Search

Umbilical cord blood (UCB) is a promising source of stem cells to use in early haematopoietic stem cell transplantation (HSCT) approaches for several genetic diseases that can be diagnosed at birth. Mucopolysaccharidosis type I (MPS-I) is a progressive multi-system disorder caused by deficiency of lysosomal enzyme α-L-iduronidase, and patients treated with allogeneic HSCT at the onset have improved outcome, suggesting to administer such therapy as early as possible. Given that the best characterized MPS-I murine model is an immunocompetent mouse, we here developed a transplantation system based on murine UCB. With the final aim of testing the therapeutic efficacy of UCB in MPS-I mice transplanted at birth, we first defined the features of murine UCB cells and demonstrated that they are capable of multi-lineage haematopoietic repopulation of myeloablated adult mice similarly to bone marrow cells. We then assessed the effectiveness of murine UCB cells transplantation in busulfan-conditioned newborn MPS-I mice. Twenty weeks after treatment, iduronidase activity was increased in visceral organs of MPS-I animals, glycosaminoglycans storage was reduced, and skeletal phenotype was ameliorated. This study explores a potential therapy for MPS-I at a very early stage in life and represents a novel model to test UCB-based transplantation approaches for various diseases.

Lo scopo di questo progetto è stato lo sviluppo preclinico di una nuova terapia per la Mucopolisaccaridosi di tipo I (MPS-I): il trapianto di cellule staminali da sangue del cordone ombelicale, eseguito in epoca neonatale. L’MPS-I è una rara malattia lisosomiale dovuta a mutazioni nel gene IDUA, che codifica per l’enzima lisosomiale α-L-iduronidasi (IDUA). L’assenza di attività iduronidasica provoca l’accumulo di glicosaminoglicani nei tessuti, causando una disfunzione multi-organo progressiva e, in particolare, gravi anomalie scheletriche. La terapia d’elezione per la MPS-I è il trapianto di cellule staminali ematopoietiche (HSCT) da donatore sano, che riduce l’accumulo dei substrati e attenua molte manifestazioni cliniche, ma non è del tutto efficace sulle anomalie ossee. L’obiettivo di questa tesi è stato quello di testare nel modello murino di MPS-I l’efficacia di una nuova strategia trapiantologica, che combinava l’intervento precoce (neonatale) e l’uso del sangue del cordone ombelicale come fonte. Infatti, è stato deciso di trattare gli animali nel periodo peri-natale, per prevenirne le manifestazioni fenotipiche, e di impiegare il sangue cordonale come fonte di cellule da trapiantare, perché il trapianto cordonale in clinica dà diversi vantaggi rispetto al trapianto di midollo osseo, in particolare in questi pazienti. Il primo risultato ottenuto in questo lavoro è stata la caratterizzazione delle proprietà fenotipiche e funzionali delle cellule cordonali murine. E’ stato poi eseguito il trapianto di tali cellule in topi adulti wild type (WT) C57BL/6: si è ottenuto un buon attecchimento a lungo termine ed è stata verificata la presenza di cellule di origine del donatore in tutti i lineages ematopoietici. Infine, è stato valutato l’esito del trapianto neonatale di cellule cordonali nei topi MPS-I: si è ottenuto un aumento di attività IDUA negli organi periferici dei topi MPS-I con attecchimento elevato (>50%), e una conseguente riduzione dei livelli di glicosaminoglicani. Il fenotipo scheletrico dei topi a 20 settimane d’età è stato dettagliatamente descritto tramite indagini radiografiche, microCT e istologiche. Le radiografie hanno rivelato che, mentre nei topi MPS-I non trattati si aveva un generale ispessimento osseo rispetto ai WT, nei topi affetti con elevato attecchimento questa anomalia non si riscontrava. Le analisi microCT e istologiche hanno dimostrato che la porzione corticale del femore dei topi MPS-I presentava irregolarità che erano invece ridotte nei topi trattati con alto attecchimento. Questi dati confermano che il trapianto neonatale di cellule del cordone ombelicale risulta una terapia efficace nel modello murino di MPS-I. In collaborazione con il Prof. Aiuti presso l’Ospedale San Raffaele-Tiget, è stato intrapreso un nuovo progetto di ricerca con lo scopo di correggere geneticamente le cellule di cordone ombelicale per ottenere livelli sovra-fisiologici di espressione dell’enzima IDUA. L’obiettivo è quello di trasdurre cellule di cordone murino MPS-I con un vettore lentivirale PGK-IDUA e di trapiantare le cellule così corrette in topi MPS-I neonati. Verificheremo se sarà possibile ottenere nei riceventi livelli di attività enzimatica più alti che nel trapianto di cellule WT, e se l’esito della terapia genica sarà quindi ancora migliore. Finora, abbiamo sviluppato una procedura per isolare dal sangue cordonale murino le cellule ematopoietiche staminali e progenitrici, che sono state in grado di ripopolare topi C57BL/6 adulti e neonati. A questo punto verranno effettuate le prime prove di infezione con vettori lentivirali GFP e IDUA su tali cellule, per trovare le migliori condizioni per il loro trapianto nei neonati MPS-I. Questi risultati potrebbero aprire la strada verso lo sviluppo di un approccio di terapia genica neonatale con cellule di cordone ombelicale geneticamente corrette nei pazienti MPS-I. The aim of this PhD project was the preclinical testing of a new possible therapeutic option for Mucopolysaccharidosis type I (MPS-I): the transplantation of umbilical cord blood (UCB) in neonatal age. MPS-I is a rare lysosomal disease due to mutations in the IDUA gene, which encodes for the lysosomal enzyme α-L-iduronidase (IDUA). The absence of IDUA activity leads to the accumulation of glycosaminoglycans (GAGs) in patients’ tissues, which causes a progressive multi-organ dysfunction, with a wide spectrum of skeletal anomalies. The first-choice therapy for MPS-I is hematopoietic stem cell transplantation (HSCT) from healthy donor, because it reduces the accumulation of substrates and it solves many clinical symptoms, but this treatment is not very effective on the skeletal defects. The aim of this thesis was to test in the murine model a novel transplantation strategy for MPS-I, combining early (neonatal) intervention and the use of murine UCB as a source. Indeed, we decided to treat our mouse model at early age, in order to prevent the anomalies, and to employ UCB cells (UCBCs) as a source for transplantation, because UCB transplantation (UCBT) has shown advantages over bone marrow transplantation in patients suffering from inherited metabolic disorders. The first result of this work was the characterization of the phenotypical and functional properties of murine hematopoietic UCBCs compared to adult bone marrow cells. Then, adult wild-type (WT) C57BL/6 mice were transplanted with UCBCs, and they reached good long-term engraftment levels, with the presence of cells of donor origin among all the hematopoietic lineages. Finally, the outcome of UCBT on neonatally-transplanted MPS-I mice was evaluated: an increase of IDUA activity was evident in the peripheral organs of high-engrafted MPS-I mice (engraftment >50%), and, consequently, GAG levels reduced. An extensive characterization of the skeletal phenotype was performed by radiographs, microCT, and histology. Radiographic images showed that MPS-I untreated mice had increased radio-opacity and diameter of the bones at 20 weeks of age, compared to WT, and these parameters were reduced in high-engrafted mice. MicroCT scans and histomorphometry revealed that the cortical region of MPS-I femurs appeared irregular, and returned to normal in treated mice with high-engraftment, confirming the benefit of neonatal UCBT on MPS-I mice. In collaboration with Alessandro Aiuti’s group at Ospedale San Raffaele-Tiget (Telethon Institute for gene therapy), a new project started with the aim of gene-correcting murine UCBCs to obtain a supra-physiological expression of IDUA enzyme. The goal is to transduce MPS-I murine UCBCs with a PGK-IDUA lentiviral vector, and to transplant gene-corrected cells into MPS-I newborns. We will verify if we can obtain in the recipients higher levels of IDUA activity than transplanting WT UCBCs, and if the outcome of gene therapy could be better than the one obtained with normal UCBCs. A method was developed to isolate hematopoietic stem and progenitor cells (HSPCs) from murine UCB and to culture them for lentiviral infection. WT untransduced UCB-HSPCs engrafted WT adult and newborn mice at high levels. We are now in the process of testing the GFP and IDUA vector on MPS-I UCBCs, to set the best conditions for their transplantation in MPS-I neonates. These results will hopefully pave the way for developing a neonatal gene therapy approach with lentivirally-corrected UCB cells in MPS-I babies.

Background aims: Cord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated. Methods: MSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. Results: We were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. Conclusions: Our results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering.

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs). The increasing interest in newborn screening procedures for LSDs underlines the need for alternative cellular and gene therapy approaches to be developed during the perinatal period, supporting the treatment of MPS patients before the onset of clinical signs and symptoms. The rationale for considering these early therapies results from the clinical experience in the treatment of MPSs and other genetic disorders. The normal or gene-corrected hematopoiesis transplanted in patients can produce the missing protein at levels sufficient to improve and/or halt the disease-related abnormalities. However, these current therapies are only partially successful, probably due to the limited efficacy of the protein provided through the hematopoiesis. An alternative explanation is that the time at which the cellular or gene therapy procedures are performed could be too late to prevent pre-existing or progressive organ damage. Considering these aspects, in the last several years, novel cellular and gene therapy approaches have been tested in different animal models at birth, a highly early stage, showing that precocious treatment is critical to prevent long-term pathological consequences. This review provides insights into the state-of-art accomplishments made with neonatal cellular and gene-based therapies and the major barriers that need to be overcome before they can be implemented in the medical community.

Neonatal bone marrow transplantation (nBMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in Mucopolysaccharidosis type I (MPS IH, Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-L-iduronidase (IDUA). MPS IH is characterized by a broad spectrum of clinical manifestations including severe progressive skeletal abnormalities. Although BMT increases the life span of MPS IH patients, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal bone marrow into newborn MPS I mice, soon after birth, can prevent skeletal dysplasia. 1- to 2-day-old mutant mice were conditioned using a single administration of 20 mg/kg busulfan and then injected via the superficial temporal vein with 2 x 106 bone marrow cells from wild type (WT) donors. Age-matched WT and untreated MPS I mice were used as controls. Transplantation of normal bone marrow cells into preconditioned MPS I and WT neonates led to a similar engraftment level at 37 weeks after nBMT (peripheral blood, median MPS I nBMT 81.30%, range from 0.80% to 95.80% vs. median WT nBMT 67.45%, range from 16.00% to 95.86%, p = 0.714). Spleen, PB and thymus cells of nBMT MPS I mice were repopulated with committed lymphoid and myeloid populations similar to the transplanted WT mice. The >50% replacement of the hematopoiesis resulted in a measurable increase in IDUA activity in visceral organs, especially in the spleen, showing a correlation between engraftment levels and enzyme activity with clearance of GAGs from blood and tissues. At the time of euthanasia (37-week-old), reconstitution of normal hematopoiesis in MPS I mice was associated with a consistent amelioration of bone pathology, as revealed by radiographic skeletal examination. Radiographic analysis has shown that the width of the humerus, radius/ulna, femur and tibia of untreated MPS I mice was significantly larger at comparison with WT littermates. For MPS I nBMT mice, long bone widths, including the humerus (p = 0.0014, vs. untreated MPS I mice), the radius/ulna (p = 0.0003, vs. untreated MPS I mice), the femur (p = 0.0003, vs. untreated MPS I mice), and the tibia (p = 0.0003, vs. untreated MPS I mice) significantly decreased, compared to untreated MPS I mice. Furthermore, several three-dimensional architectural parameters in femurs such as trabecular number and separation, cortical thickness and bone mineral volume were analyzed by micro-CT, resulting in a significant difference between untreated and nBMT MPS I mice. All examined nBMT MPS I mice displayed bone parameter values comparable to WT mice, confirming that nBMT mice had significant improvements in skeletal phenotype approaching complete normalization of each parameter tested. Histologically, in MPS I cortical bone, osteocytes were increased and contained vacuoles, likely reflecting GAGs storage. Histological amelioration of these features was consistently observed in femurs of all nBMT mice with a definite reduction in both hyperosteocytosis and lysosomal vacuolization, confirming that the perinatal treatment of the disease can positively impact the skeletal phenotype in MPS I. We also evaluated KS levels in the blood as a biomarker of MPS with skeletal dysplasia. Normalization of blood KS level strongly supports the notion that nBMT corrects the pathological and clinical bone lesions in nBMT MPS I mice. Our findings demonstrate that nBMT prevents some of the relevant abnormalities of the skeletal pathology in the MPS I mouse model. Moreover, improvements in bone parameters correlated with high levels of bone marrow-derived cell engraftment in multiple hematopoietic compartments, suggesting that the early and complete restoration of normal hematopoiesis can have significant impact on the bone development of newborn MPS I mice. Future clinical trials are needed to confirm our findings. Disclosures No relevant conflicts of interest to declare.