Modificazione genica di cellule staminali distrofiche allo scopo di trapianto autologo nella distrofia muscolare di Duchenne

Nella Distrofia Muscolare di Duchenne, sono molto colpiti sia i muscoli scheletrici che cardiaci. La terapia cellulare \ue8 un approccio promettente; le cellule staminali, infatti, possono essere isolate da un donatore sano o, quando possibile dallo stesso paziente. Nel primo caso le cellule saranno trapiantate in un regime di soppressione immunitaria, mentre nel secondo caso, le cellule dovranno essere geneticamente corrette prima del trapianto nello stesso paziente da cui sono state derivate. Il nostro laboratorio ha messo a punto la tecnica di \u201cexon skipping\u201d per trovare nuovi strumenti finalizzati al trattamento della DMD. Abbiamo, per questo, trattato cani GRMD poich\ue9 rappresentano il modello animale clinicamente pi\uf9 simile alla distrofia muscolare umana. Abbiamo isolato cellule CD133+ da biopsie muscolari di cani GRMD di un anno di et\ue0 caratterizzati da un fenotipo clinico intermedio e grave. La scelta di utilizzare cani anziani \ue8 giustificata dalla necessit\ue0 di mettere a punto una terapia che non sia efficace solo in soggetti giovani ma anche in pazienti adolescenti o adulti. I cani sono stati trattati con le proprie cellule CD133+ trasdotte con un vettore lenti virale U7exon6-8 in grado di saltare gli esoni 6-8. Tutti i cani hanno ricevuto 2 iniezioni arteriose sistemiche. Dopo l\u2019iniezione, abbiamo effettuato diverse misure funzionali: claim Stairs (Time), nuoto, 6 minute walking test (6MWT). I cani iniettati con cellule CD133+ non ingegnerizzate e i cani non trattati sono sopravvissuti un anno circa dall\u2019inizio dello studio, mentre i cani iniettati con le cellule ingegnerizzate sono sopravissuti per due anni dopo la prima iniezione. L\u2019insieme di questi dati ci conferma che l\u2019iniezione con le cellule trasdotte aumenta la sopravvivenza dei cani. L\u2019espressione della distrofina nelle biopsie era variabile, compresa fra 2 = 7% ed era visibile dopo un anno dalla prima iniezione. L\u2019analisi di Western Blot delle biopsie muscolari ha confermato la presenza di diverse quantit\ue0 di distrofina, fino a circa il 6% in peso del muscolo. I risultati preclinici ottenuti nei cani, ci ha por- Progetti di Ricerca Telethon - Malattie Neuromuscolari Responsabile TORRENTE YVAN Telethon grant N. GGP09292 Totale \u20ac 98.100 Num Centri: 1 Durata (anni): 2 Anno d\u2019inizio: 2009 PROGETTI DI RICERCA TELETHON 62 tati a giudicare promettente un approccio terapeutico che vede la combinazione di terapia genica e cellulare nel trattamento della DMD. Ci sono, per\uf2, diverse mutazioni che causano la DMD, questo approccio focalizzato sulla singola mutazione offre l\u2019opportunit\ue0 di un trattamento di terapia genica e cellulare personalizzata non solo nella DMD ma anche nella terapia di diverse malattie genetich

Muscular dystrophies therapies by engineered stem cells

In DMD, skeletal and cardiac muscles are affected, leading to wheelchair dependency, respiratory failure and premature death. Recent advances have pointed out a variety of possible therapeutic approaches. A combination of these strategies might enhance the possibility of successful therapy. We isolated CD133+ stem cells from muscle biopsies of GRMD dogs. Five dogs one year old, Jo\ue3ozinho, Kak\ue0, characterized by a mild clinical phenotype and Marcinho, Danielzihno and Valdihno, characterized by a severe clinical phenotype, were treated with their own trasnduced U7exon 6-8 CD133+ stem cells. Old GRMD dogs are well characterized in term of clinical history and no variation among different dogs in the progression of the disease are observed from this age. All dogs received 2 arterial systemic injections (100 x106 cells each) through a catheter that was introduced in the left femoralis artery and reached the aortic arch at the level of the left subclavia. From a clinical point of view, Kaka had bad performance and was walking with difficulties before injection (at the age of 11 months). Marcihno was in worst condition since unable to walking and full of retraction as well Danielzihno. Valdihno showed the same clinical condition of Marcihno and Danielzhino. In order to test whether morphological and biochemical changes, we performed three different functional measures: claim Stairs (Time), swimming, 6 minute walking test (6MWT), quantitative values. After the injection, all treated dogs had a clinical performance improvement. After the first injection, all dogs had no detectable anti-dystrophin antibodies and its circulating lymphocytes did not react to transduced CD133+ stem cells. All transplanted animals were analysed at different times (6, 12 months after injection); most of the biopsies in all muscles had a morphological amelioration when compared to untreated dogs. Dystrophin expression in the biopsies was variable, ranging from 2 to 65 7% in several biopsies of the injected legs. The percentage of dystrophin expressing fibers ranged from 1 up to 7%, in 2 distant sections of 3 different biopsies each of selected muscles from Kaka and Marcihno. Western blot analysis of extracts from different biopsies of the same muscles confirmed the presence of different amount of dystrophin, varying from an undetectable signal to around 6% of a wt canine muscle. Two dogs, Cipo and Ze, received their own muscle-derived CD133+ stem cells Dando, Weiss & Colucci Ltd, Lawrence House, Lower Bristol Road, Bath BA2 9ET, United Kingdom Dando, Weiss & Colucci Limited Cambridge Office P a g e | 4 without lentiviral transduction. No clusters of dystrophin positive myofibers and clinical modifications of the performance. However, they appear stable during the last year of observation. In the group of untreated GRMD dogs, (Valzihno and Caipirihno) died of pneumonia and other complications during the follow up. This is the first demonstration of a clinical effect in old GRMD dogs that regained walking ability after autologous transplantation of engineered stem cell

Adaptive immune response impairs the efficacy of autologous transplantation of engineered stem cells in dystrophic dogs

Duchenne muscular dystrophy (DMD) is the most common genetic muscular dystrophy, affecting 1 in 5000 male births. It is caused by mutations in the dystrophin gene, leading to absence of muscular dystrophin and a progressive degeneration of skeletal muscle and loss of function. Individuals with DMD exhibit progressive muscle weakness leading to the permanent use of a wheelchair in young adolescents, and to respiratory and heart failure in young adults. We have previously demonstrated that the exon skipping method safely and efficiently drives to the re-expression of a functional dystrophin in dystrophic CD133+ stem cells injected SCID/mdx mice. Golden Retriever dystrophic dogs (GRMD) represent the best pre-clinical model of DMD, mimicking the human pathology in many genotypic and phenotypic aspects, including the inter-individual heterogeneity. Here, we assess the capacity of serially and intra-arterially delivered autologous engineered dystrophic canine CD133+ stem cells of restoring dystrophin expression in GRMD. This is the first demonstration of five-year follow up study, showing initial clinical amelioration followed by stabilization in mild and severe affected GRMD dogs. However, the occurrence of T-cell response in three GRMD dogs, consistent with a memory response boosted by the exon skipped-dystrophin protein, suggests an adaptive immune response against dystrophin

Stem cell therapy of muscular dystrophies using exon skipping approach in GRMD dogs

In DMD, skeletal and cardiac muscles are affected. A combination of different therapeutic approaches might enhance the possibility of successful therapy. We isolated CD133+ cells from muscle biopsies of GRMD dogs. Two dogs characterized by a mild clinical phenotype and three characterized by a severe clinical phenotype, were treated with their own transduced U7exon 6-8 cells. Old GRMD dogs are well characterized in term of clinical history. All dogs received 2 arterial systemic injections through a catheter that was introduced in the left femoralis artery and reached the aortic arch at the level of the left subclavia. We performed three different functional measures: claim Stairs (Time), swimming, 6 minute walking test (6MWT). After the injection, all treated dogs had a clinical performance improvement. After the first injection, all dogs had no detectable anti-dystrophin antibodies and its circulating lymphocytes did not react to transduced CD133+ cells. All transplanted animals were analysed at different times; most of the biopsies in all muscles had a morphological amelioration when compared to untreated dogs. Dystrophin expression in the biopsies was variable, ranging from 2 to 65 7% in several biopsies of the injected legs. The percentage of dystrophin expressing fibres ranged from 1 up to 7%, in 2 distant sections of different biopsies each of selected muscles from the dogs. Western blot analysis from different biopsies of the same muscles confirmed the presence of different amount of dystrophin, varying from an undetectable signal to around 6% of a wt canine muscle. Two dogs received their own CD133+ cells without lentiviral transduction. In the group of untreated GRMD dogs, two dogs died of pneumonia and other complications during the follow up. This is the first demonstration of a clinical effect in old GRMD dogs that regained walking ability after autologous transplantation of engineered stem cells

Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs

Duchenne muscular dystrophy is the most common genetic muscular dystrophy. It is caused by mutations in the dystrophin gene, leading to absence of muscular dystrophin and to progressive degeneration of skeletal muscle. We have demonstrated that the exon skipping method safely and efficiently brings to the expression of a functional dystrophin in dystrophic CD133+ cells injected scid/mdx mice. Golden Retriever muscular dystrophic (GRMD) dogs represent the best preclinical model of Duchenne muscular dystrophy, mimicking the human pathology in genotypic and phenotypic aspects. Here, we assess the capacity of intra-arterial delivered autologous engineered canine CD133+ cells of restoring dystrophin expression in Golden Retriever muscular dystrophy. This is the first demonstration of five-year follow up study, showing initial clinical amelioration followed by stabilization in mild and severe affected Golden Retriever muscular dystrophy dogs. The occurrence of T-cell response in three Golden Retriever muscular dystrophy dogs, consistent with a memory response boosted by the exon skipped-dystrophin protein, suggests an adaptive immune response against dystrophi

Myosin Motors and Not Actin Comets Are Mediators of the Actin-based Golgi-to-Endoplasmic Reticulum Protein Transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport