Per quanto riguarda le terapie, possiamo notare come l'ERT possa ridurre molti dei parametri alterati, ma non può ripristinare completamente la condizione di WT. Inoltre, ERT non ha determinato alterazioni nei campioni di cervello, confermando che l'enzima non può attraversare la barriera ematoencefalica. Il trattamento con Genisteina non ha mostrato alterazioni significative rispetto ai soggetti I2S KO non trattati. In conclusione, confrontando i risultati ottenuti da topi WT e topi I2S KO, osserviamo valori più elevati per ciascun indice considerato. Possiamo anche evidenziare come l'aumento dei disaccaridi solfatati in posizione 2,4s indichi un accumulo di DS in questi organi, specifico della MPSII. Da questi risultati si può proporre l'analisi dell'indice di solfatazione 2,4s nel plasma, come strumento utile nella diagnosi di MPSII. Inoltre, come nei pazienti con MPSII, la terapia ERT può ridurre significativamente l'accumulo di GAG. Questi risultati rafforzano ulteriormente la coerenza di questo modello animale con la patologia MPSII. Tuttavia, ERT non è in grado di indurre la normalizzazione totale dei parametri. Al contrario, la terapia con genisteina non ha mostrato differenze significative nei soggetti I2S KO, in nessuno dei parametri considerati. Nella fase successiva, analizzeremo diversi organi. Inoltre, avremo più indicazioni sull'efficacia di diverse terapie, fornendo le basi per futuri studi clinici sull'uomo.

ANALISI DEI GLICOSAMINOGLICANI DI FLUIDI E ORGANI BIOLOGICI IN UN MODELLO ANIMALE DI MUCOPOLISACCARIDOSI II (SINDROME DI HUNTER): EFFETTO DEI TRATTAMENTI CON TERAPIA ENZIMA SOSTITUTIVA E GENISTEINA / Veronica Mantovani , 2023 May 19. 34. ciclo, Anno Accademico 2020/2021.

ANALISI DEI GLICOSAMINOGLICANI DI FLUIDI E ORGANI BIOLOGICI IN UN MODELLO ANIMALE DI MUCOPOLISACCARIDOSI II (SINDROME DI HUNTER): EFFETTO DEI TRATTAMENTI CON TERAPIA ENZIMA SOSTITUTIVA E GENISTEINA

MANTOVANI, VERONICA
2023

Abstract

Mucopolysaccharidoses II is an X-linked recessive disorder caused by lysosomal enzyme iduronate-2-sulfatase (I2S) deficiency. The lack of I2S causes glycosaminoglycans (GAGs) such as chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) to accumulate in all body tissues, causing organ abnormalities. In severe cases, this leads to death during the teenage years. In MPS II, enzyme replacement therapy (ERT) appears to be the most effective treatment. However, ERT is unable to cross the blood-brain barrier and shows several limitations. The isoflavone genistein has been studied as a potential therapy for the MPS because of its putative ability to inhibit GAG synthesis and subsequent accumulation. Furthermore, genistein can cross the blood-brain barrier in murine models, but previous studies showed variable outcomes. This project is focused on GAGs qualitative and quantitative characterization in a murine MPS II model (I2S knock-out) non-treated, treated with ERT or treated with genistein, analyzed at different weeks after the beginning of therapy. GAGs were extracted from plasma, urine, liver and brain samples of 225 mice, according to a standardized protocol. The crude GAGs fraction was digested with specific enzymes to isolate Hyaluronic Acid (HA), CS, DS and HS disaccharide units. Disaccharides were lyophilized and tagged with a fluorophore. Finally, derivatized disaccharides were separated and quantified by capillary electrophoresis interfaced with laser-induced fluorescence. Compared to wild type (WT), the I2S KO samples showed from the first weeks significant differences for all the analyzed parameters. In these subjects, CS-DS, HS and HA concentration, as well as total GAGs concentration, increased significantly compared to WT subjects and remains high during all the analyzed weeks. We observe, higher GAGs concentration, CS / HS imbalance towards HS, increase in charge density, and alteration in the sulfation profile. In particular, CS-DS was mainly sulfated in the 2,4s position, compared to the 6s position which was prevalent in WT subjects. These differences are particularly pronounced in liver samples, suggesting a higher GAGs accumulation in solid organs. As for the therapies, we can see how ERT can reduce many of the altered parameters, but it cannot restore the WT condition completely. Moreover, ERT did not show significant alterations in the brain samples, confirming that the enzyme cannot cross the blood-brain barrier. Genistein treatment, showed no significant alterations, either quantitatively or qualitatively, compared to non-treated I2S KO subjects. Comparing the results obtained from WT mice and I2S KO mice we observe higher values of each index considered. We can also evidence how the increase in disaccharides sulfated in position 2,4s indicates an accumulation of DS in plasma, urine, liver and brain, specific of MPSII. From these results, we can propose the analysis of 2,4s sulfation index in plasma, as a useful tool in the diagnosis of this pathology. Moreover, as in MPSII patients, ERT therapy can significantly reduce GAGs accumulation. These findings further strengthen the coherence of this animal model to MPSII pathology. However, ERT is not able to induce total normalization of the parameters. On the contrary, genistein therapy did not show significant differences in I2S KO subjects, in any of the parameters considered. Therefore, this therapy would not seem to benefit the patient. In the next stage, we will also analyze different organs. Furthermore, we will have more indications of the effectiveness of different therapies in reducing GAGs accumulation, providing the basis for future clinical trials on humans.
GLYCOSAMINOGLYCAN ANALYSIS OF BIOLOGICAL FLUIDS AND ORGANS IN AN ANIMAL MODEL OF MUCOPOLYSACCHARIDOSIS II (HUNTER SYNDROME): EFFECT OF TREATMENTS WITH ENZYME REPLACEMENT THERAPY AND GENISTEIN
19-mag-2023
VOLPI, Nicola
File in questo prodotto:
File Dimensione Formato  
Tesi di Dottorato in Clinical and Experimental Medicine - Veronica Mantovani.pdf

embargo fino al 18/05/2026

Descrizione: Tesi definitiva Mantovani Veronica
Tipologia: Tesi di dottorato
Dimensione 2.94 MB
Formato Adobe PDF
2.94 MB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

Licenza Creative Commons
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1305627
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact